PM 7/98 (4) Specific Requirements For Laboratories Preparing Accreditation For A Plant Pest Diagnostic Activity

Bulletin OEPP/EPPO Bulletin (2019) 49 (3), 530–563 ISSN 0250-8052. DOI: 10.1111/epp.
12629
European and Mediterranean Plant Protection Organization

Organisation Européenne et Méditerranéenne pour la Protection des Plantes PM 7/98 (4)
Diagnostics
PM 7/98 (4) Specific requirements for laboratories preparing

accreditation for a plant pest diagnostic activity
Specific scope Specific approval and amendment

This guideline includes specific quality management First approved in 2009–09.
requirements for laboratories preparing for accreditation First revision approved in 2014–04.
according to the ISO/IEC Standard 17025 General A second revision was prepared to incorporate the con-
requirements for the competence of testing and calibration clusions and recommendations of the Workshop on Flexible
laboratories (2017, references to relevant parts of ISO/IEC Scope 2017-06-26/28. Second revision approved 2018-09.
Standard 17025 are included1). It should be noted that in Third revision approved 2019-09.
EPPO Standards the verb ‘should’ carries the highest level
of obligation and is the equivalent of ‘shall’ in the ISO
Standard.
laboratories, however, need to extend their scope to cover

1. Introduction
more of their regular diagnostic activities.
Many laboratories in the EPPO region establish quality This is now possible with accreditation under a flexible
management systems (also referred to as management sys- scope. In this Standard, scopes of accreditation are
tems or quality systems) and apply for accreditation. Two described in section 3 and requirements for flexible scope
EPPO Standards have been developed on these topics. The in section 6.
Standard PM 7/84 Basic requirements for quality Accreditation against the ISO/IEC Standard 17025 is
management in plant pest diagnostic laboratories was granted by national accreditation bodies, so it is important
adopted in 2007 and revised in 2018 (EPPO, 2018a). It that laboratories develop good communication procedures
describes basic requirements to support laboratories con- and establish regular contact with their national accredita-
ducting plant pest diagnostics in designing their quality tion body throughout the process.
management system. PM 7/98 (current Standard) describes This document does not deal with health and safety mat-
requirements for laboratories applying for accreditation. ters. Laboratory practices should conform to national health
Compared to its previous versions, which included cross- and safety regulations.
references to PM 7/84, it is now a standalone document. It
reflects the requirements of the revised ISO/IEC Standard
2. Terms and definitions
17025 General requirements for the competence of testing
and calibration laboratories (ISO/IEC, 2017). The main Definitions of terms used in this standard are included in
addition compared to the previous version of PM 7/98 is a PM 7/76 Use of EPPO diagnostic protocols (EPPO,
more comprehensive guidance on risk management. 2018b).
Until recently, laboratories usually applied for accredita- In this Standard, ‘test’ refers to the application of a
tion for only a small number of pests/test/matrix combina- method to a specific pest and a specific matrix. The meth-
tions for which they carry out routine testing, and not for ods concerned include the following: bioassay methods,
all pests which they are likely to test for. Many biochemical methods, fingerprint methods, isolation/extrac-
tion methods, molecular methods, morphological and mor-
phometrical methods, pathogenicity assessment and
1
A new ISO 17025 was approved on 13 Dec 2017 and its imple- serological methods. Most test results are given in qualita-
mentation will be required by 01 Dec 2020 tive terms (test positive or negative or undetermined). It is
530 ª 2019 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 49, 530–563

PM 7/98 (4) 531
recognized that some tests will generate quantitative data In this section, management requirements covered in sec-
(e.g. optical density for ELISA, number of cells for IF, Ct tions 4 to 8 of ISO 17025 are described.
or Cq values for real-time PCR, measurements for morpho-
logical features, etc.). However, such quantitative data in
4.1. General requirements (based on ISO 17025, 2017,
most cases are used to assign a qualitative result to the test
point 4)
(positive/negative/undetermined).
As stated in PM 7/84, ‘In the context of a plant pest • Laboratory activities should be undertaken impartially,
diagnostic activity, results of one or more tests can be com- structured and managed so as to safeguard impartiality.
bined to contribute to a diagnosis’. • Risk to impartiality should be identified and prevented,
Terminology varies between different international stan- e.g. possible conflicts of interest between personnel and
dards. A comparison table maintained by the EPPO Secretariat activities performed.
and the Panel on Diagnostics and Quality Assurance is avail- • Confidentiality of results to customer and of customer’s
able at https://upload.eppo.int/download/221odbcdc6308. information is guaranteed. However, findings of regulated
pests or new pests and associated customer information
should be reported to the NPPO (including a requirement
3. Scope of accreditation: fixed and flexible
for customers from other countries to report such findings
scope
to the NPPO of their country). The customers will be
A laboratory can be accredited for different scopes: fixed notified of the information provided.
and/or flexible.
A fixed scope defines clearly and unambiguously the
4.2. Structural requirements (based on ISO 17025,
range of tests covered by the laboratory’s accreditation (e.g.
2017, point 5)
immunofluorescence test for the detection of Clavibacter
michiganensis subsp. sepedonicus on potato tubers). How- • The laboratory should be a legal entity, or a defined part
ever, a fixed scope does not readily allow new or modified of a legal entity, that is legally responsible for its labora-
tests to be added to a laboratory’s scope, even when the tory activities. A government laboratory is deemed to be
competence of the laboratory in performing and validating a legal entity on the basis of its governmental status.
related tests has already been evaluated by an accreditation • The laboratory should identify management that has over-
body. Any change in the tests included in a fixed scope of all responsibility for the laboratory and have personnel
accreditation is allowed after appropriate assessment and who have the authority to carry out their duties.
decision by the accreditation body. Although applications • Appropriate resources are available to conduct the plant
for an extension to the scope can be made at any time, the pest diagnostic activity, for example: personnel, facilities
timescales involved may actually prevent quick reactions to and equipment (see also section 5 ‘Technical require-
clients’ demands. Consequently, the concept of flexible ments’).
scope has been developed. A flexible scope of accreditation • The facilities and all activities should be described,
allows a laboratory to report the results of certain tests as including temporary or mobile facilities and the cus-
accredited, prior to an audit by the accreditation body. tomer’s facilities (laboratories at border inspection points
Requirements for both types of scopes are provided in sec- or onsite testing). This should be documented, and the
tions 4 and 5, whereas additional requirements for flexible quality documents should be archived (see also below).
scope are in section 6. Descriptions of scopes are provided • Responsibilities and tasks of personnel are clearly defined
in Guideline for the formulation of scopes of accreditation (e.g. by organizational flow charts) and appropriately
for Laboratories (ILAC-G, 1804/2010). assigned.
• Procedures and instructions are documented to the extent
necessary to ensure consistent application of the labora-
4. Management requirements
tory activities and the validity of the results.
The laboratory should establish, implement and maintain a • The laboratory management (e.g. the institute manager)
quality management system that is capable of supporting should commit to bringing into effect the goals of quality
and demonstrating the consistent achievement of the management with effective communication within the
requirements of this Standard and assuring the quality of laboratory and with customers, and to continually improv-
the laboratory results. Two options for management systems ing the effectiveness of its quality management system.
are provided in ISO 17025: 2017: the management system
is either established in accordance to ISO 9001 (8.1.3
4.3. Resource requirements (based on ISO 17025, 2017,
Option B of the ISO Standard) or following the require-
point 6)
ments described in points 8.2 to 8.9 of ISO 17025 (8.1.2
Option A). The choice of options by the laboratory should Requirements for personnel, facilities and equipment are
be discussed with the accreditation body. Option A is presented in sections 5.2, 5.3 and 5.5, respectively, of this
addressed in this EPPO Standard. EPPO Standard.
ª 2019 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 49, 530–563

532 Diagnostics
Requirements for externally provided products and ser- The laboratories should consider the risks and opportuni-
vices (based on ISO 17025, point 6.6) are provided below. ties, and define actions to address these.
• Purchased supplies (e.g. equipment) and services (calibra-
tion services, proficiency testing services, testing services,
4.6. Risk management
e.g. sequencing) should be appropriate for the intended
use, based on an established assessment procedure (ISO The revised ISO Standard 17025 places more emphasis on
points 6.6.2 and 6.6.3). risk-based thinking leading to risk management. The
• Any subcontracted testing work should be authorized in Panel on Diagnostics and Quality assurance recommends
accordance with EPPO Standard PM 7/130 Guidelines on that in order to perform risk management, the processes
the authorization of laboratories to perform diagnostic and objectives of the laboratory should be identified at
activities for regulated pests. operational (e.g. performing testing) and strategic (e.g. staff
• The laboratory should inform the customer when specific management) levels. The risk and opportunity analysis
laboratory activities under the scope of accreditation are should be conducted to identify the critical points of the
performed by external providers and gain the customer’s processes using tools such as strength, weaknesses, opportu-
approval (ISO point 7.1.1. c). nities and threats (SWOT) analysis, mind mapping or the
failure mode and effects analysis (FMEA) tool. The risk
management should be proportional to the potential impact
4.4. Process requirements (based on ISO 17025, 2017,
on the test validity and the effectiveness of the actions
point 7)
should be evaluated. The Supporting Information for this
• A process is in place to review requests, tenders and con- Standard presents more details on the risk management
tracts for their feasibility (including the selection of tests), illustrated with examples (see section Supporting Informa-
and this review should be documented. tion S1).
• When a test is requested by a customer, the laboratory The risk analysis before performing validation or verifi-
should inform the customer when it is considered to be cation is described in section 5.4.3.
inappropriate. Deviations requested by the customer A risk analysis for nonconforming work should be per-
should not impact the integrity of the laboratory or the formed to determine the risk level of the nonconforming work
validity of the results. and if there is an impact on previous activities and test results.
• A process is in place to deal with complaints. Based on the results of the risk analysis, subsequent action
• A process is in place to record, analyze and correct any should be taken (e.g. acceptability of the nonconformity or
deviation from procedures or requirements of the cus- recall of the test results and notification to the customer).
tomer. Information on nonconforming work should be recorded.
• Laboratories should have access to the data and informa-
tion necessary to perform laboratory activities. The infor-
5. Technical requirements (ISO 17025, 2017,
mation management systems should be protected from
points 6 and 7)
unauthorized access and safeguarded against tampering
and losses.
5.1. General
• The laboratory information management system should
be validated (commercial software can be considered as These technical requirements include resource and process
sufficiently validated). Whenever there are any changes, requirements (ISO 17025, 2017, points 6 and 7).
including laboratory software configuration or modifica- Many factors determine the reliability of the test results.
tions to commercial off-shelf software, they should be These factors include:
authorized, documented and validated before implementa- • personnel
tion. • facilities and environmental conditions
• plant pest diagnostic tests
• equipment
4.5. Management systems requirements (based on ISO
• reference materials/cultures
17025, 2017, point 8)
• sampling
ISO 17025 point 8 provides details on the management sys- • sample handling.
tem requirements. Some additional notes are provided below.
• The management system should document the policies
5.2. Personnel (ISO 17025, 2017, points 5.2 and 6.2)
and objectives of the laboratory to fulfil the requirements
of ISO 17025. Examples of policies are staff recruitment The laboratory management should define and ensure the
and training, and purchase of material. Examples of competence and expertise of those who perform each speci-
objectives are to include, improve or maintain compe- fic stage of the plant pest diagnostic activity and their com-
tence, to increase the number of tests per year, and to petence to use the equipment. The laboratory management
meet demands of customers or changes in equipment. should also ensure that the laboratory personnel, whatever

PM 7/98 (4) 533
their status (e.g. students, staff seconded from another orga- and reliability of the test results. Failures resulting from
nization), carry out their work in an impartial manner and deviating environmental conditions should be documented
respect confidentially requirements. and corrective actions recorded (see Appendix 2).
Personnel performing specific tasks should be qualified Measures should be taken to ensure good housekeeping
on the basis of appropriate education, training, experience in the laboratory. Space should be sufficient to allow work
and/or demonstrated skills (see examples in Appendix 1). areas to be kept clean and tidy. Clothing appropriate to the
Staff undergoing training should be appropriately super- testing being performed should be worn, especially when
vised and authorized. Staff records should be maintained, working in microbiological and molecular laboratories.
including records concerning the date on which authoriza-
tion and/or competence to perform a specific task is con-
5.4. Diagnostic tests
firmed, and training records. A procedure should be put
into place to review, ensure and monitor competence; this 5.4.1. General
is especially critical after long absences. The laboratory should use appropriate tests and procedures
for all analyses performed within its scope. (ISO 17025,
2017, point 7.2.1.1). These include sampling, handling,
5.3. Facilities and environmental conditions (ISO
transport, where relevant, storage, preparation and testing
17025, 2017, point 6.3)
of samples. It is expected that plant pest diagnostic labora-
Laboratory facilities should be such as to enable correct tories will have an understanding of the biology of organ-
performance of the plant pest diagnostic activities. isms and take this into account when subsampling and/or
Depending on the type of testing being performed, differ- when preparing the sample for testing. Equipment, reagents
ent steps of plant pest diagnostic activities may be com- and consumable materials should be appropriate for the
bined in a working area, provided that necessary intended use.
precautions are taken to avoid cross-contamination result- All instructions, standards, technical manuals and refer-
ing from samples, reference materials and facilities (see ence data relevant to the work of the laboratory should be
Appendix 2). Specific guidance on handling quarantine kept up to date and made readily available to personnel.
organisms has been developed (see Table 1 in EPPO Stan- Deviation from tests should occur only if documented, tech-
dard PM 3/64 Intentional import of live organisms that nically justified, authorized by an appropriate person and
are plant pests or potential plant pests and EU Directive accepted by the customer (ISO 17025, 2017, point 7.2.1.7).
2008/61/EC).
A laboratory usually comprises testing facilities and 5.4.2. Selection of tests
ancillary facilities (entrances, corridors, storage rooms, toi- The laboratory should select tests that are suitable accord-
lets, archives, etc.). Separate locations or clearly separated/ ing to the circumstances of use (see EPPO Standard PM 7/
designated working areas are recommended for the follow- 76 Use of EPPO diagnostic protocols). Tests described in
ing: the legislation (e.g. European Union or national legislation)
• reception of samples are mandatory for the countries concerned. If no test is
• preparation of samples (segregate location for samples mandatory, tests published as international, regional or
likely to be highly contaminated or powdery, e.g. soil national standards should, preferably, be used. Whenever
samples, plants infected by fungi, insects or mites, tubers such tests are not available or whenever performance could
with soil) be improved, laboratory-developed or adapted tests can be
• testing of samples considered (ISO 17025, 2017, points 7.2.1.1 and 7.2.1.4).
• storage of samples The laboratory should ensure that it selects the latest
• appropriate disposal of material and waste valid edition of a test, unless it is not appropriate or possi-
• maintenance of reference materials/cultures ble to do so.2 When necessary, the test description should
• preparation and storage of media, buffers and reagents. be supplemented with additional details to ensure consistent
Different activities can be separated by time. The work application in the laboratory (ISO 17025, 2017, point
area should be appropriately disinfected between different 7.2.1.3).
samples and/or activities. Specific requirements are men- Tests used under accreditation should be validated. Vali-
tioned in Appendix 2. dation is carried out to provide objective evidence that the
The laboratory should be appropriately equipped to test is suitable for the circumstances of use. If the test is
ensure proper storage, testing and containment of samples. not validated, it should undergo a validation process within
Access to the laboratory should be restricted to autho- the laboratory (see section 5.4.4). When a validated test is
rized personnel who should be aware of the intended use of already available, the laboratory should provide objective
a particular area and restrictions imposed on working in
such areas.
The laboratory should monitor, control and record envi- 2
A laboratory may continue to use a previous version of a test if it is
ronmental conditions where they may influence the quality still appropriate for the circumstances of use.

534 Diagnostics
Idenfy organism,
matrix, test
Risk analysis to idenfy which performance criteria need

Intended use (including input
specific needs of the client)
to be evaluated and to what extent

Define adequate performance
characteriscs input
Review of validaon or other input

Part (A) data already available
Review of possible altered

condions when performing a input
test with validaon data
available
Test envisaged not appropriate,

select a new test or develop a Documentaon of
new test or consider a change in conclusions
intended use
Not all relevant Relevant performance

All relevant performance
performance characteriscs not
characteriscs available
characterisc available available
Verificaon Combinaon of validaon

process (Fig 2) and verificaon (Fig. Validaon process
Part (B) (Fig. 3) 3) processes (Fig. 2)
Fig. 1 Outline of the process for preparation for accreditation of a plant pest diagnostic test (including risk analysis). [Colour figure can be viewed at
wileyonlinelibrary.com]
evidence that it can operate the test according to the estab- and the choices made should be justified. The general pro-
lished performance characteristics (see section 5.4.5) (ISO cess for risk analysis before performing validation and/or
17025, 2017, points 7.2.1.5 and 7.2.2). verification is described below and in Fig. 1, part A.
Before performing a validation or verification process,
the laboratory should perform a risk analysis as described Tests can be characterized based on the following perfor-
in section 5.4.3 to identify the extent of validation and/or mance criteria:
verification to be performed. analytical sensitivity
analytical specificity (inclusivity and exclusivity)
5.4.3. Risk analysis before performing validation and/or selectivity
verification (Fig. 1) repeatability
To identify which performance criteria need to be evaluated, reproducibility.
and to what extent, the laboratory should conduct a risk anal- In addition, robustness may indicate the degree of insen-
ysis for each performance criterium to identify critical ones sitivity of the test to deviations in the implementation, cir-
in order to obtain a reliable result of the test. This risk analy- cumstances and quality of the materials (e.g. age and
sis should be documented by the laboratory (see Appendix 3) condition of samples, different reagents) that occur in

PM 7/98 (4) 535
practice. Robustness often is included in the reproducibility. Review of altered conditions (examples of questions and
A separate evaluation of the robustness will often not be factors to consider).
necessary as it is part of the development of a test. • Sustainability of supply of the reagents/chemicals.
The scope of the test, e.g. detection and/or identification • Change of reagents.
of organism x in matrix y by method z, should be identi- • Change of equipment.
fied. The following points could be considered as input for
a risk analysis. Output of risk analysis (Fig. 1, part B).
At the end of the risk analysis, the laboratory will have
Intended use of the test (examples of questions and fac-
either identified and documented the extent of validation
tors to consider).
(see section 5.4.4) and/or verification (see section 5.4.5) to
• Description of the intended use (screening, on-site testing,
be performed or will need to select or develop a new test.
confirmation of a previous test result).
Examples of output of the risk analysis are presented
• Did the client express a specific expected level of perfor-
below.
mance or a specific intended use (on a contaminated area
• Transferable skills: If the data from a test using the same
for screening, on specific hosts, cost of analysis)?
method is transferable from a test for another pest (con-
• What are the impacts of the results (e.g. outbreak or sur-
sider if matrices are comparable), this could mean that
vey in non-contaminated area)?
the extent of validation can be reduced (e.g. for selectiv-
• Which statistical significance (e.g. level of confidence in
ity, repeatability, reproducibility). Example: experience
the test) is needed (impact on budget, repetitions etc.)?
with real-time PCR for Flavescence doree would allow
• In the case of a wide host range or polyphagous pest,
the extent of validation for repeatability and reproducibil-
which hosts should be considered for validation?
ity for Bois Noir to be reduced.
• Which matrices should be tested?
• Analytical sensitivity: If the quantity of target in the sam-
• Are there specific species/strains or populations to be
ple is not a limiting factor, the extent of validation for
detected (e.g. specific species/strains or population pre-
analytical sensitivity can be reduced. Example: Identifica-
sent in the country)?
tion on pure culture by PCR, as long as there is no inhi-
• Which possible cross-reactions have to be considered
bition effect (excess of matrix).
(e.g. consider local conditions to define species to be
• Analytical specificity: If the test cannot distinguish
tested for cross-reactions, such as specific populations,
between genera or species within a genus, then inclusivity
species or hosts present in an area)? Which cross-reac-
and exclusivity evaluations can be reduced. Example:
tions can be accepted?
Nematode extraction methods are not specific for one
• Which performance characteristics for analytical sensitiv-
species or one genus.
ity and analytical specificity (inclusivity and/or exclusiv-
• Reagents: If the choice of reagents is critical for the per-
ity) are needed?
formance of a test, a change of reagent (or lots/batches of
• What level of flexibility is needed for the use of the test
reagent) or reagent supplier may influence the perfor-
(e.g. network of laboratories as end users)?
mance of a test. In such a case, a verification of the per-
Constraints of the laboratory (examples of questions and formance of the reagent should be done.
factors to consider). • Validation after significant change: If the laboratory
• What is the availability of reference material (pest makes a significant change to a validated test (e.g. testing
related, matrix related)? outside the original scope), this ‘new’ test has to be vali-
• What level of flexibility is needed for the use of the test dated. If a minor change to a validated test is made by
(e.g. diversity of equipment available such as a PCR the laboratory, a judgement as to whether such a change
machine with rotor or plate)? requires validation or verification should be made and
• What is the availability of resources to perform the vali- documented. Any change should be authorized by an
dation (budget, staff, equipment/reagents)? appropriate person and if relevant the customer and the
• What are the time constraints? Accreditation Body should be informed.
Review of validation data available (examples of ques-

tions and factors to consider). 5.4.4. Validation of tests (ISO 17025, 2017, point 7.2.2)
• Are validation data available for the same test and/or sim- (Fig 1, part B and Fig 2)
ilar tests that could be transferable (e.g. EPPO database 5.4.4.1. Validation of tests other than morphological and
on diagnostic expertise, validation section http://dc.eppo. morphometrical tests. As mentioned in ISO 17025, 2017
int/validationlist.php, publications)? ‘the laboratory shall record [among other factors] the vali-
• Tests included in EPPO Diagnostic protocols are not all dation procedure used [...] and the results obtained’ (point
validated, but EPPO Panels on Diagnostics considered 7.2.2.4).
that those presented in Appendix 4 give appropriate confi- The general process for validation is described below
dence regarding repeatability and reproducibility. (see also Fig. 2).

536 Diagnostics
The validation procedures described here, and the details • Plan and perform the validation for individual perfor-
given in Tables A2–A7 (in Appendix 5) should be regarded mance criteria or in a combined test setup (see Fig. 2).
as general guidance according to which a test can be vali- • Present the results in a validation report with a conclusion
dated. Figures given in these tables are based on the validaon whether the test meets the requirements identified (see
tion experience of experts from EPPO Panels dealing with Appendix 3).
diagnostics. Test performance studies can be a valuable part o Performance characteristics are met: the test is
of the validation process. validated.
Validation process o Performance characteristics are not met:
• Consider the output of the risk analysis and define a vali- ▪ Adjust the test and perform a new evaluation for the
dation plan. relevant performance criteria.
• Consider the technical requirements to determine analytical ▪ If the test cannot be adjusted, the test cannot be
sensitivity, analytical specificity (inclusivity and exclusivity), validated for the originally intended use (in specific
selectivity, reproducibility and repeatability performance cases adapting the intended use of the test may be con
values by consulting the guidelines in Tables A2–A7 as sidered).
required. Then define the type and composition of samples A comparison of a test (A) with a validated test (B) is an
needed for the validation. Validation is to be performed with alternative means of validation which may be suitable in
reference material (see definition in PM 7/76) and/or spiked certain situations (see Appendix 6). This can only demon-
samples. When using cultures or isolates for biological tests, strate that, for example, test A is as good as validated test
care should be taken that they have a proven virulence. B with respect to selected performance criteria.
Validaon process
Validaon plan resulng from risk

analysis and taking into account
tables A2 to A7 (Appendix 5).
Evaluaon of the criteria selected

from risk analysis from among the
following:
• Analycal sensivity,
• Analycal Specificity
• Inclusivity
• Exclusivity
• Selecvity
• Repeatability
• Reproducibility
• Robustness.
Validaon report
Required Required
values values
met not met
Adjust the test and

Test Test not perform new evaluaon
validated validated for the relevant
performance criteria
Fig. 2 Validation process. [Colour figure can be viewed at wileyonlinelibrary.com]

PM 7/98 (4) 537
Additional information • Plan and perform the verification for individual perfor-
Collected data and results of laboratory-performed vali- mance criteria or in a combined test setup.
dations (in particular related to reproducibility), as well as • Present the results in a verification report with a conclu-
results of interlaboratory comparisons, can also provide an sion on whether or not the laboratory meets the require-
indication of the robustness of the test. ments identified.
o Performance values are met: the laboratory can perform
5.4.4.2. Validation for morphological and morphometrical the test.
tests. It is acknowledged that the procedures for morpho- o Performance values are not met:
logical and morphometrical tests are ultimately a judgement ▪ If deviation from conditions described in the validated
based on expert opinion. Validation therefore may not fol- test affects test results, investigate the reasons for this devi-
low the same procedures as for the other tests. Guidance ation. Correct, verify the test again or validate if required
for the validation of morphological and morphometrical following the procedure described in section 5.4.4.
tests is given in Appendix 7. This guidance is applicable ▪ Investigate whether the minor changes that have been
for these methods irrespective of the field they are used in introduced in the test are the cause. If it is not the case,
(entomology, nematology, mycology, botany, etc.). The lab- seek external guidance (e.g. contact the author of the test).
oratory should be able to justify the selection of morpho- Adjustments should then be made and relevant steps
logical or morphometrical tests made, in particular for repeated. If other reasons for deviation have been observed
those not described in international standards or peer-re- (e.g. staff errors) corrective action should be taken and doc-
viewed journals. umented.
▪ If the cause cannot be understood or modifications cannot
5.4.5. Verification of the performance of the laboratory to be made to allow performance values to be met, the labora-
undertake a specified test (ISO 17025, 2017, point 7.2.1.5) tory cannot operate the test according to the established
5.4.5.1. Verification process for tests other than performance criteria.
morphological and morphometrical tests.
General
5.4.5.2. Verification process for morphological and
Verification provides objective evidence that the labora-
morphometrical tests. The laboratory should confirm that it
tory is competent to perform a validated test according to
can properly carry out the validated morphological and/or
the relevant performance characteristics. Verification can
morphometrical identification. Such verification can be
also be done by successfully participating in a proficiency
achieved by taking part in a proficiency test or by having a
test or test performance study, provided that these allow the
number of samples identified in the laboratory and then
requirements in Table 1 to be fulfilled.
confirmed by an independent specialist or another validated
The general process for verification is described below
test (e.g. PCR, sequencing).
(see also Fig. 3).
Verification process
• Consider the outcome from the risk analysis and prepare a 5.4.6. Final result
verification plan. Perform the validated test as described or Use of a ‘Statement on test validation and/or verification’
with minor changes to take into account local conditions form (Appendix 3) can be valuable for summarizing the
(e.g. suppliers of reagents or equipment, unless it is specifi- results.
cally required in the validated test) to evaluate whether the
laboratory meets the performance characteristics from the 5.4.7. Evaluation of measurement uncertainty (ISO 17025,
validation data (see guidelines in Table 1). Selectivity gen- 2017, point 7.6)
erally does not need to be verified, but for serological meth- The laboratory should attempt to identify the factors influ-
ods, for example, selectivity may need to be verified to encing the uncertainty of a test, such as staff, equipment
evaluate the impact of different batches of antisera. and biological properties (i.e. serotypes, pathotypes).
Table 1. Guidance on the verification of performance criteria
Performance criteria Verification method (artificial subsamples prepared from one sample may be used)
Analytical sensitivity Analyse at least eight samples at the established limit of detection (for viruses, viroids and
phytoplasma this should be at low level).
This can be combined with repeatability/reproducibility.
Analytical specificity (inclusivity and exclusivity) Select a few of the most relevant targets (e.g. different strains, populations) for inclusivity and
non-targets for exclusivity. Tests should be performed at medium levels of organisms.
Repeatability Perform at least three simultaneous tests on the same material with low levels of target.
Reproducibility As for repeatability but at different moments, when possible with different operators and when
relevant with different equipment.

538 Diagnostics
Verification plan resulting Verification process

from risk analysis
Perform test
Verification report
Adjust
Values Values
met not met
Cause
Investigate reasons
understood/
//external
externaladvice
advice can modify
Cause not
understood/
cannot
modify
Test verified Test not verified
Fig. 3 Verification process. [Colour figure can be viewed at wileyonlinelibrary.com]
Repeatability and reproducibility will provide information reference materials and data, reagents (e.g. Mastermix,
on the level of uncertainty of the test result. Whenever pos- antisera, etc.) and consumables (e.g. pipette tips, slides,
sible, appropriate measures should be taken to control this plates, tubes). In this EPPO Standard reference material and
uncertainty. If no measures are taken, the reasons for this data are covered in point 5.6.
should be recorded and the client should be made fully The laboratory should have access to the equipment
aware of the uncertainty surrounding the test. required for testing and this should be operated by compe-
Although in most cases tests used for plant pest diagnosis tent personnel. Equipment, whether under the laboratory’s
provide qualitative results, these qualitative results may be permanent control or not, should be listed and a programme
based on measurement (e.g. morphometrical data, number should be documented and implemented for the handling,
of cells). This measurement may be just one part of the storage, transport, maintenance, verification, calibration and
diagnostic process, but if this is critical for a final result its corrective action of key equipment which significantly
uncertainty should be estimated. Two examples of labora- affects the results.
tory reports identifying critical points in the process are Equipment that has been subjected to overloading or mis-
provided in Appendix 8. handling, or gives suspect results, or has been shown to be
defective or outside specified limits should be taken out of
service, clearly labelled or marked, and appropriately stored
5.5. Equipment (except reference material and data)
until it has been repaired and shown to perform correctly
(ISO 17025, 2017, point 6.4)
when appropriate or disposed of (e.g. for reagents, consum-
Note: In ISO 17025, equipment includes, but is not limited ables). The laboratory should examine the effect of such
to, instruments (microscopes, thermocyclers, ELISA-read- deviation and initiate the management of nonconforming
ers, pipettes, etc.), software, measurement standards, work procedure (see section 4.6).

PM 7/98 (4) 539
5.5.1. Calibration and verification programmes (ISO • to validate or verify tests

17025, 2017, points 6.4.7 and 6.5) • to enable comparison of tests.
Frequency of calibration and verification should be planned
and reviewed when necessary. This can be based on risk anal- 5.6.1. Biological reference material
ysis (examples and recommendations can be found in Appen- If possible, certified biological reference material should be
dix 9). Calibrations may be performed in-house by using used, from which biological reference material, and subse-
certified or appropriate reference material provided by a com- quently working material, can be produced, but such mate-
petent producer. Calibrations should have traceability to rial is not always available. Laboratories may also produce
International System of Units (SI) whenever possible. Only their own biological reference material from which working
qualified personnel should perform calibration and verifica- material is derived. In order to maintain confidence in the
tion programmes, using procedures appropriate to the status of biological reference material verification of iden-
intended use. Calibration and verification may also be per- tity and purity should be carried out according to defined
formed externally by specialized, competent companies. procedures and schedules (including, as applicable, mor-
Documents on external and internal calibration and veri- phology, pathogenicity, virulence, antigenic properties,
fication of performance (including when the next calibration molecular properties, etc.). The laboratory should have pro-
is due) should be maintained and made available within the cedures for safe handling, transport, storage and use of bio-
laboratory. Equipment should be appropriately labelled (see logical reference material to prevent contamination or
Appendix 10). deterioration and to protect their integrity.
Working material derived from biological reference
5.5.2. Maintenance of equipment material (e.g. reference cultures) from an international col-
Up-to-date instructions on the use and maintenance of lection should be kept separate from the original material.
equipment (including any relevant manuals provided by the
manufacturer) should be readily available for use by the 5.6.2. Other sources, including reference data
appropriate laboratory personnel. Maintenance of essential These include books, pictures, slides collections, morpho-
equipment should be carried out at specified intervals logical identification keys, scientific literature and sequence
preferably based on risk as determined by factors such as databases that can be used to support diagnosis. Reference
the rate of use and age/complexity of the equipment, and data should be kept up to date and readily available.
this maintenance documented (see Appendix 11 for guid-
ance on maintenance of equipment).
5.7. Sampling (ISO 17025, 2017, point 7.3)
5.5.2.1. Records (ISO 17025, 2017, point 6.4.13). Records Sampling is a procedure in which material is collected out-
should be maintained for equipment significant to the tests side a laboratory to perform a test. A sample should be rep-
performed. Depending on the type and sensitivity of equip- resentative of the material under test and should be selected
ment, and the conditions required by the manufacturer to based on knowledge of the distribution of the pest to be
ensure failure-free running, the records should include: detected. Such a representative sample may not always be
• the identity of the item of equipment available: if so, this should be documented. Sampling usu-
• the current location ally involves targeting symptomatic plants or plant parts.
• the manufacturer’s name, type identification Correct sampling is an operation that requires careful
• the manufacturer’s instructions attention. Not all laboratories are involved in sampling.
• dates, results and copies of reports and certificates of all Laboratories involved in sampling should have a sampling
calibrations or verifications, adjustments, date of next cal- process (both plan and procedure) for collecting samples to
ibration or verification where appropriate be followed whenever practicable. This process should
• maintenance carried out to date and the maintenance plan address the factors to be controlled and be based on appro-
where appropriate priate statistical tests.
• damage, malfunction and repair of the equipment. The laboratory should have procedures for recording rel-
evant data relating to sampling whether the process is per-
formed by the laboratory staff or by the customer.
5.6. Reference material and reference data (ISO 17025,
2017, points 6.4, 6.5, 6.6 and 7.2.1.2)
5.7.1. Records of sampling (ISO 17025, 2017, point 7.3.3)
Reference material provides essential traceability in calibra- Details of sampling should be recorded and communicated
tion and testing and are used, for example: to the appropriate personnel. Records should include the
• to monitor performance of detection and identification following:
tests - sampling procedure
• to demonstrate the accuracy of results - date and time of sampling
• to calibrate or verify equipment - data to identify and describe the sample (batch number,
• to monitor laboratory performance species, etc.)

540 Diagnostics
- the name of the person who performed sampling deterioration, incorrect temperature, torn packaging or defi-
- the equipment used cient labelling, or when a sample does not conform to the
- environmental or transport conditions description provided, or if the test required is not described
- sampling location in sufficient detail, the laboratory should consult with the
- deviations, additions or exclusions from the documented customer before deciding whether to test or refuse the sam-
sampling procedure. ple. In any case, the facts and the results of discussions
should be recorded.
5.8. Sample handling (ISO 17025, 2017, point 7.4) Samples awaiting testing should be stored under suitable
conditions to minimize changes to any pest populations pre-
The laboratory should have procedures for safe transporta- sent and to protect them from cross-contamination. Storage
tion, receipt, handling, protection, storage, retention and/or conditions should be defined and recorded when necessary.
disposal of samples, including all provisions necessary to Where samples have to be returned to the customer, care is
protect the integrity of the sample and to protect the inter- required to ensure that they are not damaged.
est of the laboratory and the customer. A procedure for the retention and disposal of samples
Subsampling by the laboratory prior to testing is consid- should be written. Samples should be stored until the test
ered to be part of the test. Subsampling should be designed results are obtained, or longer if required (e.g. for potential
taking into account uneven distribution of pests. complementary analysis).
The laboratory should have a system for uniquely identi- A laboratory should have procedures in place to treat
fying samples. The system should be designed and operated contaminated samples after testing to conform to national
so as to ensure that samples cannot be confused physically or international regulations for quarantine and other plant
or when referred to in records or other documents. The sys- pests. The procedures should also be designed to minimize
tem should, if appropriate, accommodate a subdivision of the possibility of contaminating the test environment or
groups of samples and the transfer of samples within and materials. Further details on confinement conditions may be
from the laboratory. The identifier of a sample should be found in EPPO Standard PM 3/64 Intentional import of live
retained as long as this sample is retained by the laboratory. organisms that are plant pests or potential plant pests.
Suggested content for a form to identify a sample is pre-
sented in Appendix 12.
5.9. Ensuring the validity of test results (ISO 17025,
Plant pests may be sensitive to factors such as tempera-
2017, point 7.7)
ture or duration of storage and transport, so it is important
to check and record the condition of the sample on receipt The validity of test results should be ensured at different
by the laboratory. If there is insufficient material in the levels, i.e. for each test and diagnostic process, as well as
sample or the sample is in poor condition due to physical for global quality control of the laboratory.
Table 2. Internal and external quality checks (ISO 17025, section 7.7)
Elements of quality control programme Level of control*
The use of reference material (e.g. closely related organisms or collection material, non-target 1st
organisms which might be naturally present in a composite material), see section 5.6
Internal/endogenous control (e.g. COX, NAD5, 18S) 1st
The use of artificially contaminated samples 1st or 2nd
Replicate testing using the same test (technical replicates or repeated testing) 1st or 2nd
Comparative testing of the same sample by different operators 1st or 2nd
Vertical audit† for a specific sample/analysis of records 2nd or 3rd
Blind testing by processing samples with known levels of pests between routine samples 2nd
Comparison of results of different tests based on different biological principles 2nd
Retesting of retained plant material or extracts thereof, water or soil samples and insect traps 2nd
(within a predetermined suitable storage time and condition of the material before retesting)
Trend analysis on 1st, 2nd and 3rd line controls (e.g. positive controls, Shewhart chart or results 2nd or 3rd
from proficiency tests) including quantitative data
Intra- or interlaboratory evaluation of documentation of the specific determinants on which 3rd
diagnoses are based (in particular for visual determination of insects, nematodes and fungi)
Interlaboratory comparisons (in particular proficiency tests) 3rd
Supporting data (e.g. contra-expertise) 3rd
Use of alternative instrumentation that has been calibrated to provide traceable results 1st or 3rd
Functional and intermediate check(s) of measuring and testing equipment 1st or 3rd
*1st line controls are used to monitor the actual performance of the test, 2nd are used for the performance of a single operator within a laboratory and
3rd line controls evaluate the performance of the laboratory.
†
Checking all steps of the diagnostic process for a particular sample.

PM 7/98 (4) 541
Internal quality management consists of compliance with by the accreditation body. However, it places more respon-
all the procedures undertaken by a laboratory for the con- sibility on the laboratory to manage its scope of accredita-
tinuous evaluation of its work. The main objective is to tion and to demonstrate that tests are valid, suitable for
ensure the consistency of results day to day and their con- circumstances of use, and performed competently and con-
formity with defined criteria. If analysis of data is found to sistently. If the laboratory decides to report a test as accred-
deviate from the defined criteria, then appropriate action ited and an audit later identifies problems with the
should be taken to prevent incorrect results from being procedures used, results may not be valid and all reports
reported. The interval between internal quality checks (de- issued based on this test may have to be withdrawn. Experi-
fined in Table 2) will be influenced by the number of actual ence with a fixed scope of accreditation is therefore valu-
tests performed. Monitoring of test validity should be able before a laboratory applies for flexible scope, as all
planned, reviewed and registered. Wherever possible posi- requirements of the ISO/IEC Standard 17025 have to be
tive/negative controls should be used: this should be the fulfilled in both cases. Nevertheless, experience with a fixed
minimum level for quality control. A quality control pro- scope of accreditation in another activity may be sufficient
gramme may also consist of different checks, as described for the direct application for a flexible scope for plant pest
in Table 2. diagnostic activities. A laboratory should contact the
A procedure should be in place for managing infre- accreditation body to discuss the possible options.
quently used tests. Operators’ transferable skills may pro- EA-2/15 (2019) defines the requirements for accreditation
vide evidence of competence in tests based on the same of flexible scopes, including the following elements:
method. Whenever possible, an external quality assessment • A clear definition of the extent and the limits of the flexi-
(such as an external proficiency programme or proficiency ble scope.
tests) should be used to demonstrate competence. The • A procedure for the management of the scope (see Fig. 4
validity of test results is influenced by both technical per- as an example and the details provided below). Appropri-
formance of personnel and test performance characteristics. ate documents should be developed to ensure traceability
If the validity of test results is called into question, it is when the procedure is applied.
important to be able to distinguish between the two. A test • A list of tests included in the flexible scope is required
may demonstrate appropriate process control but poor diag- and maintained by the laboratory.
nostic performance or vice versa. The frequency and means to inform the accreditation
body of changes to the flexible scope should be agreed.
Information should be available to the client that the test is
5.10. Reporting the results (ISO 17025, 2017, point 7.8)
performed under flexible scope at the contract review stage.
See EPPO Standard PM 7/77 Documentation and reporting The flow diagram in Fig. 4 outlines the procedure for
on a diagnosis (EPPO, 2019). management of the flexible scope and includes the require-
ments stated in EA-2/15. Before adding a new test to the
scope, the laboratory should first confirm that this test fits
6. Additional requirements for flexible scope
with the current scope of accreditation. If not, the labora-
A flexible scope of accreditation allows a laboratory to tory should communicate with its accreditation body to
undertake certain tests and to report the results as accred- extend its scope of accreditation before including this new
ited, even though these tests are not explicitly stated in the test.
laboratory’s scope, but in a specific list of tests under One of the most important steps in the procedure is the
accreditation (EA Requirements for the Accreditation of authorization to add or delete a test from the accreditation
Flexible Scopes, EA-2/15, 2019). Examples of situations scope. This responsibility should be clearly defined.
where the need for flexible scope may arise are: Before authorizing the update of the list of accredited
• fast addition/deletion of tests under accreditation to tests, the laboratory should review the process leading to
answer urgent demands addition/deletion of a specific test to the scope of accredita-
• optimization of a given test tion by examining the relevance and the completeness of
• modification of an existing test to broaden its applicabil- the documentation (e.g. forms are duly completed). Audit-
ity (e.g. to deal with new matrices or similar pests) ing the process can serve as a review.
• inclusion of a test equivalent to one already covered by The laboratory should identify the consequences for the
accreditation. laboratory activities (e.g. appropriate resources, plans to
The concept of flexible scope encompasses a degree of adjust its organisation, modification of the quality manage-
flexibility which is usually agreed in consultation with the ment system). The quality management system should be
accreditation body. Nevertheless, it should be noted that updated. This may include revision of documents (e.g. stan-
this degree of flexibility has a varying interpretation at the dard operation procedures), update of internal quality con-
national level. The experience in plant pest diagnostic labo- trols (e.g. controls, participation in proficiency testing),
ratories so far is that a flexible scope is helpful as it allows maintenance of the competence (e.g. training of staff) and
a laboratory to be accredited for new tests prior to an audit updating the audit program.

542 Diagnostics
Potenal change in the list Potenal change in the

of tests list of tests
(addion) (deleon)
Availability of
necessary resources,
no qualified staff,
definion of
responsibilies?
Communicaon
Fits the scope of
with AB to no
accreditaon?
extend the scope
Risk assessment
validaon / verificaon
(see secon 5.4)
no Performances fit for purpose?
Resources sll available to perform

no the test?
Review the process

Consequences on laboratory acvies?
Update Quality Management System1**
AC* no Authorize?
yes
Update Quality Management

System2**
Update the list of tests

Included in the scope
Communicate with
End of process
accreditaon body and clients
1in case of addion of a test

2in case of deleon of a test
* AC= Analysis of the causes and correcve acons
**see details in the text
Fig. 4 Example of a procedure for the management of flexible scope. AB, accreditation body. [Colour figure can be viewed at
wileyonlinelibrary.com]

PM 7/98 (4) 543
Acknowledgements For readers looking at the paper or pdf version of this arti-
cle, please see the html version to access the supporting
This revision was initiated and worked on by the Panel on
information.
Diagnostics and Quality Assurance and finalized by an
Expert Working Group composed of Ms Anthoine, Ms
Bokuma, Ms de Krom, Ms Ermakovich, Ms Santala and Ms References (for the latest version of EPPO
Weekes. The EPPO Secretariat would like to thank all the Standards, please consult EPPO Global
experts involved and in particular Ms Anthoine and her team Database or the EPPO website)
from ANSES (FR) for the great support for the organization
AFNOR (1995) XP V03–111. Agricultural and food products analysis
of the Workshop on the revision of PM 7/98 (Maisons-Alfort, Protocol for the intra-laboratory evaluation of an alternative method
FR, 2019-02-11/13) which provided input for the preparation of qualitative analysis against a reference method. Association
of this revision. The final revision was reviewed by the mem- Francßaise de Normalisation, La Plaine Saint-Denis (FR).
bers of the Panel on Diagnostics and Quality Assurance. The EA (2019) EA-2/15 Requirements for the Accreditation of Flexible
previous revision of PM 7/98 was prepared by an Expert Scopes. http://www.european-accreditation.org/publication/ea-2-
15-m [accessed on 14 June 2019]. European Association for
Working Group composed of Ms Anthoine, Ms Baginska,
Accreditation.
Ms Dreo, Mr Inghelbrecht, Mr K€onig, Ms Reisenzein, Ms EPPO (2019) PM 7/77 (2) Documentation and reporting on a diagnosis.
Santala, Ms van der Blom, Ms Weekes and Mr Werkman. It EPPO Bulletin 49, 527–529.
was reviewed by the EPPO Panel on Diagnostics and Quality EPPO (2018a) PM 7/84 (2) Basic requirements for quality
Assurance. The EPPO Secretariat would also like to thank management in plant pest diagnosis laboratories. EPPO Bulletin 48,
Ms Edema and her team from the National Reference Center 378–386.
(NL) for the great support for the organization of the Work- EPPO (2018b) PM 7/76 (5) Use of EPPO diagnostic protocols. EPPO
Bulletin 48, 373–377.
shop on Flexible Scope which provided input for the prepara-
Hughes KJD, Griffin RL, Tomlinson JA, Boonham N, Inman AJ &
tion of this revision. It was reviewed by the members of the Lane C (2006) Development of a one step real-time PCR assay for
Panel on Diagnostics and Quality Assurance. diagnosis of Phytophthora ramorum. Phytopathology 96, 975–981.
ILAC-G18:04 (2010) Guideline for the Formulation of Scopes of
Accreditation for Laboratories, EA.
Supporting Information ISO (2016) ISO 16140–2 Microbiology of the food chain – method
S1: Supporting information on process approach and risk validation – Part 2. Protocol for the validation of alternative
(proprietary) methods against a reference method. Available on
management.
www.iso.org [accessed on 01 Feb 2019].
S2: Supplementary Information - List of tests included in ISO/IEC (2017) Standard 17025. General requirements for the
EPPO Diagnostic protocols that are widely used competence of testing and calibration laboratories. Available on
S3: Supporting information Examples of laboratory www.iso.org [accessed on 01 February 2018].
reports on the critical points in the diagnostic process and Petter F & Suffert M (2010) Survey on the use of tests mentioned in
relating to uncertainty of measurement. EPPO diagnostic protocols. EPPO Bulletin 40, 5–22.
Appendix 1 – Expertise and competence

An expert will have a combination of deep knowledge in a specific field, longstanding experience and particular cognitive
skills.
A competent person will be able to demonstrate that he/she can perform a particular task successfully.
For example, for the selection of morphological or morphometrical methods expertise is required. For the use of the
selected tests the laboratory should confirm that its staff is competent to carry out the morphological and/or morphometrical
identification.
Examples of factors to consider when evaluating expertise or competence can be found in Table A1.

544 Diagnostics
Table A1. Examples of factors to consider when evaluating expertise or competence
Expertise (in a specific field) Competence (for a particular task)
Education/training – diplomas/certificates Education/training – diplomas/certificates
Peer evaluation Interlaboratory comparison (in particular, proficiency testing
Proven track record – successful outcomes Blind samples
Measure of esteem, e.g. member of international Internal controls (including data trending where possible) –
Working Group or Panels, journal editor, reviewer, validation data
technical expert, keynote speaker, invited expert, technical assessor
Publications – relevant to the area of work Contra-expertise inside or outside the laboratory
Annual review/validation Audit (both internal and external)
Continued professional development (CPD) leading to CPD

a professional qualification (e.g. in the UK Royal Society
of Biology – chartered biologists/plant health professional)
Appendix 2 – Environmental monitoring and avoidance of contamination

The laboratory should ensure that environmental conditions, laboratory arrangements and working procedures are such as to
minimize the risk of cross-contamination through air, surfaces, equipment, personnel, etc. Contamination can be minimized
or avoided in the following ways:
• Laboratory equipment should not routinely be moved between different areas inside the laboratory.
• Where relevant a documented vector control programme should be in place.
• Reference materials/cultures should be stored in a separate location in the laboratory.
• Housekeeping and cleaning procedures should be defined, implemented and documented.
• Hygienic working procedures (e.g. use of “sticky” carpets when appropriate, use of gloves disinfectants, filter tips for pip-
ettes, disposable plastic ware) should be defined and implemented.
• Careful handling of samples.
The laboratory should monitor the quality of laboratory air and surfaces of relevant areas at regular intervals. The moni-
toring can be done by using air settlement plates (e.g. plate count agar or other appropriate non-selective plates), contact
plates (for even surfaces) or swabbing (for other surfaces and equipment), and insect traps. Buffers exposed to air or surfaces
can also be tested. For laboratories working on nematodes, the normal hygienic procedures ensure that contamination is
avoided.
Specific additional requirements for molecular laboratories
• Dedicated PCR work areas should be organized for (a) nucleic acid extraction and purification, (b) preparation of master-
mix, (c) addition of sample to the mastermix, (d) nucleic acid amplification and (e) analysis of amplification products. It is
highly recommended to have at least three distinct rooms. Preparation of mastermix, nucleic acid extraction, and/or analy-
sis of amplification products should not be performed in the same room.
• Dedicated equipment (including pipettes) should be used in each work area. Dedicated laboratory coats should preferably
be used in each work area (at least a specific coat for mastermix preparation) and gloves should be worn.
• Tubes containing amplification reaction products should not be opened within work areas used for nucleic acid extraction
or mastermix/reaction mixture preparation.

PM 7/98 (4) 545
Specific guidelines for monitoring contamination with bacteria and fungi
Air settlement plates, preferably three in each area to be monitored, should be exposed to air contaminants for a definite
time (30 min recommended), closed and incubated for 3 days (at approximately 30°C) or 5 days (at room temperature).
Contact plates should be exposed on the surfaces to be monitored for 15 s (recommended), closed and incubated as
above.
The acceptable level of cfu/plate/area (background counts) for bacteria or fungal colonies should be defined by the
laboratory according to the testing being carried out and according to the special requirements of the environment (e.g.
clean rooms). Environmental monitoring should be documented, corrective actions described and performed if needed
and recorded. Cleaning should be intensified if needed and new samples taken after corrective actions have been per-
formed.
Appendix 3 – Statement on test validation and/or verification

Test name
Scope of test
Intended use of the test
Summary of the risk analysis
Performance criteria Selected for validation Selected for verification Not selected Where to find information
Analytical sensitivity
Analytical specificity Inclusivity
Analytical specificity Exclusivity
Selectivity
Repeatability
Reproducibility
Robustness
Report on validation
Additional comments
Documentation for the validity of the test and the requirements that the test should meet are available in the laboratory. Doc-
umentation includes laboratory books and other information as indicated below, which shows how procedures have been val-
idated in this study.
Performance criteria NA A B C D Where to find documentation
Diagnostic sensitivity
Analytical specificity: Inclusivity
Analytical specificity: Exclusivity
Diagnostic Specificity:
(continued)

546 Diagnostics
Table (continued)
Performance criteria NA A B C D Where to find documentation
Selectivity
Repeatability
Reproducibility
Robustness
NA, not applicable; A, data from own laboratory experiments; B, data from interlaboratory comparison; C, information from
manufacturers; D, external information (literature, etc.); other information (optional).
Report on verification
Description of changes
Documentation for the verification of the test and the requirements that the test should meet are available in the laboratory.
Documentation includes laboratory books and other information which shows how the verification has been performed in
this study.
Performance characteristics Meet requirements of the Where to find

Performance criteria obtained validated test (yes/no) documentation
Analytical specificity: Inclusivity
Analytical specificity: Exclusivity
Repeatability
Reproducibility
On the basis of the above statement the validity and/or verification of the test is judged suitable for the scope of the test.
Person responsible for carrying out the test

Name in block capitals:
Signature, date:
Authorising person
Name in block capitals:
Signature, date:

PM 7/98 (4) 547
Appendix 4 – List of tests included in EPPO Diagnostic protocols that are widely used
A survey carried out in 2008 and repeated in 2013 on the use of tests included in EPPO Diagnostic Protocols (Petter & Suf-
fert, 2010) showed that those presented in this appendix are widely used. Consequently, EPPO Panels on Diagnostics consid-
ered that these tests give appropriate confidence regarding repeatability and reproducibility. A laboratory implementing these
tests should at least produce or collect validation data regarding analytical sensitivity and analytical specificity.
Tests must have been used in a minimum of two laboratories and for a minimum of eight samples in each laboratory to
be considered widely used. Please note that molecular tests include nucleic acid extraction.
Since the surveys, several protocols have been updated and new tests added. However, only the tests that included
protocols at the time of the surveys are listed here as we do not have data on the frequency of use of the tests in the
subsequently revised protocols.
The list of tests included in EPPO Diagnostic Protocols that are widely used is provided as supplementary information
(see section Supplementary Information S2).
Appendix 5 – Tables giving detailed guidance for the validation process by field
(bacteriology, botany, entomology, mycology, nematology, virology and phytoplasmology)
Instructions for the use of the tables3
Comment on the figures

The figures given in these tables are based on the validation experience of experts from EPPO Panels dealing with diagnos-
tics. Deviations from this guidance may be possible or necessary depending on pest/matrix combination. Validation for mor-
phological and morphometrical methods for all fields are described in Appendix 7.
General note on analytical sensitivity

Whenever possible, the limit of detection (as defined in PM 7/76) of a test should be determined. Nevertheless, this limit
cannot always be established absolutely while detecting plant pests. There are organisms that cannot be cultured (obligate
pathogens), which cannot be quantified (fungi), which are only present in the plant or which cannot be purified (e.g. phyto-
plasmas). For this reason, exact concentrations of these organisms cannot or can hardly ever be established accurately and
so estimates have to be used. Even with those that can be purified (many bacteria and viruses), the concentration can only
be estimated (e.g. cfu or mg per mL). This estimation is often based on an indirect measurement. Where applicable, serial
dilutions should be carried out until an end point is achieved.
Analytical sensitivity refers to a specific set of test parameters which should be stringently defined and standardized, for
example brand of PCR reagents (in particular DNA polymerase), PCR cycle conditions, the number of microscope fields to
read in the IF test, the OD threshold in the ELISA test, brand of ingredients for the medium (in particular antibiotics and
preparation of stock solutions) and incubation conditions, e.g. stage of test plants, inoculation method and incubation condi-
tions.
General note on replicates

The number of replicates (given in the tables below) does not refer to the number of technical repetitions (e.g. duplicate/trip-
licate reactions which are carried out as standard for ELISA tests or real-time PCR runs).
3
It should be noted that during country consultation comments were received on these tables. The Panel on Diagnostics and Quality Assurance considered
these during their 2018 meeting. They considered that it was not possible to address the changes proposed at this stage (additionally, comments were made
for some disciplines only and these tables have been designed to be as harmonized as possible). It was concluded that these comments will be addressed
during the next revision. A consultation of the specialized Panels will be organized to gather feedback about the relevant tables and tables will also be
prepared for proteomic methods.

548 Diagnostics
Bacteriology
When making dilution series of bacterial extracts users of the Standard should be aware that bacteria cells may cluster,
therefore making dilution series less reliable. To limit this effect, extracts should be vortexed thoroughly.
Table A2. Bacteriology (see also the instructions for the use of the tables)
Method for extraction of target organism from matrix

Extraction of the target bacterium from the matrix can only be validated in combination with a test.
Analytical Whenever possible, perform at least three experiments with serial dilutions of diseased tissues in the healthy sample selected.
sensitivity Alternatively, samples may be produced by adding infected material with known cell density of the target bacterium to the
matrix (detection of latent infection or contamination). If consistent results are not obtained after thr
ee series, additional series should be prepared and tested.
Analytical This parameter is not applicable. Extraction of the target organism from a sample is per definition non-specific.
specificity
Selectivity This parameter is not applicable. Extraction of the target organism from a sample is per definition non-selective.
Repeatability Use at least one sample with low concentration of target and make at least three subsamples (extractions). Assess extraction
efficiency by the relevant test method. If consistent results are not obtained, additional samples should be extracted.
Reproducibility As for repeatability, but with different operator(s) if possible, on different days and with different equipment when relevant or
possible.
Molecular methods, e.g. PCR, real-time PCR, LAMP

This step also includes methods for the isolation of DNA from the matrix.
Analytical Analyse at least three series of spiked sample extracts with a range of 101–106 cells of the target organism per mL.
sensitivity Preferentially, this is done by making decimal diluted cell suspensions of the target bacterium in the sample extracts. Determine
the lowest cell density giving a positive test result.
If consistent results are not obtained after three series, then additional series should be prepared and tested.
Analytical Inclusivity: analyse strains of the target bacterium covering genetic diversity, different geographic origin and hosts.
specificity Exclusivity: analyse a set of non-target bacteria, in particular those associated with the matrix.
For both inclusivity and exclusivity use cell suspensions of pure cultures at approximately 106 cells per mL and use
antiserum/antibodies at their working dilution.
Selectivity Determine whether variations in the matrix (e.g. by using different hosts of the same family, different cultivars of the host plant)
affect the test performance.
Repeatability Analyse at least three replicates of spiked sample extracts with a low concentration. If consistent results are not obtained,
additional replicates should be prepared and tested.
Reproducibility As for repeatability, but with different operator(s) if possible, on different days and with different equipment when relevant.

PM 7/98 (4) 549
Table A2. (continued)
Serological methods: IF and ELISA
sensitivity Preferentially, this is done by making decimal diluted cell suspensions of the target bacterium in the sample extracts.
Determine the lowest cell density giving a positive test result at the working dilution of the antiserum/antibodies.
If consistent results are not obtained after three series, additional series should be prepared and tested.
Analytical Inclusivity: define specificity of antibodies on strains of the target bacterium covering genetic diversity, different geographic
specificity origin and hosts. Exclusivity: define specificity of antibodies on a set of non-target bacteria, in particular those associated
with the matrix.
For both inclusivity and exclusivity use cell suspensions of pure cultures at approximately 106 cells per mL and use
antiserum/antibodies at their working dilution.
Selectivity Determine whether variations in the matrix (e.g. by using different hosts of the same family, different cultivars of the host
plant) affect the test performance.
Repeatability Analyse at least three replicates of spiked sample extracts with a low concentration. If consistent results are not obtained,
Reproducibility As for repeatability, but with different operator(s) if possible and on different days and with different equipment when
relevant.
Plating methods: selective isolation
Determine the lowest cell density giving a positive test result.
Analytical Inclusivity: define specificity of the culture medium on strains of the target bacterium covering genetic diversity, different
specificity geographic origin and hosts.
Exclusivity: define specificity for a set of non-target bacteria, in particular those associated with the matrix.
For both inclusivity and exclusivity use a cell suspension at approximately 106 cells per mL and analyse by dilution plating.
Selectivity Determine whether variations in the matrix affect the test performance.
Repeatability Analyse at least three replicates of spiked sample extracts with a low concentration. If consistent results are not obtained
Reproducibility As for repeatability, but with different operator(s) if possible and on different days and with different equipment when
relevant.

550 Diagnostics

Bioassay: selective enrichment in host plants
Determine the lowest cell density giving a positive test result. This implies isolation from test plants with or without
symptoms of infection.
Analytical Inclusivity: define specificity of the bioassay on strains of the target bacterium covering genetic diversity, different geographic
specificity origin and hosts.
For both inclusivity and exclusivity use a cell suspension at approximately 106 cells per mL.
Selectivity Determine whether variations in the matrix [e.g. by using different cultivars including the most susceptible cultivar(s)] affect
the test performance.
Repeatability Analyse at least three replicates of sample extracts with a low concentration and use the host plants determined in the
specificity test. If consistent results are not obtained additional replicates should be prepared and tested.
Reproducibility As for repeatability but with different operator(s) if possible, on different days and with different equipment when relevant.
Pathogenicity test
Analytical This parameter is not relevant for the pathogenicity test, which is generally performed with cell suspensions of approximately
sensitivity 106 cells per mL. However, analytical sensitivity may be considered when inoculating in different growth stages of the host
plant.
Analytical Inclusivity: define specificity of the pathogenicity test on a set of strains of the target bacterium covering genetic diversity,
specificity different geographic origin and hosts.
Exclusivity: define specificity on a set of non-target bacteria, in particular those associated with the matrix.
For both inclusivity and exclusivity use cell suspensions of approximately 106 cells per mL.
A positive result implies expression of symptoms and re-isolation of the target bacterium (Koch’s postulates).
Selectivity Determine whether using different cultivars of the host plant affects the test performance.
Repeatability Analyse at least three replicates of a set of strains of the target bacterium covering variability in identification tests and
virulence. Use cell suspensions of approximately 106 cells per mL.
A positive result implies expression of symptoms and re-isolation of the target bacterium (Koch’s postulates).
Reproducibility As for repeatability but with different operator(s) if possible and on different days and with different equipment when
relevant.
Fingerprint methods: protein profiling, fatty acid profiling and DNA profiling
Analytical Determine the minimum quantity of harvested bacteria from selected culture media to perform a reliable analysis.
sensitivity Test parameters should be stringently defined and standardized, e.g. culture medium, stage of culture for harvesting of cells.
Analytical Inclusivity: define specificity of the fingerprint method on strains of the target bacterium covering genetic diversity, different
specificity geographic origin and hosts.
For both inclusivity and exclusivity provide markers for differentiation at subspecies or pathovar level.
For both inclusivity and exclusivity test results can be supported by in silico comparison with data in relevant databases.
Selectivity Not applicable.
Repeatability Analyse at least three replicates of the protein/fatty acid/DNA extract.
Reproducibility As for repeatability but with different operator(s) if possible, on different days and with different equipment when relevant.

PM 7/98 (4) 551
Botany
Table A3. Botany (see also the instructions for the use of the tables)

Extraction is always validated by a test.
Analytical The method should be able to extract a sufficient quantity of the target organism to allow it to be analysed further. The
sensitivity percentage of invasive alien plant seeds that is recovered by the extraction method should be determined from a minimum of
three samples.
specificity
Selectivity This parameter is generally not applicable. Extraction of the target organism from a sample is per definition non-selective.
efficiency by the relevant test method. If consistent results are not obtained, additional samples should be extracted.
Molecular methods, e.g. barcoding, PCR

This step also includes methods for isolation of DNA from the matrix.
Analytical Determine the sufficient quantity of the target from which a detectable amount of target DNA can be obtained.
sensitivity Perform at least three experiments. If consistent results are not obtained after three series, additional series should be prepared
and tested.
Analytical Inclusivity: analyse a range of target organism(s), covering genetic diversity, different geographic origin and hosts.
specificity Exclusivity: analyse relevant non-target organism(s), in particular those associated with the matrix. The concentration of
nucleic acid should be high enough to maximize the possibility of cross-reaction but remain realistic.
For both inclusivity and exclusivity, the test results can be supported by ‘in silico’ comparison of probe/primer sequences to
sequences in genomic libraries.
Selectivity Determine whether variations of the matrix affect the test performance.
Repeatability Analyse at least three replicates of sample extracts with a low concentration. If consistent results are not obtained, additional
replicates should be prepared and tested.
Reproducibility As for repeatability, but with different operator(s) if possible, on different days, and with different equipment when relevant.
Entomology (and acarology)

Table A4. Entomology and acarology (see also the instructions for the use of the tables)

Not applicable for insects and mites if they are received as individual specimens
In other cases, extraction is always validated by a test.
sensitivity percentage of insects that is recovered by the extraction method should be determined from a minimum of three samples.
specificity
Selectivity This parameter is generally not applicable. Extraction of the target organism from a sample is per definition non-selective.
Repeatability Use at least one sample with low level of target and make at least three subsamples (extractions). Assess extraction efficiency
by observation/ morphological detection.

552 Diagnostics
Molecular methods, e.g. Barcoding, PCR.

Analytical Prepare a certain number of individuals. This number varies depending on the genus, species and stage. Determine the
sensitivity minimum number of individuals or part of individuals to be detected. Perform at least three experiments. If consistent results
are not obtained after three series, additional series should be prepared and tested.
Selectivity Not applicable for insects and mites if they have been previously isolated from the matrix.
However, if the molecular test is used as a detection test, determine whether variations of the matrix (e.g. soil, plant) affect
the test performance.
Mycology (including oomycetes)

Table A5. Mycology (see also the instructions for the use of the tables)
Method for extraction/isolation/baiting of target organism from matrix

Extraction is always validated by another method.
Analytical The method should be able to extract/isolate/bait a sufficient quantity of the target organism to allow it to be cultured or
sensitivity analysed further. Whenever possible, perform at least three experiments with serial dilutions of diseased tissues or features in
the healthy sample selected. If consistent results are not obtained after three series, additional series should be prepared and
tested.
Extract/isolate/bait the target from at least three samples (naturally infected or artificially infected samples). This may include
washing procedure and membranes to trap spores.
Analytical Although some level of specificity can occur using this method, this parameter is not applicable as the specific detection of the
specificity organism is mostly based on the subsequent method (e.g. morphological identification, PCR).
Selectivity If relevant, determine whether variations of the matrix (e.g. type of soil, texture or organic matter content) may affect the
performance of the test.
efficiency by the relevant test method. If consistent results are not obtained additional samples should be extracted.

PM 7/98 (4) 553

Analytical Determine the minimum quantity of target (e.g. number of conidia, weight of infected material in healthy material) from
sensitivity which a detectable amount of target DNA can be obtained. Perform at least three experiments with serial dilutions, preferably
in host plant DNA. If consistent results are not obtained after three series, additional series should be prepared and tested.
Analytical Inclusivity: analyse a range of target organisms covering genetic diversity, different geographic origin and hosts. Exclusivity:
specificity analyse relevant non-target organisms (e.g. phylogenetically close fungi) that might be present in the sample and
sample extract. The concentration of nucleic acid should be high enough to maximize the possibility of cross-reaction but
remain realistic.
For inclusivity and exclusivity, the test results can be supported by ‘in silico’ comparison of probe/primer sequences to
Selectivity Determine whether variations of the matrix (e.g. by using different host plants and plant tissues) affect the test performance.
Serological methods: ELISA
Analytical Determine the minimum quantity of target from which a positive test result can be obtained at the working dilution of the
sensitivity antiserum/antibodies by performing at least three experiments with serial dilutions of known number of conidia or known
weight of infected material diluted with healthy material.
Analytical Inclusivity: define specificity of antibodies on strains of the target organism covering genetic diversity, different geographic
Exclusivity: define specificity on a set of non-target organisms, in particular those associated with the matrix.
Selectivity Determine whether variations of the matrix (e.g. by using different susceptible host species or plant tissues) affect the test
performance.
Repeatability Analyse at least three replicates of sample extracts with a low concentration determined by the results from the analytical
sensitivity experiments. If consistent results are not obtained, additional replicates should be prepared and tested.
Bioassay: Pathogenicity test
Analytical Determine the necessary quantity of matrix or matrix extract (e.g. grams of leaves, soil) to produce symptoms. Perform three
sensitivity experiments with dilution series.
Analytical Inclusivity: define specificity of the bioassay on strains of the target fungi covering genetic diversity, different geographic
Exclusivity: define specificity for a set of non-target fungi, in particular those associated with the matrix.
Selectivity Determine whether variations of the matrix (e.g. by using different host plants or plant tissues) affect the test performance.
Repeatability Analyse at least three replicates of sample extracts with a low concentration and use the host plants determined in the
specificity test. If consistent results are not obtained, additional replicates should be prepared and tested.

554 Diagnostics
Nematology
Table A6. Nematology (see also the instructions for the use of the tables)

Extraction is always validated by a test.
sensitivity percentage of nematodes that is recovered by the extraction method should be determined from a minimum of three samples.
specificity
Selectivity Determine whether variations of the matrix (e.g. type of soils for cysts extractors) affect the test performance.
efficiency by the relevant test method.

Analytical Prepare a number of individuals. This number varies depending on the genus, species and stage. Determine the minimum
sensitivity number of individuals or part of individuals to be detected or identified.
Perform at least three experiments. If consistent results are not obtained after three series, then additional series should be
prepared and tested.
Selectivity Not applicable for nematodes if they have been previously isolated from the matrix.
However, if the molecular test is used as a detection test, determine whether variations of the matrix (e.g. soil, plant) affect the
test performance.
Biochemical methods, e.g. enzyme electrophoresis, protein profiling

This step also includes sample preparation.
Analytical Determine the minimum number of individuals to be detected to perform a reliable analysis with a minimum of three
sensitivity samples whenever possible. The lowest number of target individuals depends on the condition of the sample (good to very
poor), the known intraspecies variability, the difficulty to interpret features, etc.
Test parameters should be stringently defined and standardized.
Analytical Inclusivity: analyse a range of target organism(s).

specificity Exclusivity: analyse non-target genus and/or species.
Selectivity Not applicable.

PM 7/98 (4) 555
Baiting or bioassay methods (including pathogenicity tests)
Analytical Determine the minimum number of individuals to produce symptoms or multiply in plant material with at least three
sensitivity repetitions.
If consistent results are not obtained, additional replicates should be prepared and tested.
Analytical Inclusivity: define specificity of the bioassay on strains of the target organism covering genetic diversity, different geographic
Exclusivity: define specificity for a set of non-target organisms, in particular those associated with the matrix.
Selectivity Determine whether variations of the matrix (e.g. by using different cultivars of the host plant) affect the test performance.
Repeatability Use at least three replicates with the minimum number of individuals to establish a population and use the host plants
determined in the specificity test. If used for a pathogenicity test, the three replicates should have the minimum number of
individuals to produce symptoms. If consistent results are not obtained, additional replicates should be prepared and tested.
Virology and phytoplasmology
Table A7. Virology and phytoplasmology (see also the instructions for the use of the tables). This table covers viruses, viroids and phytoplasmas

This step also includes methods for extraction of RNA/DNA from the matrix.
Analytical Because the concentration of viruses, viroids and phytoplasmas is never known, determine the maximum dilution of RNA/
sensitivity DNA detected. Perform at least three experiments with serial dilutions of infected sample in the healthy sample selected. If
(relative consistent results are not obtained after three series, additional series should be prepared and tested.
sensitivity)
Analytical Inclusivity: analyse a range of variants/strains of the target covering genetic diversity, different geographic origin and hosts.
specificity Exclusivity: analyse relevant non-targets, in particular those that might be present in the matrix. The concentration of nucleic
acid should be high enough to maximize the possibility of cross-reaction but remain realistic.
For both inclusivity and exclusivity, the test results can be supported by ‘in silico’ comparison of primer/probe sequences to
Repeatability Analyse at least three replicates of sample extracts with a low (relative) concentration. If consistent results are not obtained,
Serological methods: ELISA and tissue print-ELISA, including sample preparation (not applicable for viroids).
Analytical Perform at least three experiments with serial dilutions of infected sample in the healthy sample selected. Determine the
sensitivity highest dilution of sample extracts which could be detected.
(relative If consistent results are not obtained after three series, additional series should be prepared and tested.
sensitivity)
Analytical Inclusivity: determine reactivity of antibodies on variants/strains of the target covering genetic diversity, different geographic
Exclusivity: determine reactivity of antibodies on a set of (relevant) non-targets, in particular those associated with the matrix.

556 Diagnostics
Bioassay: plant test (e.g. mechanical or insect inoculation) and grafting
Analytical Determine the maximum dilution of infected sample in the healthy sample to produce symptoms or multiply in plants. This is
sensitivity only an estimation of dilutions that can be used. Perform three series with dilution steps.
(relative If consistent results are not obtained, additional replicates should be prepared and tested.
sensitivity) Not relevant for grafting.
Analytical Inclusivity: determine reactivity of the bioassay on variants/strains of the target covering genetic diversity, different geographic
Exclusivity: determine reactivity of the bioassay on a set of non-targets, in particular those associated with the matrix.
Repeatability Analyse at least three replicates of sample extracts with an appropriate dilution determined in the sensitivity test and select
host plants on the basis of the results of the above performance criteria. If consistent results are not obtained, additional
Biochemical methods: e.g. electrophoresis, R-PAGE
Analytical Perform at least three experiments with serial dilutions of infected sample in the healthy sample selected. Determine the
sensitivity highest dilution of sample extracts which could be detected.
(relative Test parameters should be stringently defined and standardized.
sensitivity)
Analytical Inclusivity: investigate intraspecific variability (not applicable for R-PAGE).

specificity Exclusivity: compare with relevant target/nucleic acids/proteins/contaminants and show differentiation can be made.
Selectivity This parameter is generally not applicable. If relevant, determine whether variations of the matrix (e.g. by using different
cultivars of the host plant) affect the test performance.
Appendix 6 – Procedure for validation of a test A by comparison with a validated test B

Comparison of a test A with a validated test B can be an appropriate validation procedure for situations when the analytical
sensitivity or analytical specificity level of the validated test B is considered adequate and when test A presents an advantage
(e.g. speed, ease of use).
It is recognized that test A may have a better sensitivity or specificity level than the validated test B and that the compar-
ison will only provide the information that the sensitivity or specificity of test A is at least at the level of the one determined
for the validated test B.
Repeatability and reproducibility should also be evaluated for test A (see Appendix 5).
The comparison of test A with the validated test B should be performed as follows:
Perform three repetitions with the target organism and three with each of the non-target organisms as indicated in
Table A8. Samples are processed with the two tests in parallel.
The number of samples indicated in this table has been determined by comparison with published standards, e.g. ISO
16140 Microbiology of food and animal feeding stuffs Protocol for the validation of alternative methods (ISO, 2016) and
AFNOR XP V03-111 Agricultural and food products analysis. Protocol for the intra-laboratory evaluation of an alternative
method of qualitative analysis against a reference method (AFNOR, 1995).

PM 7/98 (4) 557
Table A8. Minimum number of samples required when comparing test A to a validated test B
Level of organism
Type of material Low/low (relative*) Medium/medium (relative*) High/high (relative*)
Isolates of pure cultures of target or samples 10† 7† 5†

spiked with target
Isolates of pure cultures of – 11–22 –

non-target(s) or samples spiked
with non-target(s)
Naturally contaminated sample with target Adequate dilution series are prepared with 15 positive samples previously identified with
organism the validated test B to reach the limit of detection of the validated test B
*For virology and phytoplasmology.

†
The total number of samples of target(s) should at least be twice the number of non-target(s).
Correlation between results obtained with the validated test B and test A should be evaluated for the different pest levels.
Results can be presented as shown in Table A9 and relative performance characteristics calculated.
Positive deviation and negative deviation need to be analysed.
Table A9. Example of results from a correlation between a validated test B and test A
Validated test B
+ – Total
+ 69 3 72
Test A PA PD
– 6 ND NA 12 18
Total 75 15 90
This table is adapted from Hughes et al. (2006). Numbers in this table are for demonstration purposes. PA, positive agreement; PD, positive devia-
tion; ND, negative deviation; NA, negative agreement. Positive (+) and negative (-) results for 90 samples tested using both tests, illustrating diagnos-
tic sensitivity [PA/(PA + ND)], diagnostic specificity [NA/(NA + PD)], and relative accuracy (PA + NA)/(PA + PD+ND + NA).
Diagnostic sensitivity = 92%, diagnostic specificity = 80%, relative accuracy = 90%.
It should be noted that relative accuracy is no longer referred to in the revised version of ISO 16140 (ISO, 2016).
Appendix 7 – Validation of morphological and morphometrical tests used in, for example,
entomology, nematology, mycology, botany and virology
This guidance is based on the validation experience of experts from EPPO Panels dealing with diagnostics.4
Morphological identification is based on expertise (see Appendix 1). Expert judgment is usually based on the use of avail-
able documentation in the form of keys, original morphological descriptions, specimens and voucher photographs, which are
recognized as reference documentation to support the identification. As these documents or supporting information have been
produced by specialists of the group(s) concerned, they are consequently considered as validated tests in the current Stan-
dard.
Examples of documents or supporting information considered as validated tests in the current Standard include the follow-
ing:
• Morphological and morphometrical tests included in International Standards such as the IPPC Diagnostic Protocols and
the EPPO Diagnostic Protocols.
• Morphological and morphometrical tests, taxon reviews, descriptions, preferably including original articles, and keys pub-
lished in peer-reviewed journals, preferably including original articles.
4
Experience with accreditation for morphological and morphometrical identification in a forensic laboratory was also taken into account.

558 Diagnostics
• Voucher specimens and type material (such as holotypes, paratypes, lectotypes and neotypes) and voucher photographs
(specimens and photographs should be identified and confirmed by an expert).
• Morphological and morphometrical tests in common usage, which are published in non-peer reviewed publications, includ-
ing electronic media (in particular for keys).
The laboratory should have the expertise to be able to select and justify the selection of morphological and morphometri-
cal methods made, in particular for those not described in international standards or peer-reviewed journals. The keys or
other documentation may need to be reviewed to ensure they are relevant/appropriate for the intended use, for example to
ensure inclusion of all necessary species (e.g. from different geographical regions/new species described).
As explained in section 5.4.5.2, the laboratory should confirm that it can properly carry out the morphological and/or mor-
phometrical identification.
Appendix 8 – Example of laboratory reports on the critical points in the diagnostic process
and relating to uncertainty of measurement
The following examples of laboratory reports on the critical points in the diagnostic process and relating to uncertainty of
measurement are provided as supporting information (see section Supporting Information S3):
Report 1: Identification of critical points and estimation of the uncertainty of measurement (courtesy of the National Insti-
tute of Biology, Slovenia, 2012).
Report 2: Detection of Flavescence doree (FD) and Bois noir (BN) by real-time PCR. Validated method: French Method
MOA006 parts A and B version 1b – Detection of phytoplasmas from 16SrV group (flavescence doree) and 16SrXII group
(bois noir)/Matrix: Vitis sp.
Appendix 9 – Calibration of equipment and verification of performance of equipment
Calibration
The information in Table A10 is provided for guidance purposes and the frequency will be based on the need, use, type and
previous performance of the equipment (in particular in relation to the drift observed between calibrations).
Table A10. Recommendations and suggested frequencies for calibration for equipment
Type of equipment Recommendation Suggested frequency
Reference thermometers and reference thermocouples (a) Full traceable re-calibration (a) Every 7 years
(b) Single point (at working temperature) (b) Annually
Spectrophotometric equipment Calibration Annually
Calibration weight(s) Full traceable calibration Every 7 years
Microscopes Traceable calibration of stage micrometer Initially
Pipettes Calibration Annually
Autoclaves (for media preparation) Calibration Annually

PM 7/98 (4) 559
Verification of performance
The information in Table A11 is provided for guidance purposes and the frequency will be based on the need, type, use and
previous performance of the equipment. Monitoring frequency should be adapted to the conditions of the laboratory with a
frequency being higher at the beginning and adapted later based on identified risk.
Table A11. Guidance on verification of performance of equipment
Temperature-controlled equipment (a) Establish stability and uniformity (a) Initially, and after repair,
(incubators, baths, refrigerators, of temperature modification
freezers, Berlese funnels, slide (b) Monitor temperature (b) Daily/each use
drying benches, etc.)
Thermocyclers Verification of efficiency Annually
Spectrophotometric Certified plate Monthly
Working thermometers and working check against reference thermometer Annually

Thermocouples at ice-point and/or working
temperature range
Sterilizing ovens (a) Establish stability and uniformity (a) Initially and after
of temperature repair/modification
(b) Monitor temperature (b) Each use
Autoclaves (for destruction) (a) Establish characteristics for typical (a) Initially and after
loads/cycles repair/modification
(b) Monitor temperature/time (b) Each use
Safety cabinets (a) Establish performance (a) Initially and after

repair/modification
(b) Check with sterility plates or (b) Weekly
swabbing
(c) Air flow monitoring (c) Yearly (with a service contract)
Laminar air-flow cabinets (a) Establish performance (a) Initially and after
repair/modification
(b) Check with sterility plates or (b) Weekly
swabbing
(c) Filters and air flow c) Yearly
Growth chambers (a) Monitor temperature, humidity and (a) Each use
light
(b) Monitor for pests using sticky (b) Weekly
plates
pH meters Adjust check using at least two Daily/each use

buffers
Balances Check zero and reading against check Daily/each use

weight
Check weight(s) Check against calibrated weight or Annually

check on balance immediately
following traceable calibration
(contined)
(continued)

560 Diagnostics
Table A11 (continued)
Stills, de-ionizers and reverse (a) Check conductivity (a) Daily

osmosis unit (b) Check for microbial contamination (b) Monthly if the treated water or the
end-use product containing the
treated water are not sterilized by
autoclaving or filtration before use
Gravimetric diluters (a) Check weight volume (weight) (a) Daily

dispensed
(b) Check dilution ratio (b) Monthly
Automatic media preparators Check sterility using chemical and As recommended by manufacturer
biological indicators
Pipettors/pipettes Check accuracy, fidelity and precision Regularly (to be defined by taking
of volume dispensed account of the frequency and nature
of use, and depending on the drift
observed)
Spiral platers (a) Establish performance against (a) Initially and annually
conventional method
(b) Check stylus condition and the (b) Daily/each use
start and end points
(c) Check volume dispensed (c) Monthly
Colony counters Check against number counted Annually

manually
Anaerobic jars/incubators Check with anaerobic indicator Each use
Additional verification to be done in the laboratory
Laboratory environment (microbial) Monitor for airborne and surface Weekly

microbial contamination using, for
example, air samplers, settle plates,
contact plates or swabbing
Laboratory environment Monitor for pests using sticky plates Every two weeks
(entomological)
Risk-based analysis examples for equipment
Examples of a risk-based analysis developed by a specific laboratory for two types of equipment are given in the Supporting
Information S1.
Appendix 10 – Equipment: identification and labelling procedures

This example document suggests the information sufficient to clearly identify equipment.
Identification procedure
Each piece of equipment should be identified by a unique code, all of which should be recorded in a specific register. Differ-
ent methods and codes can be used, and they will depend on the system implemented by the quality assurance department
of each laboratory. The two following methods may be used:
• The identification code is composed of 5 alphanumeric characters: 3 letters referring to the equipment type, and 2 numbers
indicating the number in a series. Example: BAL02 represents the second (02) balance (BAL) in the laboratory. The main
advantage of this coding method is that the code indicates the type of equipment to which it refers.

PM 7/98 (4) 561
• The material is identified by a unique specific serial number. Example: material n°250, whatever it may be, is the 250th
piece of material registered and identified in the laboratory. Although this system is very easy to apply, it is not possible
to have an idea of the type of equipment concerned from its number.
Labelling procedure
Each piece of equipment should be permanently labelled with its unique code. This label should not be modified or
removed.
It is therefore often suggested that the equipment is etched with its unique code. The code should be positioned to be
easily read without needing to handle the equipment. Care should be taken when etching equipment to avoid damaging it.
A temporary label may also mention the date when the next calibration, verification or maintenance is due.
Appendix 11 – Guidance on maintenance of equipment and environment

Table A12 is provided for guidance purposes and the frequency will be based on the need, use, type and previous perfor-
mance of the equipment.
Table 14. Guidance on maintenance of equipment and environment
Incubators (for microbiological purposes) Clean and disinfect internal surfaces Monthly
Incubators (for other than microbiological Clean and disinfect internal surfaces Every 3 months
purposes)
Refrigerators, freezers, ovens Clean and disinfect internal surfaces Annually
Centrifuges (a) Service (a) Annually

(b) Clean and disinfect (b) Each use
Autoclaves (a) Make visual checks of gasket, clean/drain (a) Regularly as recommended by
chamber manufacturer
(b) Full service (b) Annually
(c) Safety check of pressure vessel (c) Annually
Safety cabinets Full service and mechanical check Annually
Laminar air-flow cabinets Service and mechanical check As recommended by manufacturer

Microscopes (a) Clean and full maintenance service (a) Annually
(b) Check eye-piece graticule (b) Every 6 months
pH meters Clean electrode Each use
Balances, gravimetric diluters (a) Clean (a) Each use

(b) Service (b) Annually
Stills Clean and de-scale As required (e.g. every 3 months)
De-ionizers, reverse osmosis units Replace cartridge/membrane As recommended by manufacturer
Anaerobic jars clean/disinfect After each use
Media dispensers, volumetric equipment, Decontaminate, clean and sterilize as Each use
pipettes and general service equipment appropriate
Spiral platers (a) Service (a) Annually

(b) Decontaminate, clean and sterilize (b) Each use
Mixers/blenders Clean Each use
(continued)

562 Diagnostics
Table 14 (continued)
Thermocyclers General service Annually
Growth chamber Clean After each use
Berlese funnels Clean Each use
Slide drying benches Clean Weekly
Laboratory (a) Clean and disinfect working surfaces (a) Daily and during use
(b) Clean and disinfect floors, sinks and basins (b) Weekly
(c) Clean and disinfect other surfaces (c) Every 3 months
Appendix 12 – Suggested form for sample identification (laboratory sheet)
Sample record form
The example of a sample record form below enables anonymous tracing of samples or batches of samples within a labora-
tory. A group of samples may be recorded as one batch when they arrive from the same client, are all of the same plant spe-
cies or plant part and the same analysis is required.
Batch identification code (if appropriate): Purpose of sampling (e.g. import, control of outbreak, survey):
Plant species:
Nature of the submitted material to analyse (e.g. plant part, isolated pest):
Analysis requested by the client:
Date of sampling:
Name of the person receiving/recording the sample:
Date of reception/recording:
Suitability of the sample for testing:
Comments (e.g. urgent, type and name of applied pesticides etc.):
Sample identification codes
Laboratory identification code (code given by the Client’s identification code (identification code given
laboratory, unique to each sample) by the client, unique to each sample)

PM 7/98 (4) 563
Analysis undertaken
Analysis protocols (used by the laboratory) Date and signature (of the operator responsible for choosing
the relevant analysis protocol)
Report of the analysis sent
Report number Date and signature (of the operator

responsible for sending the report)

PM 7/98 (4) Specific Requirements For Laboratories Preparing Accreditation For A Plant Pest Diagnostic Activity

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Bulletin OEPP/EPPO Bulletin (2019) 49 (3), 530–563 ISSN 0250-8052. DOI: 10.1111/epp.

European and Mediterranean Plant Protection Organization

PM 7/98 (4) Specific requirements for laboratories preparing

Specific scope Specific approval and amendment

laboratories, however, need to extend their scope to cover

530 ª 2019 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 49, 530–563

ª 2019 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 49, 530–563

ª 2019 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 49, 530–563

ª 2019 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 49, 530–563

Risk analysis to idenfy which performance criteria need

to be evaluated and to what extent

Review of validaon or other input

Review of possible altered

Test envisaged not appropriate,

Not all relevant Relevant performance

Veriﬁcaon Combinaon of validaon

ª 2019 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 49, 530–563

Review of validation data available (examples of ques-

ª 2019 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 49, 530–563

Validaon plan resulng from risk

Evaluaon of the criteria selected

Adjust the test and

ª 2019 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 49, 530–563

Table 1. Guidance on the verification of performance criteria

ª 2019 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 49, 530–563

Verification plan resulting Verification process

Test verified Test not verified

Fig. 3 Verification process. [Colour figure can be viewed at wileyonlinelibrary.com]

ª 2019 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 49, 530–563

5.5.1. Calibration and verification programmes (ISO • to validate or verify tests

ª 2019 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 49, 530–563

Elements of quality control programme Level of control*

ª 2019 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 49, 530–563

ª 2019 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 49, 530–563

Potenal change in the list Potenal change in the

no Performances ﬁt for purpose?

Resources sll available to perform

Review the process

Update Quality Management

Update the list of tests

1in case of addion of a test

ª 2019 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 49, 530–563

Appendix 1 – Expertise and competence

ª 2019 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 49, 530–563

Table A1. Examples of factors to consider when evaluating expertise or competence

Expertise (in a specific field) Competence (for a particular task)

Education/training – diplomas/certificates Education/training – diplomas/certificates

Peer evaluation Interlaboratory comparison (in particular, proficiency testing

Proven track record – successful outcomes Blind samples

Annual review/validation Audit (both internal and external)

Continued professional development (CPD) leading to CPD

Appendix 2 – Environmental monitoring and avoidance of contamination

Specific additional requirements for molecular laboratories

ª 2019 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 49, 530–563

Specific guidelines for monitoring contamination with bacteria and fungi

Appendix 3 – Statement on test validation and/or verification

Analytical specificity Inclusivity

Analytical specificity Exclusivity

Performance criteria NA A B C D Where to find documentation

Type of material Low/low (relative) Medium/medium (relative) High/high (relative*)