Animal Health Research Institute

Alex food inspection lab

Detection of VIBRIOPARAHEAMOLYTICUS spp. using real time

PCR

Code No. Test Procedure ( )

Version No. 1
Issue Date 23/ 1/2022
No. of Pages 7

Preparation Revision Approval

Name Dr\Mohamed ali Dr\amr seif Dr\ahmed aiad
Position Analyst Head of PCR unit Technical
manager
Sign
Date

) ‫( جدول التعديالت‬

‫اعتمادات الوثيقة‬ ‫التاريخ‬ ‫رقم‬ ‫بيان التعديل‬ ‫إصدا مراجع‬

\ ‫إصدار‬ ‫الصفحة‬ ‫ة‬ ‫ر‬
‫مراجعة و‬ ‫إعداد‬
‫مراجعة‬
‫اعتماد‬

CONTENTS

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Animal Health Research Institute
Alex food inspection lab

1. Purpose:
2. Scope:
3. Applicability.
4. Environment.
5. Equipment And Media.
6. Precautions.
7. Procedure.
8. Retention And Disposal.
9. Method Validation.
10. Test Report
11 Assuring the test result
12. Appendices.
13 Distribution List.

1. Purpose:
Detection of VIBRIOPARAHEAMOLYTICUS spp . using real time pcr .

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Animal Health Research Institute
Alex food inspection lab

2. Scope:

A horizontal method for the detection of VIBRIOPARAHEAMOLYTICUS

spp. by real time pcr according to : ISO ISO 20837;2006,20836:2021

3. Applicability.
This procedure shall be applied by all personnel who authorized to
Carry out this test.
4. Environment.
 No air currents
 No dusty atmosphere
 Atmospheric confederation .22±2 and 45-50% humidity
5. Equipments and Media.
5.1. Equipments
- THERMAL CYCLER
- VORTEX
-CENTRIFUGE
-THERMAL BLOCK
-automatic pipette

5.2 kits

DNA extraction kits

VIBRIOPARAHEAMOLYTICUS detection kit

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Animal Health Research Institute
Alex food inspection lab

6. Precautions.
 Avoid unnecessary talking
 Wear a laboratory coat to protect your cloths
 Do not put any thing in or near your mouth
 Wash your hands with soap and water.
 Operation should not be carried out in direct sunlight.
7.Procedure
7.1.Enrichment and sample treatment for bacteria
Food samples should be enriched according to the corresponding International
Standards or other appropriate standards. Other enrichment media found to be more
PCR compatible could be used, if they have been shown, through validation, to
have performance at least comparable to those described in International Standards.
Some enrichment media recommended in International Standards contain less PCR-
inhibitory substances than others, which should be carefully considered in
connection with the choice of sample preparation method.
For some products, special care should be taken to suppress the growth of
competing background microorganisms (e.g. by addition of selective chemicals or
antibiotics). Least destructive methods, such as a simple dilution, centrifugation,
protein digestion, filtration, density centrifugation, immunomagnetic separation,
etc. may be tried. In the case of lack of PCR response, more rigorous methods such
as boiling, the use of chelating agents or harsh chemicals, such as chloroform and
ethanol, or kits with similar actions may be tried. Simple physical methods may be
used to reduce the fat content of high-fat samples. Chelating agents may be used to
reduce the high calcium content of dairy products which can be inhibitory.
7.2 Nucleic acid extraction
7.2.1 DNA extraction
7.2.1.1 DNA release and purification
Several DNA extraction principles may be combined. For example, the following
steps may be carried out:
a) degrade the proteins in the cell extract with proteases (e.g. Proteinase K) and
RNA with ribonucleases;

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Animal Health Research Institute
Alex food inspection lab

b) precipitate the resulting peptides with organic solvents (e.g. a mixture of phenol
and chloroform) to leave the DNA in the aqueous phase;
c) purify the DNA solution and concentrate further by ethanol precipitation in the
presence of monovalent cations;
d) collect the precipitated DNA by centrifugation;
e) wash the DNA with ethanol and resuspend in buffer [e.g.
tris(hydroxymethyl)aminomethane/EDTA buffer(Tris-EDTA buffer) or Tris
buffer].
A DNA co-precipitant such as glycogen, polyethylene glycol (PEG) or transfer
RNA (t-RNA) may be used to improve the DNA recovery during the precipitation
steps. Only co-precipitants without any nuclease activity and without PCR
inhibitors/competitors, and without any sequence homology with potential PCR
targets under study may be used.
NOTE
Using vacuum freeze dryers to dry the DNA pellets obtained after a precipitation
step can cause cross
contamination.
DNA may be released by thermal cell disruption (e.g. by boiling for 10 min). After
boiling, centrifuge the chilled sample and use the supernatant for PCR. Before
boiling, to facilitate cell disruption, enzymatic treatment may be applied (e.g.
lysozyme, mutanolysin, for use with Gram-positive bacteria) followed by a
protease/proteinase incubation. Other methods such as vigorous agitation with
beads may be required when the organism has a particularly tough cell wall (e.g.
Mycobacterium spp.).
Any other method including commercial kits may be used for nucleic acid
extraction provided the results are at least comparable.
7.3. Alternative of DNA extraction method
DNA extraction procedures of commercially available extraction kits manuals
could be followed according to manufacturer instructions.
PCR amplification
Amplification of specific nucleic acid sequences can occur in vitro through a
reaction catalysed by a DNA polymerase in the presence of oligonucleotide primers
and deoxyribonucleoside triphosphates in a defined reaction buffer. An important
prerequisite for amplification of the target sequence is that there is no inhibition of
the DNA polymerase in the reaction. Amplification of DNA is a cyclical process

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Alex food inspection lab

consisting of
a) denaturation of double-stranded DNA into single-stranded nucleic acid by means
of heating,
b) annealing of oligonucleotide primers to the complementary target sequence on
both of the single strands of DNA at a suitable temperature, and
c) extension of the primers with deoxyribonucleoside triphosphates by a DNA
polymerase at a suitable temperature.
RNA can be detected using PCR if the sequence has first been transcribed into a
complementary DNA sequence by a reverse transcriptase.
.2 Detection and/or confirmation of PCR products

For real time PCR analysis, the detection occurs simultaneously with amplification.
Confirmation of the identity of the PCR product should be undertaken by an
appropriate method other than size determination, for example by the following:
a) by DNA sequencing of the PCR product;
b) by hybridization of the PCR product with specific DNA
probes( VIBRIOPARAHEAMOLYTICUS )
c) by carrying out restriction analysis of the PCR product; the length of the
fragments after restriction shall correspond to the expected length of the target
DNA sequence after restriction.
A positive result may also or alternatively be confirmed by using a standardized
microbiological cultural and confirmatory method as described by appropriate
International Standards.

Controls
The quality, integrity and amount of the DNA template influences the outcome of
the PCR, and hence the analytical results obtained.
Because of the risk of obtaining false positive and/or false negative results,
appropriate controls shall be included in each PCR run. The frequency of use shall
be determined as part of the laboratory quality assurance programme. An internal or
external amplification control shall be used in every PCR run.
If available and appropriate, reference materials and reference cultures should be
used a positive and negative controls.
For the purposes of this International Standard, the controls to be used are given in
ISO 22174:2005, 9.3 and Table 1.

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Animal Health Research Institute
Alex food inspection lab

Alternative of realtime PCR amplification and detection method

Rt.PCR amplification and detection procedures of commercially
VIBRIOPARAHEAMOLYTICUS kits manuals could be followed according to
manufacturer instructions.
8. Method Validation
The test procedure validation is not needed because the procedure is
derived from .iso ISO 20837;2006,20836:2021.
9. Test Report .
The final laboratory report has a ( form 20-1)
10. Assuring the quality of test results
The laboratory applied the quality control program to insure the quality
10.1 Internal quality control :
 Spiked samples
 Repeatability
 Duplicate tes
11. Distribution List :
 Technical Manger specialty
 Staff of pcr unit.

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