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    Mycobacterium Tuberculosis, Airborne 0900

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    JM Safety Perú S.A.C. JM Safety Perú S.A.C.
    This document summarizes a method for detecting airborne Mycobacterium tuberculosis particles using polymerase chain reaction (PCR) and a microplate reader. Filters are used to sample air at a flow rate of 4 L/min or higher, then placed in stripping solution to extract any particles. PCR is performed on the solution using a Roche kit, with results read on a microplate reader. The method can detect approximately 20 or more M. tuberculosis particles and provides a qualitative positive/negative result.

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    Mycobacterium Tuberculosis, Airborne 0900

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    JM Safety Perú S.A.C. JM Safety Perú S.A.C.
    16 views4 pages
    This document summarizes a method for detecting airborne Mycobacterium tuberculosis particles using polymerase chain reaction (PCR) and a microplate reader. Filters are used to sample air at a flow rate of 4 L/min or higher, then placed in stripping solution to extract any particles. PCR is performed on the solution using a Roche kit, with results read on a microplate reader. The method can detect approximately 20 or more M. tuberculosis particles and provides a qualitative positive/negative result.

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    NIOSH 0900

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    0900

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    This document summarizes a method for detecting airborne Mycobacterium tuberculosis particles using polymerase chain reaction (PCR) and a microplate reader. Filters are used to sample air at a flow rate of 4 L/min or higher, then placed in stripping solution to extract any particles. PCR is performed on the solution using a Roche kit, with results read on a microplate reader. The method can detect approximately 20 or more M. tuberculosis particles and provides a qualitative positive/negative result.

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    16 views4 pages

    Mycobacterium Tuberculosis, Airborne 0900

    Uploaded by

    JM Safety Perú S.A.C. JM Safety Perú S.A.C.
    This document summarizes a method for detecting airborne Mycobacterium tuberculosis particles using polymerase chain reaction (PCR) and a microplate reader. Filters are used to sample air at a flow rate of 4 L/min or higher, then placed in stripping solution to extract any particles. PCR is performed on the solution using a Roche kit, with results read on a microplate reader. The method can detect approximately 20 or more M. tuberculosis particles and provides a qualitative positive/negative result.

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    © All Rights Reserved
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    of 4

    MYCOBACTERIUM TUBERCULOSIS, AIRBORNE 0900

    METHOD: 0900, Issue 1 EVALUATION: N/A Issue 1: 15 January 1998

    BIOLOGICAL INDICATOR OF: exposure to M. tuberculosis

    SYNONYMS: TB, tubercle bacilli

    SAMPLING MEASUREMENT

    SAMPLER: FILTER TECHNIQUE: POLYMERASE CHAIN REACTION


    (PFTE filter, 37-mm) (PCR)/ MICROPLATE READER [2]

    FLOW RATE-RANGE: 4 L/min or higher [1] ANALYTE: M. tuberculosis

    RECOMMENDATION: if the airborne particle concentration is WAVELENGTH: 450 nm


    low, sample for at least 8 hours and/or
    use high volume sampling; in the QUALITY
    laboratory studies, sampling times were CONTROL: 3 laboratory negative PCR controls, all
    10 min should read less than 0.25 absorbance units,
    2 laboratory positive controls, both should
    PRESERVATIVE: none read 2.0 absorbance units or greater

    SHIPMENT: follow CDC guidelines for interstate RANGE: purified M. tuberculosis H37Ra DNA, 1-300
    shipment of human pathogen (42 CFR copies; H37Ra mycobacteria, approximately
    Part 72); may ship at ambient 4 to 1950 particles (all higher ranges will test
    temperature positive)

    SAMPLE ESTIMATED LOD: approximately 20 mycobacteria particles


    STABILITY: 1 week or more @ ambient (from air samples)
    temperature; indefinitely @ -20 C

    CONTROLS: 2 or more field blanks

    APPLICABILITY: This is a qualitative method which permits the detection of airborne M. tuberculosis particles. It will detect approximately
    20 or greater M. tuberculosis particles. This method does not indicate how many particles were detected.

    INTERFERENCES: Positive interferences, M. bovis, M. bovis BCG; negative interferences, metals, and other unknown airborne
    particulate matter (note: to detect suspected negative interferences, spike field samples which resulted in negative readings with M.
    tuberculosis H37Ra DNA or H37Ra particles and re-run assay. Alternately, the Roche positive control may be used instead of H37Ra).

    OTHER METHODS: The measurement technique was originally developed by Roche Diagnostic Systems for the analysis of clinical
    samples [2]. Various other M. tuberculosis detection methods are now available, such as Gen-Probe [3,4] and Digene [5].

    NIOSH Manual of Analytical Methods (NMAM), Fourth Edition


    MYCOBACTERIUM TUBERCULOSIS, AIRBORNE: METHOD 0900, Issue 1, dated 15 January 1998 - Page 2 of 4

    REAGENTS: EQUIPMENT:

    1. Filter stripping solution containing 1% Triton 1. Sampler: polytetrafluoroethylene (PTFE)


    X-100 in 10 mM Tris-HCl, pH 8.0. Alternately, filter, 37-mm, 1.0-µm pore size, with
    the Sputum Wash solution (Roche) may be cellulose support pad in plastic, three-piece
    used. filter cassette. (Costar #130810).
    2. Roche reagent kit, the AMPLICOR 2. Sampling pump, 4 L/min or greater, with
    Mycobacterium tuberculosis test, containing flexible connecting tubing.
    various solutions and controls. 3. Petri dishes, Polystyrene, 50-mm (Gelman
    3. Bleach (5.25% sodium hypochlorite) #7242, or equivalent).
    4. Forceps and/or tweezers.
    5. Disposable gloves.
    6. Platform clinical rotator.
    7. Serological pipets, sterile, disposable,
    2.0-mL.
    8. Microcentrifuge tubes, 2.0-mL.
    9. Microcentrifuge.
    * See SPECIAL PRECAUTIONS 10. Heating block or water bath.
    11. PCR thermocycler and accessories.
    12. Microplate reader.
    13. Microplate washer.
    14 Microplate incubator.
    15. Aerosol barrier pipettor tips or positive
    displacement micropipettors.
    16. Sample bags, size 10.2 X 15.2 cm.

    SPECIAL PRECAUTIONS: Entering rooms contaminated with airborne TB is a health risk. Appropriate
    respiratory protection should be worn [6]. Filter samples should be opened in a biosafety cabinet. Once
    the samples are heated for the specified time period, there is little risk to the laboratory worker conducting
    the analysis. The TB PCR method is extremely sensitive. Therefore, segregation of the various
    laboratory activities is essential in order to prevent the generation of false positives.

    SAMPLER ASSEMBLY:

    1. Assemble each filter cassette in a clean environment wearing disposable, preferably powder free or
    low powder, gloves.
    a. With forceps, insert support pad and PTFE filter into lower section of cassette.
    b. Attach middle and top cassette sections and insert plugs.
    c. Place shrinkable sealing band around cassette and air dry.
    d. Place each filter cassette into a sample bag for transport (one sampler/bag).

    SAMPLING:

    2. Calibrate each pump with a representative filter cassette in line.


    3. Sample closed-face at a flow rate of 4 L/min or greater. Thesampling time period depends on the
    suspected concentration. If the concentration is unknown, sample for long time periods (hours).
    NOTE: If the airborne particle concentration is unknown, assume the particles are present at a low
    concentration.
    4. Reinsert cassette plugs, place each filter cassette in a sample bag (one cassette /bag), and pack
    securely for shipment. Ship at ambient temperatures, according to CDC guidelines for shipment of
    human pathogens (42 CFR Part 72).

    SAMPLE PREPARATION:

    NIOSH Manual of Analytical Methods (NMAM), Fourth Edition


    MYCOBACTERIUM TUBERCULOSIS, AIRBORNE: METHOD 0900, Issue 1, dated 15 January 1998 - Page 3 of 4

    5. Place 2.0 mL of filter stripping solution in a 50-mm Petri dish.


    6. Remove the PTFE filter with a forceps or tweezers.
    7. Wet both sides of the filter by touching each side of the filter to the stripping solution and then place
    the filter (one filter/dish), sample side up, in the dish. Cover tightly.
    8. Place the dishes on the platform clinical rotator and strip the filters for 30 min. The stripping solution
    should move back and forth across the surface of the filter.
    9. Transfer the stripping solution from each filter to a 2.0-mL microcentrifuge tube.
    10. Centrifuge at 12,500 X g for 10 min and decant the supernatant into a beaker containing bleach.
    (Residual stripping solution should be removed from the microcentrifuge tubes).
    11. Add 100 µL Roche lysis reagent to each microcentrifuge tube, close lids tightly and heat at 60 C for
    45 min.
    12. Follow steps described in the AMPLICORMycobacterium tuberculosis test booklet containedin the
    Roche reagent kit.

    CALIBRATION AND QUALITY CONTROL:

    13. Calibrate the PCR thermocycler and microplate reader according to the manufacturer’s instructions.
    14. Prepare positive and negative controls as described in the Roche reagent booklet.
    NOTE: Negative and positive controls are included in the Roche reagent kit.
    Include 3 negative controls and 2 positive controls each time the test is performed, randomizing the
    positions of these samples in the test.
    Discard the run:
    a. If one or more of the negative control values exceeds 0.25 absorbance units.
    b. If either of the positive control values falls below 2.0 absorbance units.

    MEASUREMENT:

    15. Mycobacterium tuberculosis is considered present in the sample if the absorbance of the unknown
    sample is equal to or greater than 0.35 absorbance units. A sample yielding an absorbance value less
    than 0.35 absorbance units is considered negative forMycobacterium tuberculosis.

    CALCULATIONS:

    16. Since this is a qualitative method (positive/negative), no special calculations are required.

    REFERENCES:

    [1] Schafer MP, Fernback JE, Jensen PA [in press]. Sampling and analytical method development for
    qualitative assessment of airborne mycobacterial species of theMycobacterium tuberculosis complex.
    Am Ind Hyg Assoc J (submitted).
    [2] Devallois A, Legrand E, Rastogi N [1996]. Evaluation of Amplicor MTB test as adjunct to smears and
    culture for direct detection ofMycobacterium tuberculosis in the French Caribbean. J Clin Microbiol
    34:1065 -1068.
    [3] Pfyffer GE, Kissling P, Wirth R, Weber R [1994]. Direct detection ofMycobacterium tuberculosis
    complex in respiratory specimens by a target-amplified test system. J Clin Microbiol 32:918-923.
    [4] Bodmer T, Gurtner A, Schopfer K, Matter L [1994]. Screening of respiratory tract specimens for the
    presence of Mycobacterium tuberculosis by using the Gen-Probe amplified Mycobacterium
    tuberculosis direct test. J Clin Microbiol32:1483-1487.
    [5] Huang T-S, Liu Y-C, Lin H-H, Huang W-K, Cheng D-L [1996]. Comparison of the Roche AMPLICOR
    MYCOBACTERIUM Assay and Digene SHARP signal system with in-house PCR and culture for
    detection of Mycobacterium tuberculosis in respiratory specimens. J Clin Microbiol34:3092-3096.
    [6] NIOSH [1996]. Protect yourself againsttuberculosis - a respiratory protection guide for health care
    workers. DHHS (NIOSH) Publication No. 96-102.

    NIOSH Manual of Analytical Methods (NMAM), Fourth Edition


    MYCOBACTERIUM TUBERCULOSIS, AIRBORNE: METHOD 0900, Issue 1, dated 15 January 1998 - Page 4 of 4

    METHOD WRITTEN BY:

    Millie P. Schafer, Ph.D., NIOSH/DPSE

    NIOSH Manual of Analytical Methods (NMAM), Fourth Edition

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