MYCOBACTERIUM TUBERCULOSIS, AIRBORNE: METHOD 0900, Issue 1, dated 15 January 1998 - Page 3 of 4
SAMPLE PREPARATION:
- Place 2.0 mL of filter stripping solution in a 50-mm Petri dish.
- Remove the PTFE filter with a forceps or tweezers.
- 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.
- 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.
- Transfer the stripping solution from each filter to a 2.0-mL microcentrifuge tube.
- Centrifuge at 12500 × g for 10 min and decant the supernatant into a beaker containing bleach. (Residual stripping solution should be removed from the microcentrifuge tubes).
- Add 100 µL Roche lysis reagent to each microcentrifuge tube, close lids tightly, and heat at 60 °C for 45 min.
- Follow steps described in the AMPLICOR Mycobacterium tuberculosis test booklet contained in the Roche reagent kit.
CALIBRATION AND QUALITY CONTROL:
- Calibrate the PCR thermocycler and microplate reader according to the manufacturer’s instructions.
- 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:- If one or more of the negative control values exceeds 0.25 absorbance units.
- If either of the positive control values falls below 2.0 absorbance units.
MEASUREMENT:
- 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 for Mycobacterium tuberculosis.
CALCULATIONS:
- 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 the Mycobacterium 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 of Mycobacterium tuberculosis in the French Caribbean. J Clin Microbiol 34:1065–1068.
[3] Pfyffer GE, Kissling P, Wirth R, Weber R [1994]. Direct detection of Mycobacterium 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 Microbiol 32:1483–1487.
NIOSH Manual of Analytical Methods (NMAM), Fourth Edition
