Page:NIOSH Manual of Analytical Methods - 0900.pdf/3

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.</li></ol>

CALIBRATION AND QUALITY CONTROL:

<li>Calibrate the PCR thermocycler and microplate reader according to the manufacturer’s instructions.</li> <li>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: <ol style="list-style-type: lower-alpha;"> <li>If one or more of the negative control values exceeds 0.25 absorbance units.</li> <li>If either of the positive control values falls below 2.0 absorbance units.</li></ol> </li></ol>

MEASUREMENT:

<li>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.</li></ol>

CALCULATIONS:

<li>Since this is a qualitative method (positive/negative), no special calculations are required.</li></ol>

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