ALKALINE DECONTAMINATION OF SPUTUM SPECIMENS ADVERSELY AFFECTS STABILITY OF MYCOBACTERIAL MESSENGER-RNA

Reverse transcriptase PCR (RT-PCR) is an important tool for Mycobacter ium tuberculosis research and diagnostics. A standard procedure using N-acetyl-L-cysteine (NALC) and NaOH has been widely adopted for digest ion and decontamination of sputum specimens for mycobacterial culture. The objective of this study was to determine the compatibility of thi s method with the recovery of RNA for RT-PCR assays. Nineteen sputum s pecimens were collected from smear-positive, pretreatment tuberculosis patients. After homogenization with NALC and glass beads, specimens w ere further processed by the addition of either NaOH, as per the stand ard decontamination protocol, or phosphate buffer. RNA was prepared by using a modified guanidine-phenol extraction method developed specifi cally for sputum sediments. DNA was isolated from the same specimens. Reverse transcriptions of alpha antigen (85B protein) mRNA and 16S rRN A were performed together, and aliquots were removed for separate PCRs . In all specimens, the 85B mRNA target was greatly diminished by trea tment with NaOH; however, the 16S rRNA target remained unaffected. Sto ring sputum specimens for 48 h at 4 degrees C before processing did no t seem to affect the integrity or yield of RNA; however, some degradat ion occurred by 72 h. Data suggest that the NaOH-NALC method for proce ssing sputum samples is not suitable for detecting mRNA targets in RT- PCR assays.