Le. Desjardin et al., ALKALINE DECONTAMINATION OF SPUTUM SPECIMENS ADVERSELY AFFECTS STABILITY OF MYCOBACTERIAL MESSENGER-RNA, Journal of clinical microbiology, 34(10), 1996, pp. 2435-2439
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.