Alkaline decontamination of sputum specimens adversely affects stability of mycobacterial mRNA.
Reverse transcriptase PCR (RT-PCR) is an important tool for Mycobacterium tuberculosis research and diagnostics. A standard procedure using N-acetyl-L-cysteine (NALC) and NaOH has been widely adopted for digestion and decontamination of sputum specimens for mycobacterial culture. The objective of this study was to determine the compatibility of this method with the recovery of RNA for RT-PCR assays. Nineteen sputum specimens were collected from smear-positive, pretreatment tuberculosis patients. After homogenization with NALC and glass beads, specimens were further processed by the addition of either NaOH, as per the standard decontamination protocol, or phosphate buffer. RNA was prepared by using a modified guanidine-phenol extraction method developed specifically for sputum sediments. DNA was isolated from the same specimens. Reverse transcriptions of alpha antigen (85B protein) mRNA and 16S rRNA were performed together, and aliquots were removed for separate PCRs. In all specimens, the 85B mRNA target was greatly diminished by treatment with NaOH; however, the 16S rRNA target remained unaffected. Storing sputum specimens for 48 h at 4 degrees C before processing did not seem to affect the integrity or yield of RNA; however, some degradation occurred by 72 h. Data suggest that the NaOH-NALC method for processing sputum samples is not suitable for detecting mRNA targets in RT-PCR assays.
Desjardin
L E
LE
Department of Pathology, University of Arkansas for Medical Sciences, Little Rock, USA.
Perkins
M D
MD
Teixeira
L
L
Cave
M D
MD
Eisenach
K D
KD
D000468:Alkalies; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012329:RNA, Bacterial / Q000032:analysis* / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics; D012680:Sensitivity and Specificity; D013183:Sputum / Q000382:microbiology*
D000468:Alkalies; D004269:DNA, Bacterial; D012329:RNA, Bacterial; D012333:RNA, Messenger