Rapid detection of viable M. tuberculosis directly from sputum by reverse transcriptase PCR targeting 85B gene

This study was carried out to standardize reverse transcriptase PCR (RT-PCR) targeting 85B gene for the rapid detection of viable Mycobacterium tuberculosis (M tuberculosis) from sputum specimens. 100 sputum samples from clinically suspected tuberculosis patients were included in the study. The sputum samples were collected in sterile diethyl pyrocarbonate (DEPC) treated containers and transported in ice to the laboratory within 2 hours to prevent degradation of RNA. The following microbiological analysis was performed on the sputum specimens: Ziehl Neelsen staining, Mycobacterial culture by BACTEC TB 460 reader and RT-PCR targeting 85B gene. Out of the 100 sputum samples, 40 sputum samples were Ziehl Neelsen stain positive, 58 sputum samples were culture and RT-PCR positive and 42 were culture and RT-PCR were negative. Among direct smear positive specimens, 3 specimens did not grow in culture and was RT-PCR negative indicating the non-viability of the acid-fast bacilli seen in the direct smear. The results of RT-PCR and culture against control group and clinically diagnosed tuberculosis patients were statistically significant by Chi square test (P < 0.001). RT-PCR targeting 85B gene of M tuberculosis standardized in our laboratory for is a rapid, reliable and sensitive technique to detect the viable M. tuberculosis directly from sputum specimens.