Development of real-time polymerase chain reaction targeting Mycobacterium tuberculosis

Abstract

Conventional tuberculosis (TB) diagnostic methods have been acid fast bacilli (AFB) stating and culture. AFB staining is simple and rapid, but has limitations in its low sensitivity. In addition, AFB includes not only Mycobacterium tuberculosis (MTB) but also other nontuberculous mycobacteria (NTM). Culture using solid media has higher sensitivity than AFB staining, but it takes 4 to 6 weeks to get the results. For these reasons, new TB diagnostic methods using various molecular techniques have been recently developed. Among those, a real-time PCR seems to be one of the best methods for TB diagnosis. So far, the most widely used genetic target for TB diagnosis using real-time PCR has been IS6110, since it has been known to be highly sensitive due to its general high copy numbers in the MTB genome. However, there have been reports showing that there exist clinical isolates which harbor no IS6110 element in the genome. If so, targeting IS6110 element as a genetic marker for TB diagnosis may generate false negative results. In addition, IS6110 is known be also present in the MTB complex which include other mycobacteria such as M. africanum, M. bovis, M. microti, and M. canetti. As an effort to develop a real-time PCR to be more sensitive and specific, a new genetic target RD9 was employed in this study. RD9 is one of the genomic regions which is known be only present in the MTB genome. After development of a new real-time PCR targeting RD9, the assay was evaluated using 65 clinical sputum specimens with culture positive results. The results showed that the sensitivity of the assay using smear positive sputum samples were 100% (50/50), while that of smear negative sputum samples were 86.7% (13/15). As a result, the overall positive rate was 96.9% (63/65) with culture positive sputum specimens. Recently, NTM infections have been increasing. Thus, in this study, a new real-time PCR was developed to detect MTB and NTM simultaneously. Subsequently, the assay was evaluated using 84 culture confirmed samples by comparing with a commercial kit which also detects MTB and NTM by the real-time PCR. The commercial kit compared in this study targets IS6110 for MTB detection. The results showed that while the assay developed in this study detected 18 MTB and 66 NTM, the other commercial kit 69 MTB and 15 NTM. The samples with different results were sequence analyzed, and the new assay was found to be more accurate than commercial kit compared in this study. The reason for this different result seems to be due to the different genetic markers used in two assays for targeting MTB detection. Targeting DNA for MTB detection has the main problem, since it cannot give information on the viability of the MTB. In order to resolve this problem, a new real-time PCR targeting mRNA marker was developed in this study. For this, mRNA of 85B gene and mRNA of rpoB gene were evaluated for their usefulness to represent viability of MTB. The 85B gene is known to be one of the essential genes for MTB infection, while the rpoB is known to be one of the essential genes for MTB survival. The viability of cells was tested using mycobacterial phage assay and culture forming unit count assay. The results showed that the 85B mRNA decreased before cell died, whereas the rpoB mRNA did not. From the results of this experiment, the rpoB mRNA was selected for the genetic marker to detect viable cells in a new real-time PCR assay for MTB detection. The assay may be useful to monitor the outcome of anti-TB chemotherapy or to screen effect of new drug on MTB cells.