Highly Sensitive Molecular Test Developed for Tuberculosis

Rapid, accurate detection of Mycobacterium tuberculosis is crucial in the diagnosis of tuberculosis (TB), but conventional diagnostic methods have limited sensitivity and specificity or are time consuming.

A new highly sensitive nucleic acid amplification (NAA) test, combined nested and real-time polymerase chain reaction (PCR) in a single tube, one-tube nested real-time PCR, was developed for detecting M. tuberculosis, which takes advantages of two PCR techniques, including real-time PCR.

Biomedical Laboratory Scientists at Yonsei University (Wonju, Republic of Korea) and their colleagues collected sputum specimens from 167 subjects having active TB and from 108 healthy subjects. Sputum specimens were liquefied and processed and the sediment were suspended in sterile saline and used for smear preparation, inoculation into Löwenstein-Jensen (LJ) media and DNA extraction. The presence of acid fast bacilli (AFB) was examined using a light microscope and the results of AFB smear were graded.

Initial identification of M. tuberculosis was confirmed using the SD TB Ag MPT64 immunochromatographic test (SD Bioline Inc.; Yongin, Korea) and the REBA Myco-ID molecular diagnostic kit (M&D Inc.; Wonju, Korea). The scientists designed a one-tube nested real-time PCR to have two sequential reactions with two sets of primers and dual probes for the IS6110 sequence of M. tuberculosis in a single closed tube. A real-time PCR assays were performed and validated using an Applied Biosystems 7500 Fast Real-time PCR System (Foster City, CA, USA).

The minimum limits of detection of IS6110 real-time PCR was 100 fg/μL and for the IS6110 one-tube nested real-time PCR it was 1 fg/μL of M. tuberculosis DNA. AdvanSure TB/NTM real-time PCR (LG Life Science; Seoul, Korea), IS6110 real-time PCR, and one-tube nested real-time PCR showed 100% sensitivity and 100% specificity for clinical M. tuberculosis isolates and nontuberculous mycobacteria (NTM) isolates. In comparison, the sensitivities of AdvanSure TB/NTM real-time PCR was 91% (152/167), 94.6% (158/167) for the single IS6110 real-time PCR, and 100% (167/167) one-tube nested real-time PCR.

The authors concluded that the new efficient, highly sensitive PCR assay, one-tube nested real-time PCR targeting IS6110 sequence that was developed for detecting M. tuberculosis, utilizes the advantages of nested PCR and real-time PCR. One-tube nested real-time PCR showed superior sensitivity to conventional real-time PCR and AdvanSure TB/NTM real-time PCR, a commercial TB real-time PCR kit. The study was published on August 30, 2014, in the journal Diagnostic Microbiology and Infectious Disease.

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Yonsei University 
SD Bioline Inc.
M&D Inc.