Development of a highly sensitive one-tube nested real-time PCR for detecting Mycobacterium tuberculosis

Yeonim Choi, Bo Young Jeon, Tae Sun Shim, Hyunwoo Jin, Sang Nae Cho, Hyeyoung Lee

Research output: Contribution to journalArticlepeer-review

16 Citations (Scopus)


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 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 advantage of two PCR techniques, i.e., nested PCR and real-time PCR. One-tube nested real-time PCR was designed to have two sequential reactions with two sets of primers and dual probes for the insertion sequence (IS) 6110 sequence of M. tuberculosis in a single closed tube. The minimum limits of detection of IS. 6110 real-time PCR and IS. 6110 one-tube nested real-time PCR were 100 fg/μL and 1 fg/μL of M. tuberculosis DNA, respectively. AdvanSure TB/non-tuberculous mycobacteria (NTM) real-time PCR, IS. 6110 real-time PCR, and two-tube nested real-time PCR showed 100% sensitivity and 100% specificity for clinical M. tuberculosis isolates and NTM isolates. In comparison, the sensitivities of AdvanSure TB/NTM real-time PCR, single IS. 6110 real-time PCR, and one-tube nested real-time PCR were 91% (152/167), 94.6% (158/167), and 100% (167/167) for sputum specimens, respectively. In conclusion, IS. 6110 one-tube nested real-time PCR is useful for detecting M. tuberculosis due to its high sensitivity and simple manipulation.

Original languageEnglish
Pages (from-to)299-303
Number of pages5
JournalDiagnostic Microbiology and Infectious Disease
Issue number4
Publication statusPublished - 2014 Dec 1

Bibliographical note

Funding Information:
This study was supported by a grant of the Korea Healthcare Technology R&D Project, Ministry of Health & Welfare, Republic of Korea ( HI10C1708 (A101750) ).

Publisher Copyright:
© 2014 Elsevier Inc.

All Science Journal Classification (ASJC) codes

  • Microbiology (medical)
  • Infectious Diseases


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