title: Development of a highly sensitive one-tube nested real-time PCR for detecting Mycobacterium tuberculosis.
abstract: 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 IS6110 real-time PCR and IS6110 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, IS6110 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 IS6110 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, IS6110 one-tube nested real-time PCR is useful for detecting M. tuberculosis due to its high sensitivity and simple manipulation.
authors.affiliation: Department of Biomedical Laboratory Science, College of Health Sciences, Yonsei University, Wonju, Republic of Korea; M&D Inc., Wonju, Republic of Korea.
authors.forename: Yeonim
authors.initials: Y
authors.lastname: Choi
authors.affiliation: Department of Biomedical Laboratory Science, College of Health Sciences, Yonsei University, Wonju, Republic of Korea.
authors.forename: Bo-Young
authors.initials: BY
authors.lastname: Jeon
authors.affiliation: Division of Pulmonary and Critical Care Medicine, University of Ulsan College of Medicine, Asan Medical Center, Seoul, Republic of Korea.
authors.forename: Tae Sun
authors.initials: TS
authors.lastname: Shim
authors.affiliation: Department of Biomedical Laboratory Science, College of Health Sciences, Yonsei University, Wonju, Republic of Korea; Department of Clinical Laboratory Sciences, College of Health Sciences, Catholic University of Pusan, Pusan, Republic of Korea.
authors.forename: Hyunwoo
authors.initials: H
authors.lastname: Jin
authors.affiliation: Department of Microbiology, College of Medicine, Yonsei University, Seoul, Republic of Korea.
authors.forename: Sang-Nae
authors.initials: SN
authors.lastname: Cho
authors.affiliation: Department of Biomedical Laboratory Science, College of Health Sciences, Yonsei University, Wonju, Republic of Korea; M&D Inc., Wonju, Republic of Korea. Electronic address: hyelee@yonsei.ac.kr.
authors.forename: Hyeyoung
authors.initials: H
authors.lastname: Lee
mesh_terms: D006801:Humans; D057230:Limit of Detection; D009169:Mycobacterium tuberculosis / Q000235:genetics* / Q000302:isolation & purification; D009170:Nontuberculous Mycobacteria / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods; D060888:Real-Time Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods* / Q000592:standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013183:Sputum / Q000382:microbiology; D014376:Tuberculosis / Q000175:diagnosis / Q000382:microbiology*
keywords: IS6110; Mycobacterium tuberculosis; One-tube nested real-time PCR