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 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 language | English |
|---|---|
| Pages (from-to) | 299-303 |
| Number of pages | 5 |
| Journal | Diagnostic Microbiology and Infectious Disease |
| Volume | 80 |
| Issue number | 4 |
| DOIs | |
| Publication status | Published - 2014 Dec 1 |
Fingerprint
All Science Journal Classification (ASJC) codes
- Microbiology (medical)
- Infectious Diseases
Cite this
}
Development of a highly sensitive one-tube nested real-time PCR for detecting Mycobacterium tuberculosis. / Choi, Yeonim; Jeon, Bo Young; Shim, Tae Sun; Jin, Hyunwoo; Cho, Sangnae; Lee, Hyeyoung.
In: Diagnostic Microbiology and Infectious Disease, Vol. 80, No. 4, 01.12.2014, p. 299-303.Research output: Contribution to journal › Article
TY - JOUR
T1 - Development of a highly sensitive one-tube nested real-time PCR for detecting Mycobacterium tuberculosis
AU - Choi, Yeonim
AU - Jeon, Bo Young
AU - Shim, Tae Sun
AU - Jin, Hyunwoo
AU - Cho, Sangnae
AU - Lee, Hyeyoung
PY - 2014/12/1
Y1 - 2014/12/1
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=84919384853&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84919384853&partnerID=8YFLogxK
U2 - 10.1016/j.diagmicrobio.2014.08.009
DO - 10.1016/j.diagmicrobio.2014.08.009
M3 - Article
C2 - 25241639
AN - SCOPUS:84919384853
VL - 80
SP - 299
EP - 303
JO - Diagnostic Microbiology and Infectious Disease
JF - Diagnostic Microbiology and Infectious Disease
SN - 0732-8893
IS - 4
ER -