Quantification of colony-forming units for M. tuberculosis complex using gyrB-based real-time PCR assay.
It is inappropriate to select insertion sequence (IS) 6110 as the amplification target for the quantitative analysis of Mycobacterium tuberculosis complex (MTC).



To develop a novel Taqman real-time polymerase chain reaction (RT-PCR) for the quantitative analysis of MTC by amplifying a single-copy PCR target.



Analytic sensitivity and specificity, repeatability and reproducibility, and standard curve were estimated by analysing 18 reference strains and 100 clinical isolates. Diagnostic sensitivity and specificity, positive predictive value (PPV) and negative predictive value (NPV) were evaluated by detecting 50 clinical specimens. Quantitative accuracies of commercial and in-house Taqman RT-PCR were also compared.



Analytic sensitivity of this method was 30 colony-forming units/ml and analytic specificity was 100%. In comparison with commercial Taqman RT-PCR (reference), diagnostic sensitivity, diagnostic specificity, PPV and NPV of the novel Taqman RT-PCR were respectively 85.7%, 94.4%, 85.7% and 94.4%. There was no significant difference between the two methods (χ(2) = 0.25, P > 0.05) and there was strong agreement between them (κ = 0.802, P < 0.05). In-house Taqman RT-PCR was more accurate than commercial assay.



The novel RT-PCR is sensitive and specific for the detection of MTC and it is also accurate for quantitative analysis of MTC.
Clinical Laboratory, The Fifth Affiliated Hospital, Nanfang Hospital, Southern Medical University, Guangzhou, People's Republic of China.
X-M
XM
Yin
Department of Laboratory Medicine, Nanfang Hospital, Southern Medical University, Guangzhou, People's Republic of China; Clinical Laboratory, Bao'an Maternity and Child Health Hospital, Shenzhen, Guangdong Province, People's Republic of China.
L-J
LJ
Wu
Department of Laboratory Medicine, Nanfang Hospital, Southern Medical University, Guangzhou, People's Republic of China.
L
L
Zheng
Department of Laboratory Medicine, Nanfang Hospital, Southern Medical University, Guangzhou, People's Republic of China.
X-R
XR
Mao
Department of Laboratory Medicine, Nanfang Hospital, Southern Medical University, Guangzhou, People's Republic of China.
S-M
SM
Zhang
Department of Laboratory Medicine, Nanfang Hospital, Southern Medical University, Guangzhou, People's Republic of China.
Q
Q
Wang
D002138:Calibration; D015169:Colony Count, Microbial / Q000592:standards; D027081:DNA Gyrase / Q000235:genetics*; D005838:Genotype; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000592:standards; D009169:Mycobacterium tuberculosis / Q000235:genetics* / Q000254:growth & development / Q000302:isolation & purification; D010641:Phenotype; D011237:Predictive Value of Tests; D060888:Real-Time Polymerase Chain Reaction / Q000592:standards; D012015:Reference Standards; D015203:Reproducibility of Results; D014376:Tuberculosis / Q000175:diagnosis* / Q000382:microbiology
D027081:DNA Gyrase