Standardization and evaluation of a tetraplex polymerase chain reaction to detect and differentiate Mycobacterium tuberculosis complex and nontuberculous Mycobacteria--a retrospective study on pulmonary TB patients.
The clinical presentation of pulmonary tuberculosis by members of Mycobacterium tuberculosis complex (MTC) and nontuberculous mycobacteria (NTM) cannot be differentiated using the available standard diagnostic procedures. A single-tube tetraplex polymerase chain reaction (T-PCR) was designed to simultaneously amplify 4 well-known DNA targets of MTC. Taguchi's protocol was followed for the optimization of the conditions and was then tested on 288 pulmonary TB patient samples. The analytical sensitivity of the T-PCR was 100 fg of purified mycobacterial DNA, and specificity was found to be 100% in being able to distinguish MTC and NTM in all the cases tested. The results correlated well when validated with hsp65 PCR restriction analysis and sequencing of the 16S-23S internal transcribed spacer region, hsp65, and rpoB. The T-PCR described here is a quick, valuable, and cost-effective tool for determining whether the causative organism is MTC or NTM, and thus is useful for disease surveillance.
Mycobacterium Research Group, Department of Molecular Microbiology, Rajiv Gandhi Centre for Biotechnology, Trivandrum 695014, India.
A K
AK
Anilkumar
G K
GK
Madhavilatha
Laiza K
LK
Paul
Indulakshmi
I
Radhakrishnan
R Ajay
RA
Kumar
Sathish
S
Mundayoor
D005798:Genes, Bacterial; D006801:Humans; D008969:Molecular Sequence Data; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods* / Q000592:standards*; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000302:isolation & purification*; D009170:Nontuberculous Mycobacteria / Q000235:genetics / Q000302:isolation & purification*; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012189:Retrospective Studies; D012680:Sensitivity and Specificity; D014397:Tuberculosis, Pulmonary / Q000175:diagnosis*
D012336:RNA, Ribosomal, 16S