title: Role of real-time PCR for detection of tuberculosis and drug resistance directly from clinical samples.
abstract: BACKGROUND
  Only a few studies done earlier in India reveal the utility of real-time PCR in detecting drug resistance in cases of pulmonary tuberculosis.
  
  
  OBJECTIVE
  The study was carried out to standardise real-time PCR (Quantitative real-time PCR, qPCR) targeting 16s RNA for the rapid detection of tuberculosis and its drug resistance from suspected TB patients.
  
  
  METHODS
  Sputum samples from 100 clinically suspected tuberculosis patients, after processing were subjected to microscopy, MGIT culture and qPCR. qPCR targeted 16sRNA for detecting Mycobacterium tuberculosis complex, KatG and rpoB genes for detection of resistance to isoniazid and rifampicin respectively. 1% proportionate method and Line probe assay (Hain Lifesciences, Nehren, Germany) were used to confirm the MDR isolates.
  
  
  RESULTS
  The study showed positivity of microscopy, culture and qPCR for M. tuberculosis as 37%, 44% and 46% respectively. Sensitivity of 100% and specificity of 96.5% in the detection of M. tuberculosis was observed for qPCR in comparison to culture. MDRTB was detected in 14 cases whereas monoresistance to rifampicin and isoniazid was detected in 1 and 3 samples respectively.
  
  
  CONCLUSIONS
  Real-time PCR targeting 16sRNA, KatG and rpoB is a sensitive, specific, rapid and reliable technique to detect pulmonary tuberculosis and its MDR status directly from the sputum samples.
authors.affiliation: Research Fellow, Department of Microbiology, Kasturba Medical College, Manipal, Manipal University, Karnataka 576104, India.
authors.forename: Parashuram
authors.initials: P
authors.lastname: Rao
authors.affiliation: Professor and Head, Department of Microbiology, Kasturba Medical College, Manipal, Manipal University, Karnataka 576104, India. Electronic address: arunkiranchawla@yahoo.com.
authors.forename: Kiran
authors.initials: K
authors.lastname: Chawla
authors.affiliation: Associate Professor, Department of Microbiology, Kasturba Medical College, Manipal, Manipal University, Karnataka 576104, India.
authors.forename: Vishnu Prasad
authors.initials: VP
authors.lastname: Shenoy
authors.affiliation: Professor, Department of Microbiology, Kasturba Medical College, Manipal, Manipal University, Karnataka 576104, India.
authors.forename: Chiranjay
authors.initials: C
authors.lastname: Mukhopadhyay
mesh_terms: D000995:Antitubercular Agents / Q000494:pharmacology; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D004351:Drug Resistance / Q000235:genetics*; D024901:Drug Resistance, Multiple, Bacterial / Q000235:genetics; D006801:Humans; D007194:India; D007538:Isoniazid; D008826:Microbial Sensitivity Tests; D009169:Mycobacterium tuberculosis / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012293:Rifampin; D012680:Sensitivity and Specificity; D013183:Sputum; D018088:Tuberculosis, Multidrug-Resistant / Q000175:diagnosis* / Q000188:drug therapy / Q000382:microbiology
chemical_list: D000995:Antitubercular Agents; D004269:DNA, Bacterial; D007538:Isoniazid; D012293:Rifampin
keywords: Drug resistance; Real time PCR; Tuberculosis