title: Comparative Evaluation of Several Gene Targets for Designing a Multiplex-PCR for an Early Diagnosis of Extrapulmonary Tuberculosis.
abstract: OBJECTIVE
  Diagnosis of extrapulmonary tuberculosis (EPTB) poses serious challenges. A careful selection of appropriate gene targets is essential for designing a multiplex-polymerase chain reaction (M-PCR) assay.
  
  
  METHODS
  We compared several gene targets of Mycobacterium tuberculosis, including IS6110, devR, and genes encoding MPB-64 (mpb64), 38kDa (pstS1), 65kDa (hsp65), 30kDa (fbpB), ESAT-6 (esat6), and CFP-10 (cfp10) proteins, using PCR assays on 105 EPTB specimens. From these data, we chose the two best gene targets to design an M-PCR.
  
  
  RESULTS
  Among all gene targets tested, mpb64 showed the highest sensitivity (84% in confirmed cases and 77.5% in clinically suspected cases), followed by IS6110, hsp65, 38kDa, 30kDa, esat6, cfp10, and devR. We used mpb64+IS6110 for designing an M-PCR assay. Our M-PCR assay demonstrated a high sensitivity of 96% in confirmed EPTB cases and 88.75% in clinically suspected EPTB cases with a high specificity of 100%, taking clinical diagnosis as the gold standard.
  
  
  CONCLUSIONS
  These M-PCR results along with the clinical findings may facilitate an early diagnosis of EPTB patients and clinical management of disease.
authors.affiliation: Centre for Biotechnology, Maharshi Dayanand University, Rohtak, India.
authors.forename: Ankush
authors.initials: A
authors.lastname: Raj
authors.affiliation: Centre for Biotechnology, Maharshi Dayanand University, Rohtak, India.
authors.forename: Netrapal
authors.initials: N
authors.lastname: Singh
authors.affiliation: Department of TB & Respiratory Medicine, Postgraduate Institute of Medical Sciences (PGIMS), Rohtak, India.
authors.forename: Krishna B
authors.initials: KB
authors.lastname: Gupta
authors.affiliation: Department of Pulmonary and Critical Care Medicine, Postgraduate Institute of Medical Sciences (PGIMS), Rohtak, India.
authors.forename: Dhruva
authors.initials: D
authors.lastname: Chaudhary
authors.affiliation: Department of Microbiology, Postgraduate Institute of Medical Sciences (PGIMS), Rohtak, India.
authors.forename: Aparna
authors.initials: A
authors.lastname: Yadav
authors.affiliation: Rajan Babu Institute of Pulmonary Medicine and Tuberculosis (RBIPMT), Delhi, India.
authors.forename: Anil
authors.initials: A
authors.lastname: Chaudhary
authors.affiliation: Rajan Babu Institute of Pulmonary Medicine and Tuberculosis (RBIPMT), Delhi, India.
authors.forename: Kshitij
authors.initials: K
authors.lastname: Agarwal
authors.affiliation: Vallabhbhai Patel Chest Institute, University of Delhi, Delhi, India.
authors.forename: Mandira
authors.initials: M
authors.lastname: Varma-Basil
authors.affiliation: Vallabhbhai Patel Chest Institute, University of Delhi, Delhi, India.
authors.forename: Rajendra
authors.initials: R
authors.lastname: Prasad
authors.affiliation: Department of Biochemistry, Postgraduate Institute of Medical Education and Research, Chandigarh, India.
authors.forename: Gopal K
authors.initials: GK
authors.lastname: Khuller
authors.affiliation: Centre for Biotechnology, Maharshi Dayanand University, Rohtak, India. pkmehta3@hotmail.com.
authors.forename: Promod K
authors.initials: PK
authors.lastname: Mehta
mesh_terms: D001431:Bacteriological Techniques / Q000379:methods; D004251:DNA Transposable Elements / Q000235:genetics; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics; D042241:Early Diagnosis; D005260:Female; D005784:Gene Amplification; D006801:Humans; D008297:Male; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012680:Sensitivity and Specificity; D014376:Tuberculosis / Q000175:diagnosis*
chemical_list: D004251:DNA Transposable Elements; D004269:DNA, Bacterial
keywords: Mycobacterium tuberculosis; PCR; diagnosis; extrapulmonary tuberculosis; multiplex-PCR