Standardization of a TaqMan-based real-time PCR for the detection of Mycobacterium tuberculosis-complex in human sputum

Am J Trop Med Hyg. 2014 Oct;91(4):709-14. doi: 10.4269/ajtmh.13-0603. Epub 2014 Aug 11.

Abstract

Real-time polymerase chain reaction (qPCR) was optimized for detecting Mycobacterium tuberculosis in sputum. Sputum was collected from patients (N = 112) with suspected pulmonary tuberculosis, tested by smear microscopy, decontaminated, and split into equal aliquots that were cultured in Löwenstein-Jensen medium and tested by qPCR for the small mobile genetic element IS6110. The human ERV3 sequence was used as an internal control. 3 of 112 (3%) qPCR failed. For the remaining 109 samples, qPCR diagnosed tuberculosis in 79 of 84 patients with culture-proven tuberculosis, and sensitivity was greater than microscopy (94% versus 76%, respectively, P < 0.05). The qPCR sensitivity was similar (P = 0.9) for smear-positive (94%, 60 of 64) and smear-negative (95%, 19 of 20) samples. The qPCR was negative for 24 of 25 of the sputa with negative microscopy and culture (diagnostic specificity 96%). The qPCR had 99.5% sensitivity and specificity for 211 quality control samples including 84 non-tuberculosis mycobacteria. The qPCR cost ∼5US$ per sample and provided same-day results compared with 2-6 weeks for culture.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • DNA Primers / genetics
  • DNA, Bacterial / genetics
  • Humans
  • Mycobacterium tuberculosis / genetics
  • Mycobacterium tuberculosis / isolation & purification*
  • Quality Control
  • Real-Time Polymerase Chain Reaction / economics
  • Real-Time Polymerase Chain Reaction / methods*
  • Real-Time Polymerase Chain Reaction / standards
  • Reference Standards
  • Sensitivity and Specificity
  • Sputum / microbiology*
  • Tuberculosis, Pulmonary / diagnosis*
  • Tuberculosis, Pulmonary / microbiology

Substances

  • DNA Primers
  • DNA, Bacterial