Highly sensitive sequence specific qPCR detection of Mycobacterium tuberculosis complex in respiratory specimens

Jennifer L. Reed, Zachary J. Walker, Debby Basu, Veronica Allen, Mark P. Nicol, David M. Kelso, Sally M. McFall*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

16 Scopus citations


Nucleic acid amplification tests for Mycobacterium tuberculosis (MTB) detection from sputum are highly sensitive and specific with smear microscopy positive specimens, but their sensitivity with smear-negative/culture-positive specimens is much lower; therefore, these tests cannot rule out a tuberculosis diagnosis. Co-extraction of PCR inhibitors may be a cause of decreased test sensitivity. Here the design and early validation of a MTB screening assay with sample preparation and qPCR methods designed to specifically address this diagnostic gap is reported. First, human genomic DNA is identified as a significant qPCR inhibitor. To circumvent this problem, a novel, streamlined sample preparation method utilizing detergent and proteolysis to thin the sputum and DNA sequence specific MTB DNA isolation was developed. Additionally, a multiplexed qPCR assay targeting two MTB complex-specific loci: the potentially multi-copy IS6110 and the single-copy senX3-regX3, combined with the cotJC gene from Bacillus atrophaeus spores amplified as a process control was developed. The limit of detection of the test was estimated to be 20 cfu/ml which is significantly lower than the Xpert® MTB/RIF assay. In a preliminary field study of 60 de-identified blinded sputa, a test sensitivity of 96% and specificity of 100% was observed when compared to the Xpert® MTB/RIF assay.

Original languageEnglish (US)
Pages (from-to)114-124
Number of pages11
StatePublished - Dec 1 2016


  • Diagnosis
  • Mycobacterium tuberculosis
  • Quantitative PCR
  • Sequence specific capture
  • Sputum

ASJC Scopus subject areas

  • Microbiology
  • Immunology
  • Microbiology (medical)
  • Infectious Diseases


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