Highly sensitive sequence specific qPCR detection of Mycobacterium tuberculosis complex in respiratory specimens. (December 2016)

• Record Type: Journal Article
• Authors: Reed, Jennifer L.; Walker, Zachary J.; Basu, Debby; Allen, Veronica; Nicol, Mark P.; Kelso, David M.; McFall, Sally M.
• Is Part Of: Tuberculosis. Volume 101(2016)
• Page Start: 114
• Page End: 124
• Publication Date: 2016-12
• Subjects: Mycobacterium tuberculosis -- Quantitative PCR -- Sequence specific capture -- Diagnosis -- Sputum; 616.995
• DOI: 10.1016/j.tube.2016.09.002
• Languages: English
• ISSNs: 1472-9792

Abstract

Summary: 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 IS 6110 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.