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

doi:10.1016/j.tube.2016.09.002

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

Biomedical Engineering

Research output: Contribution to journal › Article › peer-review

22 Scopus citations

Abstract

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 language	English (US)
Pages (from-to)	114-124
Number of pages	11
Journal	Tuberculosis
Volume	101
DOIs	https://doi.org/10.1016/j.tube.2016.09.002
State	Published - Dec 1 2016

Funding

This work was funded by the Northwestern Global Health Foundation and Quidel Corporation. The study sponsors played no role in the design of any of the studies, in the collection, analysis and interpretation of data; in the writing of the manuscript; nor in the decision to submit the manuscript for publication.

Keywords

Diagnosis
Mycobacterium tuberculosis
Quantitative PCR
Sequence specific capture
Sputum

ASJC Scopus subject areas

Microbiology (medical)
Infectious Diseases
Microbiology
Immunology

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

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10.1016/j.tube.2016.09.002

Cite this

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title = "Highly sensitive sequence specific qPCR detection of Mycobacterium tuberculosis complex in respiratory specimens",

abstract = "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{\textregistered} 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{\textregistered} MTB/RIF assay.",

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author = "Reed, {Jennifer L.} and Walker, {Zachary J.} and Debby Basu and Veronica Allen and Nicol, {Mark P.} and Kelso, {David M.} and McFall, {Sally M.}",

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AU - Reed, Jennifer L.

AU - Walker, Zachary J.

AU - Basu, Debby

AU - Allen, Veronica

AU - Nicol, Mark P.

AU - Kelso, David M.

AU - McFall, Sally M.

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N2 - 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.

AB - 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.

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

Abstract

Funding

Keywords

ASJC Scopus subject areas

UN SDGs

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