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

Content Provider	World Health Organization (WHO)-Global Index Medicus
Author	Reed, Jennifer L. Walker, Zachary J. Basu, Debby Allen, Veronica Nicol, Mark P. Kelso, David M. McFall, Sally M.
Description	Country affiliation: United States Author Affiliation: Reed JL ( Center for Innovation in Global Health Technologies (CIGHT), Department of Biomedical Engineering, Northwestern University, Evanston, IL 60208, USA.); Walker ZJ ( Center for Innovation in Global Health Technologies (CIGHT), Department of Biomedical Engineering, Northwestern University, Evanston, IL 60208, USA.); Basu D ( Center for Innovation in Global Health Technologies (CIGHT), Department of Biomedical Engineering, Northwestern University, Evanston, IL 60208, USA.); Allen V ( Division of Medical Microbiology, Department of Pathology, University of Cape Town and National Health Laboratory Service, Cape Town, South Africa.); Nicol MP ( Division of Medical Microbiology, Department of Pathology, University of Cape Town and National Health Laboratory Service, Cape Town, South Africa.); Kelso DM ( Center for Innovation in Global Health Technologies (CIGHT), Department of Biomedical Engineering, Northwestern University, Evanston, IL 60208, USA.); McFall SM ( Center for Innovation in Global Health Technologies (CIGHT), Department of Biomedical Engineering, Northwestern University, Evanston, IL 60208, USA. Electronic address: s-mcfall@northwestern.edu.)
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.
File Format	HTM / HTML
ISSN	14729792
Journal	Tuberculosis
Volume Number	101
e-ISSN	1873281X
Language	English
Publisher	Elsevier
Publisher Date	2016-12-01
Publisher Place	Great Britain (UK)
Access Restriction	One Nation One Subscription (ONOS)
Subject Keyword	Discipline Pulmonary Medicine
Content Type	Text
Resource Type	Article
Subject	Infectious Diseases Immunology Microbiology Microbiology (medical)

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