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®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.
authors.affiliation: Center for Innovation in Global Health Technologies (CIGHT), Department of Biomedical Engineering, Northwestern University, Evanston, IL 60208, USA.
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Reed
authors.affiliation: Center for Innovation in Global Health Technologies (CIGHT), Department of Biomedical Engineering, Northwestern University, Evanston, IL 60208, USA.
authors.forename: Zachary J
authors.initials: ZJ
authors.lastname: Walker
authors.affiliation: Center for Innovation in Global Health Technologies (CIGHT), Department of Biomedical Engineering, Northwestern University, Evanston, IL 60208, USA.
authors.forename: Debby
authors.initials: D
authors.lastname: Basu
authors.affiliation: Division of Medical Microbiology, Department of Pathology, University of Cape Town and National Health Laboratory Service, Cape Town, South Africa.
authors.forename: Veronica
authors.initials: V
authors.lastname: Allen
authors.affiliation: Division of Medical Microbiology, Department of Pathology, University of Cape Town and National Health Laboratory Service, Cape Town, South Africa.
authors.forename: Mark P
authors.initials: MP
authors.lastname: Nicol
authors.affiliation: Center for Innovation in Global Health Technologies (CIGHT), Department of Biomedical Engineering, Northwestern University, Evanston, IL 60208, USA.
authors.forename: David M
authors.initials: DM
authors.lastname: Kelso
authors.affiliation: Center for Innovation in Global Health Technologies (CIGHT), Department of Biomedical Engineering, Northwestern University, Evanston, IL 60208, USA. Electronic address: s-mcfall@northwestern.edu.
authors.forename: Sally M
authors.initials: SM
authors.lastname: McFall
mesh_terms: D004269:DNA, Bacterial / Q000032:analysis; D006801:Humans; D008403:Mass Screening / Q000379:methods; D025202:Molecular Diagnostic Techniques / Q000379:methods; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000302:isolation & purification*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity; D013048:Specimen Handling / Q000379:methods; D013183:Sputum / Q000382:microbiology*; D014397:Tuberculosis, Pulmonary / Q000175:diagnosis*
chemical_list: D004269:DNA, Bacterial
keywords: Diagnosis; Mycobacterium tuberculosis; Quantitative PCR; Sequence specific capture; Sputum