title: A two-tube combined TaqMan/SYBR Green assay to identify mycobacteria and detect single global lineage-defining polymorphisms in Mycobacterium tuberculosis.
abstract: We have developed a novel real-time PCR assay to identify and perform preliminary genotyping of mycobacteria in a manner tailored to our local service. Within a single thermocycler run, mycobacterial 16S rDNA and the Mycobacterium tuberculosis global lineage-defining RD750 polymorphism are targeted in separate reaction tubes, each of which includes both TaqMan and SYBR Green chemistries. The results of this 16S-RD assay differentiate M. tuberculosis complex (MTBC) from nontuberculous mycobacteria (NTM) and recognize whether or not MTBC isolates belong to the East African-Indian lineage, the single most frequently isolated global MTBC lineage in our service. If required, NTM amplicons may be sequenced to provide more specific identities. We report the technical performance of this assay on 88 mycobacteria-positive cultures and discuss its use in the initial management of mycobacterial infections. The 16S-RD assay correctly identified all 70 MTBC-positive cultures and 17 NTM-positive cultures while contemporaneously recognizing 26 MTBC isolates as within and 44 outside the East African-Indian lineage. In artificial samples, the combined assay also showed limited potential to detect mixed mycobacterial infections (MTBC/NTM) and tuberculosis infections involving more than one global MTBC lineage. The approach we have established can be readily tailored to targets of particular value for any mycobacterial diagnostic service, thereby optimizing the value of the results for local clinical and public health management of mycobacterial infections.
authors.affiliation: Department of Infection, Immunity, and Inflammation, The Medical School, University of Leicester, Leicester, United Kingdom.
authors.forename: Eddy S G
authors.initials: ES
authors.lastname: Cheah
authors.forename: Joanne
authors.initials: J
authors.lastname: Malkin
authors.forename: Robert C
authors.initials: RC
authors.lastname: Free
authors.forename: Su-Min
authors.initials: SM
authors.lastname: Lee
authors.forename: Nelun
authors.initials: N
authors.lastname: Perera
authors.forename: Gerrit
authors.initials: G
authors.lastname: Woltmann
authors.forename: Hemu
authors.initials: H
authors.lastname: Patel
authors.forename: Patrick T
authors.initials: PT
authors.lastname: Kimmitt
authors.forename: Rebecca J
authors.initials: RJ
authors.lastname: Smith
authors.forename: Kumar
authors.initials: K
authors.lastname: Rajakumar
authors.forename: Michael R
authors.initials: MR
authors.lastname: Barer
mesh_terms: D015373:Bacterial Typing Techniques / Q000295:instrumentation / Q000379:methods*; D052160:Benzothiazoles; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics; D003959:Diamines; D005456:Fluorescent Dyes / Q000378:metabolism*; D006801:Humans; D009169:Mycobacterium tuberculosis / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D009930:Organic Chemicals / Q000378:metabolism*; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*; D011110:Polymorphism, Genetic; D011804:Quinolines; D012336:RNA, Ribosomal, 16S / Q000032:analysis / Q000235:genetics; D019914:Taq Polymerase / Q000378:metabolism*
chemical_list: D052160:Benzothiazoles; D004269:DNA, Bacterial; D003959:Diamines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D011804:Quinolines; D012336:RNA, Ribosomal, 16S; C098022:SYBR Green I; D019914:Taq Polymerase