title: Combination of multiplex PCR with denaturing high-performance liquid chromatography for rapid detection of Mycobacterium genus and simultaneous identification of the Mycobacterium tuberculosis complex.
abstract: A new assay with the combination of multiplex polymerase chain reaction and denaturing high-performance liquid chromatography analysis was developed for simultaneous detection of Mycobacterium genus and identification of the Mycobacterium tuberculosis complex (MTC). Targeting at genus-specific 16S rRNA sequence of Mycobacterium and specific insertion elements IS6110 and IS1081 of MTC, the assay was validated with 84 strains covering 23 mycobacteria species and 30 strains of non-mycobacteria species. No cross reactivity was observed. Clinical application was carried out on 198 specimens (155 human sputum and 43 bovine tissue samples) and compared with culture. The multiplex assay detected all culture-positive (36 in number) and 35.2% (57/162) culture-negative specimens. The molecular assay was fast that could be completed within 1 h on purified DNA, with the limit of detection as 0.8-1.6 pg per reaction on DNA template. This work provided a useful laboratory tool for rapid identification of Mycobacterium and differentiation of MTC and nontuberculous mycobacteria.
authors.affiliation: Technical Center, Guangdong Entry-Exit Inspection and Quarantine Bureau, Guangzhou 510623, China. ychenru@sina.com
authors.forename: Ru
authors.initials: R
authors.lastname: Chen
authors.forename: Xiao-Bo
authors.initials: XB
authors.lastname: Gao
authors.forename: Zhi-Hui
authors.initials: ZH
authors.lastname: Liu
authors.forename: Xiao-Bing
authors.initials: XB
authors.lastname: Shen
authors.forename: Ai-Zhen
authors.initials: AZ
authors.lastname: Guo
authors.forename: Yan-Yu
authors.initials: YY
authors.lastname: Duan
authors.forename: Zhi-Ling
authors.initials: ZL
authors.lastname: Liu
authors.forename: Xiao-Wei
authors.initials: XW
authors.lastname: Wu
authors.forename: Dao-Zhong
authors.initials: DZ
authors.lastname: Zhu
mesh_terms: D001431:Bacteriological Techniques / Q000379:methods*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004251:DNA Transposable Elements; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D009169:Mycobacterium tuberculosis / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012680:Sensitivity and Specificity; D013997:Time Factors; D014376:Tuberculosis / Q000175:diagnosis* / Q000382:microbiology*
chemical_list: D004251:DNA Transposable Elements; D012336:RNA, Ribosomal, 16S
keywords: DHPLC; Multiplex PCR; Mycobacterium; The Mycobacterium tuberculosis complex