Detection of <I>Mycobacterium tuberculosis</I> in Clinical Specimens by Polymerase Chain Reaction Method

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  • BI Hsiao Gang
    First Department of Internal Medicine, Faculty of Medicine, University of the Ryukyus
  • SAITO Atsushi
    First Department of Internal Medicine, Faculty of Medicine, University of the Ryukyus Research Center of Comprehensive Medicine, Faculty of Medicine, University of the Ryukyus
  • KOIDE Michio
    First Department of Internal Medicine, Faculty of Medicine, University of the Ryukyus Research Center of Comprehensive Medicine, Faculty of Medicine, University of the Ryukyus
  • ISHIMINE Tomohiko
    First Department of Internal Medicine, Faculty of Medicine, University of the Ryukyus
  • FUTENMA Mitsuhiko
    First Department of Internal Medicine, Faculty of Medicine, University of the Ryukyus
  • YAMASHIRO Yuko
    First Department of Internal Medicine, Faculty of Medicine, University of the Ryukyus
  • KUSANO Nobuchika
    First Department of Internal Medicine, Faculty of Medicine, University of the Ryukyus
  • KAWAKAMI Kazuyoshi
    First Department of Internal Medicine, Faculty of Medicine, University of the Ryukyus

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Other Title
  • PCR法による臨床材料からの<I>Mycobacterium tuberculosis</I>の同定
  • PCR法による臨床材料からのMycobacterium tuberculosisの同定〔英文〕
  • PCRホウ ニ ヨル リンショウ ザイリョウ カラ ノ Mycobacteri
  • Detection of Mycobacterium tuberculosis in Clinical Specimens by Polymerase Chain Reaction Method

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Abstract

An insertion sequence repeated multiple times in the chromosome of Mycobacterium tuberculosis was used as a target for amplification using the polymerase chain reaction (PCR) assay for detecting Mycobacterium tuberculosis in chinical specimens. The sequences of primers were 5'-CCTGCGAGCGTAGGCGTCGG-3'(primer 1) and 5'-CTCGTCCAGCGCCGCTTCGG-3'(primer 2). One cycle of amplification consisted of denaturing at 94°C for 2 min, primer annealing at 68°C for 2 min, and extension at 72°C for 2 min. DNA (5 fg) extracted from M. tuberculosis was detected by gel electrophoresis and Southern blot hybridization after 40 cycles of amplification. The amplification products were not obtained by DNA extracted from M. kansasii, M. intracellulare, M. avium, M. fortuitum, Escherichia coli, Klebsiella Pneumoniae, Pseudomonas aeruginosa, Legionella pneumophila and Staphylococcus aureus; only from the M. tuberculosis complex. PCR results were compared with conventional cultural, pathological and microscopic findings in the detection of M. tuberculosis in 112 clinical specimens. There were 25 specimens that were positive for M. tuberculosis by cultural or pathological examination, of which 20 (80%) were positive by PCR. PCR detected the organism in 5 (83%) of 6 smear-positive specimens and 15 (79%) of 19 smear-negative specimens in which culture or pathology revealed M. tuberculosis. In addition, 2 smear-negative specimens and 8 smear-negative and culture-negative specimens were positive by PCR. These 10 samples were collected from the patients suspected as having tuberculosis by the clinical diagnosis based on the clinical history, characteristic radiographs, a positive PPD skin test and the effectiveness of anti-tuberculous drugs. There were no false positive samples according to the PCR. PCR assay may be used to detect M. tuberculosis in clinical specimens and has significant potential as a rapid, sensitive and specific assay for diagnosis of infection caused by M. tuberculosis.

Journal

  • Kansenshogaku Zasshi

    Kansenshogaku Zasshi 69 (3), 272-279, 1995

    The Japanese Association for Infectious Diseases

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