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Molecular Technologies Used in Detecting of Sensitive and Isoniazid-Resistant Mycobacterium tuberculosis

机译：敏感和耐异烟肼结核分枝杆菌检测的分子技术

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摘要

Two variants for the detection of single nucleotide polymorphisms in codon 315 of the katG gene of Mycobacterium tuberculosis (MTB) (mutations in this gene are associated with resistance to isoniazid, which is an antituberculosis drug of the first line) have been developed. Two sets of primers, either of which included an additional competitive blocking primer with a 3'-terminal phosphate group (in order to prevent nonspecific amplification), permitted the identification of the most frequent AGC→ ACC and AGC → AGA point mutations in codon 315 of the katG gene. Conduction of PCR with a set of two primers, one of which contained five LNA monomers, permitted the detection of any of the six known mutations in codon 315 of the katG gene and, thereby, for the discrimination between isoniazid-sensitive and isoniazid-resistant MTB. The purity and structure of the 17 bp long primers containing LNA-modified nucleotides were characterized by time-of-flight MALDI mass spectrometry, and the 17 bp duplex formed by two LNA-containing complementary oligonucleotides was analyzed by thermal denaturation. The molecular genetic test systems created for differentiating between the wild-type MTB isolates and isoniazid-resistant MTB (an antituberculosis drug of the first line) can be used in clinical laboratories equipped with standard PCR devices; such systems permit the shortening of the time required for the detection of isoniazid resistance of MTB: from 1 -3 months by the standard bacteriological methods to 1 -3 days by PCR.
机译：已经开发出了两种用于检测结核分枝杆菌（MTB）katG基因第315位密码子单核苷酸多态性的变体（该基因的突变与对异烟肼的耐药性有关，异烟肼是第一线的抗结核药物）。两组引物（均包含具有3'末端磷酸基团的竞争性封闭性引物（以防止非特异性扩增））允许鉴定密码子315中最频繁的AGC→ACC和AGC→AGA点突变的基因。用一组两个引物（其中一个包含五个LNA单体）进行PCR，可以检测katG基因第315位密码子中的六个已知突变中的任何一个，从而区分异烟肼敏感和耐异烟肼山地车通过飞行时间MALDI质谱分析了含有LNA修饰核苷酸的17 bp长引物的纯度和结构，并通过热变性分析了由两个含LNA的互补寡核苷酸形成的17 bp双链体。用于区分野生型MTB分离株和耐异烟肼MTB（第一线抗结核药）的分子遗传学检测系统可用于配备标准PCR装置的临床实验室；这样的系统可以将检测MTB的异烟肼抗性所需的时间从标准细菌学方法的1 -3个月缩短到PCR的1 -3天。

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    《Cytology and genetics》 |2011年第6期|p.362-372|共11页
  • 作者

    O. Yu. Limanskaya; O. P. Limanskii;

  • 作者单位

    Mechnikov Institute of Microbiology and Immunology, National Academy of Medical Sciences of Ukraine, 14-16 Pushkinskaya St., Kharkiv, 61057 Ukraine,National Scientific Center Institute of Experimental and Clinical Veterinary Medicine, National Academy of Agrarian Sciences of Ukraine, 83 Pushkinskaya St., Kharkiv, 61023 Ukraine;

    Mechnikov Institute of Microbiology and Immunology, National Academy of Medical Sciences of Ukraine, 14-16 Pushkinskaya St., Kharkiv, 61057 Ukraine;

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