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Nested PCR-Linked Capillary Electrophoresis and Single-Strand Conformation Polymorphisms for Detection of Macrolide-Resistant Mycoplasma pneumoniae in Beijing, China ▿

机译:巢式PCR联用毛细管电泳和单链构象多态性检测北京大环内酯耐药肺炎支原体

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摘要

Mycoplasma pneumoniae is usually susceptible to macrolides, but macrolide-resistant strains have been found frequently in recent years. Mutations in domain V of the 23S rRNA gene of M. pneumoniae interfere with the binding of macrolides to rRNA and mediate macrolide resistance. In this study, we developed a rapid and inexpensive method that combines nested PCR (nPCR), single-strand conformation polymorphisms (SSCPs), and capillary electrophoresis (CE) to detect macrolide-resistant mutants directly from throat swabs. nPCR was used to specifically amplify M. pneumoniae 23S rRNA gene fragments containing mutations, and amplicons were analyzed by CE-SSCP for macrolide resistance mutations, with results confirmed by sequencing. From January to December 2009, 665 throat swabs were collected in Beijing, China, yielding 110 samples that tested positive for M. pneumoniae by nPCR and serological testing. We randomly selected 64 positive throat swabs for CE-SSCP analysis. The A2063G mutation was found in 57 samples, and a coexisting T2611C mutation was identified in 1 sample. An A2063T mutation was identified in 1 sample. The total mutation rate was 91%. All mutant samples identified by nPCR-CE-SSCP were sequenced. The nPCR-CE-SSCP method could identify macrolide-resistant mutants directly from clinical samples. This is the first report of an nPCR-CE-SSCP assay for the detection of dominant mutations that confer macrolide resistance on M. pneumoniae. This approach would allow clinicians to choose appropriate therapy rapidly and could be used as a screening method for genetic mutations related to antibiotic resistance.
机译:肺炎支原体通常易受大环内酯类药物的侵害,但近年来发现了对大环内酯类耐药的菌株。肺炎支原体23S rRNA基因的结构域V中的突变会干扰大环内酯类与rRNA的结合并介导大环内酯类药物的耐药性。在这项研究中,我们开发了一种快速且廉价的方法,该方法结合了巢式PCR(nPCR),单链构象多态性(SSCP)和毛细管电泳(CE)来直接从咽拭子检测大环内酯类耐药突变体。使用nPCR特异性扩增包含突变的肺炎支原体23S rRNA基因片段,并通过CE-SSCP分析扩增子的大环内酯类抗性突变,并通过测序确认了结果。 2009年1月至2009年12月,在中国北京收集了665份咽拭子,得到110个样本,这些样本通过nPCR和血清学检测呈阳性。我们随机选择64个阳性咽拭子进行CE-SSCP分析。在57个样本中发现了A2063G突变,在1个样本中发现了共存的T2611C​​突变。在1个样品中鉴定出A2063T突变。总突变率为91%。对所有通过nPCR-CE-SSCP鉴定的突变体样品进行测序。 nPCR-CE-SSCP方法可以直接从临床样品中鉴定抗大环内酯的突变体。这是nPCR-CE-SSCP检测法的首次报道,该检测法用于检测对肺炎支原体具有大环内酯类耐药性的显性突变。这种方法将使临床医生能够迅速选择适当的治疗方法,并可用作与抗生素耐药性相关的基因突变的筛选方法。

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