氨基糖苷类药物耐药基因armA实时荧光定量聚合酶链反应检测方法的建立
Establishment of real-time PCR assay to detect resistance gene armA
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摘要: 目的 建立一种快速、准确检测细菌中armA耐药基因的TaqMan实时荧光定量-聚合酶链反应(real-time fluorescence quantitative-polymerase chain reaction,real-time PCR)方法.方法 根据armA基因设计特异的引物及探针,在多种常见致病菌中检测其特异性;使用阳性质粒标准品评价该方法的灵敏度;使用粪便模拟标本验证该方法的应用性.结果 本方法特异性好,建立的armA耐药基因real-time PCR 方法标准曲线,确定其对质粒标准品的灵敏度为4.07×101拷贝/ml.应用该方法对粪便模拟标本进行检测,其检测下限为1.3×104 cfu/ml.结论 本研究建立了armA耐药基因荧光定量PCR检测方法,有望在耐药基因监测中推广使用.Abstract: Objective To establish a TaqMan real-time PCR assay for the detection of resistance gene armA. Methods We designed the primers and probe according to armA gene and evaluated its specificity by using common pathogens. The sensitivity of the assay was evaluated by using different concentrations of recombinant standard plasmid. The simulated fecal specimens made by mixing different concentrations of armA-positive Klebsiella pneumonia with feces were used to evaluate the applicability of the assay in clinical laboratories. Results The results demonstrated that this assay had good specificity. The sensitivity of the assay was found to be 4.07×101copy/ml by standard curve analysis. The lower threshold of real-time PCR assay of the simulated fecal specimens was 1.3×104 cfu/ml. Conclusion The real-time PCR assay established in our study can be used in the detection of armA in resistance gene surveillance.
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