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

目的建立一种快速、准确检测细菌中arm A耐药基因的Taq Man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arm A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arm A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1.3×104cfu/ml。结论本研究建立了arm A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。

Establishment of real-time PCR assay to detect resistance gene armA

Objective To establish a Taq M an real-time PCR assay for the detection of resistance gene arm A. Methods We designed the primers and probe according to arm A gene and evaluated its specificity by using common pathogens.The sensitivity of the assay w as evaluated by using different concentrations of recombinant standard plasmid. The simulated fecal specimens made by mixing different concentrations of arm A-positive Klebsiella pneumonia w ith feces w ere 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 w as found to be 4. 07 × 10^1 copy / ml by standard curve analysis.The low er threshold of real-time PCR assay of the simulated fecal specimens w as 1. 3 × 10^4 cfu / ml. Conclusion The real-time PCR assay established in our study can be used in the detection of arm A in resistance gene surveillance.