耐药基因bla_(TEM-1)、bla_(IMP-3)、fosA3多重重组聚合酶扩增(RPA)快速检测方法的建立和应用

Establishment and application of rapid detection method for bla_(TEM-1),bla_(IMP-3) and fosA3 genes by multiple recombination polymerase amplification(RPA)

为了建立同时检测多种耐药基因的快速检测方法,本研究以bla_(TEM-1)、bla_(TEM-1)、fosA3基因保守序列设计特异性引物,经优化反应条件,建立恒温检测上述3种耐药基因的多重重组聚合酶扩增(RPA)方法。条件优化结果显示,所建立的RPA方法在引物浓度为480 mmol/L,37℃恒温反应20 min时检测效果最佳。该方法对携带bla_(TEM-1)、bla_(TEM-1)、fosA3基因的菌株能扩增出条带,阴性菌株无条带,特异性高;对bla_(TEM-1)、bla_(IMP-3)基因的最低检出限为1.8×10^(1)拷贝/μL,对fosA3基因的最低检出限为1.8×10^(2)拷贝/μL,敏感性较强。用多重RPA方法对浙江省内水产养殖环境及生物体中分离到的400株菌株进行检测,养殖用水分离菌株的bla_(TEM-1)、bla_(TEM-1)和fosA3检出率分别为81.00%(81/100),11.00%(11/100)和29.00%(29/100);水产动物体内分离菌株的bla_(TEM-1)、bla_(IMP-3)和fosA3检出率分别为73.67%(221/300),1.33%(4/300)和23.33%(70/300),与常规PCR基本一致。建立的RPA方法操作简单、反应快速、检测限低、结果可靠,适用于多种耐药基因的快速检测,为快速摸清养殖产业链各环节的耐药基因谱,减少耐药基因传播,保障食品安全提供了技术支撑。

In order to establish a simple and rapid molecular detection method for multidrug resistance genes(bla_(TEM-1),bla_(TEM-1),fosA3),the recombinant polymerase amplification(RPA)method for detecting the above three multidrug resistance genes was established by optimizing the reaction conditions.The optimized test results show that the RPA method has the best detection effect under the conditions of 37℃and 20 min with 480 mmol/L primer concentration;the specificity test results showed that,only the positive strains were positive,with strong specificity;and the lowest detection limit of the RPA method for bla_(TEM-1)and bla_(IMP-3)gene was 1.8×10^(1)copies/μL,and for fosA3 gene was 1.8×10^(2)copies/μL,indicating the high sensitivity of this method.The detection rates of bla_(TEM-1),bla_(IMP-3)and fosA3 in samples of aquaculture water were 81.00%(81/100),11.00%(11/100)and 29.00%(29/100).The detection rates of blaTEM-1,blaIMP-3and fosA3in samples of aquatic animals were 73.67%(221/300),1.33%(4/300)and 23.33%(70/300)for the 400strains isolated from aquaculture environment and organisms in Zhejiang Province,which were basically consistent with PCR.The RPA method established in this study is simple,rapid,low detection limit and reliable,which is suitable for rapid detection of multidrug resistance genes.It provides technical support for the rapid identification of antibiotic-resistant gene spectrum,the reduction of antibiotic-resistant gene transmission,and the protection of food safety.