双重RT-RPA法快速检测沙门氏菌及其1类整合酶基因

以重组酶聚合酶扩增法(recombinase polymerase amplification, RPA)为技术基础,建立了一种能单管同时快速鉴定沙门氏菌及其耐药相关1类整合酶基因的双重荧光定量重组酶聚合酶扩增(duplex real-time recombinase polymerase amplification, RT-RPA)方法。该方法以沙门氏菌特异毒力基因fimY和细菌耐药相关1类整合酶基因intI1为靶标序列,设计特异性RPA引物与exo探针,建立双重RT-RPA方法。结果显示,在fimY引物终浓度320 nmol·L^-1,intI1 引物终浓度400 nmol·L^-1,fimY探针终浓度60 nmol·L^-1,intI1探针终浓度100 nmol·L^-1,反应温度37 ℃,反应20 min时,双重RT-RPA扩增效率最高,且特异性好。灵敏度试验显示,沙门氏菌检测灵敏度为1.29×10¹ CFU·mL^-1,intI1检测灵敏度为1.60×10¹ CFU·mL^-1。实际样品检测试验中,前期从生猪养殖场、屠宰场、超市和农贸市场筛选到61株沙门氏菌(2株携带intI1基因)、555株大肠埃希菌(均携带intI1基因),用建立的双重RT-RPA方法对上述菌株进行检测,可以同时鉴定出沙门氏菌及intI1基因。该方法与传统培养方法和聚合酶链式反应(polymerase chain reaction, PCR)法相比,具有特异性强、灵敏度高、速度快(检测时间20 min)等优点,为快速鉴定携带耐药相关整合酶基因intI1的沙门氏菌提供了准确有效的方法,可为耐药有害微生物的快速鉴定奠定基础。

Development of a duplex real-time recombinase polymerase amplification (RT-RPA) assay for efficient detection of Salmonella and drug-resistant related class 1 integron

Based on the technology of recombinase polymerase amplification (RPA), this study established a duplex real-time recombinase polymerase amplification to identify Salmonella and its drug-resistant class 1 integrase genes in a single tube rapidly. The RPA primers and exo probes were designed by virulence gene fimY and the class 1 integrase gene intI1 to establish a dual RT-RPA method. The results showed the reaction was good and specific when the final concentration of the primers of fimY and intI1 were 320 nmol·L^-1 and 400 nmol·L^-1, respectively. And the final concentration of probe of fimY and intI1 were 60 nmol·L^-1 and 100 nmol·L^-1, respectively. The RPA amplification was initiated by incubating the reaction mixture at 37 ℃ for 20 min. The sensitivity test showed that the detection sensitivity of Salmonella was 1.29×10¹ CFU·mL^-1, and the detection sensitivity of intI1 was 1.60×10¹ CFU·mL^-1. In this study, 61 Salmonella strains (2 integrase-positive strains) and 555 integrase-positive Escherichia coli strains were isolated from pig farms, slaughterhouses, supermarkets and markets in the early. And the dual RT-RPA method could detect the gene of fimY and intI1 in a single tube. Compared with the traditional method, the dual RT-RPA method had the advantages of high specificity, high sensitivity and high speed (20 min), which provided an accurate and efficient method to detect Salmonella and intI1 gene, laying the foundation for the detection of resistant and harmful microorganisms rapidly.