关键词: 双重荧光定量重组酶聚合酶扩增, 快速检测, 沙门氏菌, 1类整合酶基因

Abstract: 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.

Key words: duplex real-time recombinase polymerase amplification, rapid detection, Salmonella, class 1 integrase gene