沙门氏菌重组酶聚合酶检测方法的建立及应用

摘 要：建立一种重组酶聚合酶扩增技术（recombinase polymerase amplification，RPA）检测沙门氏菌（Salmonella）的方法。本研究依据沙门氏菌侵袭蛋白A基因（invA）的保守序列设计特异性引物，通过对反应时间的优化，建立的RPA方法在38?℃水浴锅中恒温反应20?min，即可实现对目的片段的有效扩增；除沙门氏菌外，其他26?种食源性致病菌均无扩增，具有良好的特异性；以沙门氏菌基因组DNA作为模板，该方法的检测灵敏度为1.1×10－3?ng/μL，与本研究应用的实时荧光聚合酶链式反应（real-time polymerase chain reaction，PCR）方法一致；人工污染实验表明，当羊肉、鸡肉和西兰花样品污染量为4?CFU/25?g，增菌8?h，即可通过RPA方法检出沙门氏菌。在人工污染实验中，RPA和PCR检测结果一致。本研究建立的沙门氏菌的RPA检测方法特异性强、操作简单、为食源性致病菌的鉴定提供了一种新的方向。

Development and Application of a Recombinase Polymerase Amplification Assay for Detection of Salmonella

The aim of the study was to develop a method for the determination of Salmonella by recombinase polymerase amplification (RPA). A specific primer pair was designed based on the conserved sequence of the invasion protein A gene (invA) of Salmonella. The reaction time was optimized and the RPA method was performed successfully at 38 ℃ for 20 min in a water bath: the target fragment was effectively amplified. The RPA reaction could specifically detect Salmonella rather than 26 other foodborne pathogens. The detection limit (LOD) of RPA was 1.1 × 10-3 ng/μL with the genomic DNA of Salmonella as a template, which was consistent with that of real-time PCR. For artificially contaminated lamp, chicken and broccoli samples with a bacterial concentration of 4 CFU/25 g, Salmonella could be detected by RPA after 8 hours of culture. Consistent results were obtained using real-time PCR. The RPA assay was specific, simple and rapid, and could represent a new direction for the detection of foodborne pathogens.