【摘要】 为建立一种快速、高效地检测猪源沙门氏菌的聚合酶螺旋反应（PSR）方法,本研究针对猪源沙门氏菌侵袭蛋白A （invA）基因设计3套特异性扩增引物,通过优化孵育温度、d NTPs和Mg²⁺浓度、Bst聚合酶浓度和反应时间初步建立了猪源沙门氏菌PSR方法。结果显示:该方法可以特异性扩增2株猪沙门氏菌,而对其它12株病原菌无扩增,特异性较强;以沙门氏菌标准株CMCC 50094为模板,并经一系列的稀释,对PSR、LAMP和qRCR方法的敏感性进行检测和比较,结果显示PSR方法和qPCR方法对菌液的最小检出量均为5×10¹cfu/mL,敏感性为LAMP方法的10倍,敏感性较高;临床样品检测结果显示,经PSR方法在132份仔猪腹泻样本中共检测出猪源沙门氏菌阳性样品18份,与常规培养方法鉴定结果一致,检出率均为13.64%。本研究建立的PSR法为猪源沙门氏菌的快速检测提供了一种可行方法。更多还原

【Abstract】 To develop a rapid and efficient polymerase spiral reaction for the detection of Salmonella in swine, three sets of specific amplification primers were designed for the Salmonella invasion protein A（invA） gene. The reaction conditions were optimized including the reaction temperature, dNTPs concentration, Mg²⁺ concentration, Bst polymerase concentration and reaction time. Two strains of porcine Salmonella and twelve other pathogens were selected for the specificity analysis. The PSR assay could only detect two strains of porcine Salmonella, but not other pathogens. The sensitivity tests of PSR, loop mediated isothermal amplification（LAMP） and real time quantitative PCR（qPCR） were carried out and compared by using the Salmonella standard strain CMCC 50094 as the template with series of dilutions. The results showed that the sensitivities of PSR and qPCR were both 5 ×10¹ cfu/mL, which were 10 times more than that of LAMP assay. For testing 132 piglet diarrheal samples, 18 of them were detected as positive by PSR. This result was the same as the result obtained from conventional identification methods. The detection rate was 13.64%. Therefore, the PSR method established in this study provides a feasible method for the rapid detection of porcine Salmonella.更多还原