交叉引物恒温扩增技术结合核酸试纸条检测沙门菌方法的建立

Establishment of a cross - priming constant temperature assay coupled with nucleic acid test strip for the detection of Salmonella

目的 建立一种快速检测沙门菌的交叉引物恒温扩增技术(CPA)-核酸试纸条法。方法 收集不同来源的15株沙门菌和非沙门菌,作为实验用菌株,使用加热煮沸法提取细菌DNA核酸;对CPA方法进行敏感性和特异性研究,并与沙门菌荧光定量PCR法进行比较与评价。结果 特异性试验结果显示,本研究建立的CPA-核酸试纸条法对15种沙门菌的检测阳性率达100%,对15株非沙门菌检测结果全部为阴性。灵敏性试验结果显示,对沙门菌的纯菌液灵敏度检测极限为1.6 cfu/ml,模拟样本的检测限达到160 cfu/g,与荧光定量PCR法一致。结论 该方法检测沙门菌具有检测时间短、扩增效率高、灵敏度高、特异性强、操作简便等特点,可应用于沙门菌快速检测。

Objective This paper aims to establish a cross-priming constant temperature amplification(CPA)-nucleic acid strip method for rapid detection of Salmonella.Methods Fifteen strains of Salmonella strains and non-Salmonella strains from different sources were collected as experimental strains,extracting bacterial DNA and nucleic acid by heating and boiling.The sensitivity and specificity of CPA method were studied and compared with that of fluorescence quantitative PCR.Results The results of specificity test showed that CPA-nucleic acid strip method established in this study can detect 15 kinds of Salmonella with a positive rate of 100%,and all 15 non-Salmonella strains were negative.The sensitivity test results showed that the detection limit of pure bacterial solution for Salmonella was 1.6 cfu/ml,and the detection limit of simulated samples was 160 cfu/g,which was consistent with the fluorescence quantitative PCR method.Conclusion This method has the characteristics of short detection time,high amplification efficiency,high sensitivity,strong specificity and simple operation.It can be applied to the rapid detection of Salmonella.