交叉引物恒温扩增结合免疫金标方法检测副溶血性弧菌

A cross-priming amplification assay coupled with immune-gold localization for the detection of Vibrio parahaemolyticus

目的用交叉引物恒温扩增技术(CPA)结合免疫金标方法快速检测副溶血性弧菌。方法用加热煮沸方法提取菌株和标本的DNA;对CPA方法进行敏感性和特异性检测,并和荧光定量PCR方法进行比较;并用建立的方法对模拟牡蛎样本进行检测。结果该方法 60℃40 min即可完成反应,且敏感性和特异性高,14株副溶血性弧菌检测结果均阳性;最低检测限为1.8 cfu/ml,模拟样本的检测限达到180 cfu/g。与荧光定量PCR检测结果一致。结论该方法具有检测时间短、扩增效率高、灵敏度高、特异性强、操作简单等特点,特别适合在基层实验室和现场快速检测。

Objective To establish a cross- priming amplification（ CPA） coupled with immunological gold- labeled strip for the rapid detection of Vibrio parahaemolyticus. Methods DNA was extracted from strains and samples by boiling them. Sensitivity and repeatability of CPA method were evaluated respectively,and then compared with the fluorescence quantitative PCR method. Oyster samples were detected by the established method. Results Reaction can complete in 40 min at 60 ℃ with this method; sensitivity and specificity of the method were high,and the detection results of Vibrio parahaemolyticus in all the 14 strains were positive. The minimum detection limit was about 1. 8 cfu / ml,and the detection limit of the sample was up to180 cfu / g,which was consistent with fluorescence quantitative PCR. Conclusion The method has the advantages of short detection time,high amplification efficiency,high sensitivity,strong specificity,simple operation,and is especially suitable for the rapid detection in laboratory or site.