RPA等温扩增技术检测副溶血性弧菌

刘小青; 严琼英; 陈国培; 王远洋; 肖承荣; 张永鑫; 陈晶

doi:10.13386/j.issn1002-0306.2020.01.019

RPA等温扩增技术检测副溶血性弧菌

Detection of Vibrio parahaemolyticus by RPA Isothermal Amplification Technology

摘要

摘要: 目的:建立副溶血性弧菌的RPA-exo荧光探针快速检测方法。方法:采用重组酶聚合酶扩增技术,以irgB为靶基因设计副溶血性弧菌的RPA-exo引物探针,对引物探针进行组合筛选,建立RPA-exo荧光探针快速检测方法,并对其特异性、灵敏度、模拟污染实验及实际应用效果进行测试。结果:建立的方法15 min可获得结果,具有高特异性,无交叉反应;灵敏度为1.0×10³ CFU/mL,DNA检测限为0.35 pg/μL,质粒检测限为1×10³ copies/μL;模拟污染实验中加入终浓度为1.36×10³ CFU/mL时,无需增菌即可被检出;实际样品检测中,10份水产品,有3份样品被检出,检测结果与国标GB 4789.7-2013结果一致。结论:本研究成功建立了副溶血性弧菌的RPA-exo快速检测方法。

Abstract: Objective:To establish a rapid RPA-exo fluorescent probe detection method for Vibrio parahaemolyticus. Methods:Using RPA technology and irgB as target gene to design RPA-exo primer and probe,the primers were combined and screened to establish a rapid method. Its specificity,sensitivity,simulated contamination experiment and practical application effect were tested. Results:The construction method results were obtained in 15 min,with high specificity and no cross-reaction.The sensitivity was 1.0×10³ CFU/mL,DNA detection limit was 0.35 pg/μL,plasmid detection limit was 1×10³ copies/μL. The added final concentration 1.36×10³ CFU/mL of Vibrio parahaemolyticus could be tested without enrichment culture in simulated contamination experiment. In the actual sample test,3 samples of 10 aquatic products were detected Vibrio parahaemolyticus. The results was consistent with the result of GB 4789.7-2013.Conclusions:A rapid RPA-exo method for the detection of Vibrio parahaemolyticus was successfully established in this study.