实时荧光跨越式滚环等温扩增结合PMA检测虾产品中的活副溶血性弧菌  

Detection of Vibrio parahaemolyticus in Edible Crustaceans by Real-time Fluorescence Saltatory Rolling Circle Amplification Combined with Propidium Monoazide

作　　者：董晶 徐慧 郭威 卢鑫 张伟[1,2,3] DONG Jing;XU Hui;GUO Wei;LU Xin;ZHANG Wei(College of Food Science and Technology,Hebei Agricultural University,Baoding 071000,China;College of Science and Technology,Hebei Agricultural University,Cangzhou 061100,China;College of Life Sciences,Hebei Agricultural University,Baoding 071000,China)

机构地区：[1]河北农业大学食品科技学院,河北保定071000 [2]河北农业大学理工学院,河北沧州061100 [3]河北农业大学生命科学学院,河北保定071000

出　　处：《食品科学》2021年第24期289-295,共7页Food Science

基　　金：国家自然科学基金面上项目(31371772);河北省自然科学基金重点项目(C2019204342);河北省重点研究开发计划项目(18275501D);中央引导地方科技发展资金项目-基础研究项目(216Z5501G);河北农业大学食品加工学科群经费资助项目(2021-06)。

摘　　要：目的:为快速检测虾产品中的活副溶血性弧菌,建立一种实时荧光跨越式滚环等温扩增(real-time fluorescent saltatory rolling circle amplification,RF-SRCA)与叠氮溴化丙锭(propidium monoazide,PMA)染料相结合的检测方法。方法:依据副溶血性弧菌的特异性基因toxR设计并筛选引物,对PMA-RF-SRCA检测方法进行特异性、灵敏度及检出限分析,并与PMA-实时聚合酶链式反应(real-time polymerase chain reaction,real-time PCR)方法进行对比。对76份样品进行检测,以GB 4789.7—2013方法为标准,对PMA-RF-SRCA方法的相对敏感性、相对特异性、相对符合率进行计算,以对本方法的实用性进行评估。结果:此方法的引物特异性良好,12株副溶血性弧菌结果均呈阳性,18株非副溶血性弧菌结果均呈阴性;PMA-RF-SRCA法灵敏度为3.2×10^(0) CFU/mL,检出限为2.08×10^(1) CFU/g,此方法的灵敏度是PMA-real-time PCR的100倍。经过实际样品检测,PMA-RF-SRCA方法的敏感性、特异性、符合率分别为100.00%、96.00%和98.68%。结论:PMA-RF-SRCA方法特异性强、灵敏度高,可用于虾产品中的活副溶血性弧菌的检测与筛查。Objective:A novel and rapid method for the detection of Vibrio parahaemolyticus in shrimp products was established by combined use of real-time fluorescent saltatory rolling circle amplification(SRCA)and propidium monoazide(PMA).Methods:Primers were designed and screened based on the V.parahaemolyticus-specific gene toxR.The specificity,sensitivity and detection limit of the PMA-RF-SRCA method were analyzed and compared with those of PMA combined with real-time polymerase chain reaction(PMA-real-time PCR).To evaluate its practicability,the relative sensitivity,relative specificity and relative accuracy of PMA-RF-SRCA were calculated using the national standard method GB 4789.7-2013 as a reference standard when it was used to detect 76 samples.Results:The specificity of the designed primers was excellent,giving positive results for 12 V.parahaemolyticus strains and negative results for 18 non-V.parahaemolytic strains.PMA-RF-SRCA was 100-fold more sensitive than PMA-RF-SRCA with a sensitivity of 3.2×10^(0) CFU/mL and a detection limit of 2.08×10^(1) CFU/g.Compared with the GB 4789.7-2013 method,the relative sensitivity,relative specificity and relative accuracy of PMA-RF-SRCA were 100.00%,96.00%and 98.68%,respectively.Conclusion:The PMA-RF-SRCA method has the advantages of strong specificity and high sensitivity,and is suitable for the detection and screening of live V.parahaemolyticus in edible crustaceans.

关 键 词：实时荧光跨越式滚环等温扩增 叠氮溴化丙锭 副溶血性弧菌 toxR基因 食品 检测 

分 类 号：TS201[轻工技术与工程—食品科学]