跨越式滚环等温扩增技术结合CRISPR/Cas12a定量检测海产品中的副溶血性弧菌  

Saltatory Rolling Circle Amplification Combined with CRISPR/Cas12a for Quantitative Detection Vibrio parahaemolyticus in Seafoods

作　　者：董换哲 苑宁 张蕴哲[1] 杨倩[1,3] 卢鑫 郭威 张伟 DONG Huanzhe;YUAN Ning;ZHANG Yunzhe;YANG Qian;LU Xin;GUO Wei;ZHANG Wei(College of Food Science and Technology,Hebei Agricultural University,Baoding 071001,China;College of Science and Technology,Hebei Agricultural University,Cangzhou 061100,China;College of Public Health,Hebei University,Baoding 071002,China;College of Life Sciences,Hebei Agricultural University,Baoding 071001,China;Hebei Key Laboratory of Analysis and Control of Zoonotic Pathogenic Microorganism,Baoding 071001,China)

机构地区：[1]河北农业大学食品科技学院,河北保定071001 [2]河北农业大学理工学院,河北沧州061100 [3]河北大学公共卫生学院,河北保定071002 [4]河北农业大学生命科学学院,河北保定071001 [5]河北省人畜共患病原微生物分析与防控重点实验室,河北保定071001

出　　处：《食品科学》2022年第14期289-295,共7页Food Science

基　　金：国家自然科学基金面上项目(32172288,31371772);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);河北省外专百人计划项目(360-0803-JSN-3YGS);河北省高等学校科学技术研究项目(QN2022073);河北省博士后科研项目(B2021005007);河北农业大学食品加工学科群经费资助项目(2021-06)。

摘　　要：将CRISPR/Cas12a系统与跨越式滚环等温扩增技术(saltatory rolling circle amplification,SRCA)相结合,建立一种快速、灵敏定量检测海产品中副溶血性弧菌(Vibrio parahemolyticus)的方法(SRCA-Cas12a)。通过SRCA扩增靶标DNA,CRISPR/Cas12a系统识别靶标DNA并裂解单链报告探针,从而建立SRCA-Cas12a检测方法。分析该方法的特异性与灵敏度,并进行加标回收实验和实际样品检测。结果显示该方法可区分副溶血性弧菌和其他食源性致病菌,特异性良好。在最优检测体系下,建立了副溶血性弧菌菌液浓度的对数与荧光信号强度的线性关系,线性方程为y=1.8472x+2.4738(R^(2)=0.9866),检出限为3.6 CFU/mL(R_(SN)=3)。在人工污染样品中副溶血性弧菌的加标回收率为95.5%~104.4%。在实际样品检测中,该方法的敏感性为100.0%、特异性为95.2%、符合率为97.2%。本研究所建立的SRCA-Cas12a方法具有特异性好、检测限低的优点,为副溶血性弧菌的快速定量检测提供了一种新策略。This study aimed to develop a rapid and sensitive method for the quantitation of Vibrio parahemolyticus in seafoods by combining the CRISPR/Cas12a system with saltatory rolling circle amplification(SRCA-Cas12a).The target DNA of V.parahaemolyticus was amplified by SRCA and recognized by the CRISPR/Cas12a system and the single-stranded reporter probe was cleaved by the CRISPR/Cas12a system.The sensitivity,specificity and spiked recovery of SRCA-Cas12a were tested.The results indicated that SRCA-Cas12a could distinguish V.parahaemolyticus from other food-borne pathogens with excellent specificity.Under optimal conditions,a linear relationship between the logarithm of the concentration of V.parahaemolyticus and the intensity of fluorescence signal was established,which was fitted as follows:y=1.8472x+2.4738(R^(2)=0.9866)and the detection limit was 3.6 CFU/mL(R_(SN)=3).The spiked recoveries of V.parahaemolyticus in artificially contaminated samples were 95.5%-104.4%.The method was applied for the detection of actual samples with a sensitivity of 100.0%,a specificity of 95.2%,and a coincidence rate of 97.2%.Hence,this developed method has the advantages of excellent specificity and low detection limit,thereby providing a new strategy for rapid quantitative detection of V.parahaemolyticus.

关 键 词：CRISPR/Cas12a 跨越式滚环等温扩增 副溶血性弧菌 定量检测 海产品 

分 类 号：TS207.4[轻工技术与工程—食品科学]