食品科学 ›› 2022, Vol. 43 ›› Issue (14): 289-295.doi: 10.7506/spkx1002-6630-20210917-218

• 安全检测 • 上一篇    

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

董换哲，苑宁，张蕴哲，杨倩，卢鑫，郭威，张伟   

  1. （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）
  • 发布日期:2022-07-28
  • 基金资助:
    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）

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

DONG Huanzhe, YUAN Ning, ZHANG Yunzhe, YANG Qian, LU Xin, GUO Wei, ZHANG Wei   

  1. (1. College of Food Science and Technology, Hebei Agricultural University, Baoding 071001, China; 2. College of Science and Technology, Hebei Agricultural University, Cangzhou 061100, China; 3. College of Public Health, Hebei University, Baoding 071002, China; 4. College of Life Sciences, Hebei Agricultural University, Baoding 071001, China; 5. Hebei Key Laboratory of Analysis and Control of Zoonotic Pathogenic Microorganism, Baoding 071001, China)
  • Published:2022-07-28

RichHTML

23

PDF (PC)

90

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=1.847 2x＋2.473 8（R2=0.986 6），检出限为3.6 CFU/mL（RSN=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。

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

Abstract: 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 = 1.847 2x + 2.473 8 (R2 = 0.986 6) and the detection limit was 3.6 CFU/mL (RSN = 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.

Key words: CRISPR/Cas12a; saltatory rolling circle amplification; Vibrio parahemolyticus; quantitative detection; seafood

中图分类号: 

京ICP备14027880号-3　京公网安备 11010202009729号
地址：北京市丰台区洋桥70号 中国食品杂志社《食品科学》编辑部　邮编：100068
电话：010-83155446/47/48/49/50　 传真：010-83155436　 Email：foodsci@126.com
本系统由北京玛格泰克科技发展有限公司设计开发　技术支持：support@magtech.com.cn