基于CRISPR/Cas蛋白的副溶血弧菌检测方法的研究

王群; 李哲; 赵林; 王紫鉴; 赵宏群; 阚飙; 逄波

doi:10.3784/j.issn.1003-9961.2020.06.012

基于CRISPR/Cas蛋白的副溶血弧菌检测方法的研究

Research on detection method of Vibrio parahaemolyticus based on CRISPR/Cas protein

摘要

摘要:
目的利用规律间隔性成簇短回文重复序列（CRISPR）免疫原理及Cas12a酶的特点构建一种快速检测副溶血弧菌（VP）的方法，实现对病原菌准确快速的检测和识别。
方法本研究通过制备纯化Cas12a蛋白，筛选构建VP的gRNA，建立CRISPR-VP荧光检测系统，根据最终荧光扩增曲线判定CRISPR-VP检测方法的有效性。
结果在CRISPR-VP检测方法中只在VP的序列存在时才会产生明显的荧光信号。
结论本实验初步建立了基于CRISPR/Cas蛋白的VP的检测方法，为后续简易检测试剂的研制提供理论依据。

Abstract:
ObjectiveTo establish a method for rapid detection of Vibrio parahaemolyticus by use the principle of clustered regularly interspaced short palindromic repeats (CRISPR) and the characteristics of Cas12a enzyme to achieve accurate and rapid detection and identification of pathogens.
MethodsIn this study, we prepared and purified Cas12a protein, screened and constructed V. parahaemolyticus guide RNA, established the CRISPR fluorescence detection system of V. parahaemolyticus, and determined the effectiveness of the CRISPR-VP detection method based on the final fluorescence amplification curve.
ResultsIn the CRISPR-VP detection method, a significant fluorescent signal will be produced only in the presence of the sequence of V. parahaemolyticus.
ConclusionThis experiment initially established the detection method of V. parahaemolyticus based on CRISPR/Cas protein, providing the preliminary conditions for the subsequent development of simple detection reagents.

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