RPA-CRISPR/Cas12a在病原微生物检测中的研究进展  

Research progress of RPA-CRISPR/Cas12a in the detection of pathogenic microorganisms

作　　者：王文涛[1,2] 赵良 侯立功 张万存 孙萌 王慧英[1] 凡雪静 陈东辉[1,2] 杨辉 WANG Wentao;ZHAO Liang;HOU Ligong;ZHANG Wancun;SUN Meng;WANG Huiying;FAN Xuejing;CHEN Donghui;YANG Hui(Children’s Hospital Affiliated of Zhengzhou University,Zhengzhou 450066,Henan,China;Henan International Joint Laboratory for Prevention and Treatment of Pediatric Diseases,Zhengzhou 450018,Henan,China;Biobank of Henan Children’s Hospital Zhengzhou Children’s Hospital,Zhengzhou 450018,Henan,China;The General Hospital of Jinshui District Zhengzhou City,Zhengzhou 450008,Henan,China)

机构地区：[1]郑州大学附属儿童医院,河南郑州450066 [2]河南省儿科病防治国际联合实验室,河南郑州450018 [3]河南省儿童医院郑州儿童医院生物样本库,河南郑州450018 [4]郑州市金水区总医院,河南郑州450008

出　　处：《微生物学通报》2024年第8期2785-2796,共12页Microbiology China

基　　金：河南省科技厅科技攻关计划(222102310109)。

摘　　要：重组酶聚合酶扩增(recombinase polymerase amplification,RPA)可以在恒温条件下高效扩增靶标序列以快速达到可以检测的水平,具有检测灵敏度高、检测速度快和设备依赖程度低等优点,是应用较广泛的恒温扩增技术之一。然而,RPA的非特异扩增会导致假阳性等问题。成簇的规律间隔短回文重复序列相关蛋白12a(clustered regularly interspaced short palindromic repeats/associated protein 12a,CRISPR/Cas12a)具有特异性酶切靶标双链功能,并且其最适温度与RPA反应温度相近。近年来,研究者将RPA与CRISPR/Cas12a联合对目的基因进行双重特异性识别,极大地提高了检测特异性,同时也进一步提升了检测的灵敏度,展示出检测特异性强、灵敏度高、适用范围广和操作简单等诸多优点,尤其在病原微生物检测方面展示出较大的应用前景。本文就RPA-CRISPR/Cas12a的技术原理、产物检测策略和在病原微生物检测等方面的研究进展进行综述,以期为RPA-CRISPR/Cas12a进一步开发、利用以及在病原微生物检测方面的应用提供借鉴。Recombinase polymerase amplification(RPA)can efficiently amplify the target sequence at constant temperatures and is praised for high sensitivity,short time consumption,and low equipment dependence.Therefore,RPA is a widely used isothermal amplification technology in recent years.However,non-specific amplification of RPA has limited its further development.The clustered regularly interspaced short palindromic repeats/associated protein 12a(CRISPR/Cas12a)is capable of specifically cleaving the double strands and has similar optimal temperature to the PRA temperature.Therefore,researchers have combined RPA with CRISPR/Cas12a to improve the detection specificity.Moreover,this method demonstrates high sensitivity,a wide application range,and simple operation.Particularly,the RPA-CRISPR/Cas12a-based approach shows a promising application prospect in the detection of pathogenic microorganisms.This paper introduces RPA-CRISPR/Cas12a regarding the principles,product detection strategies,and application,providing a reference for the further development and application of RPA-CRISPR/Cas12a in the detection of pathogenic microorganisms.

关 键 词：重组酶聚合酶扩增 成簇的规律间隔短回文重复序列相关蛋白12a 病原微生物 实时检测 肉眼观测 现场检测 

分 类 号：Q78[生物学—分子生物学] R446.5[医药卫生—诊断学]