猫疱疹病毒1型RPA-Cas12a-LFD检测方法的建立及初步应用  

RPA-Cas12a-LFD Based Nucleic Acid Detection for Feline Herpesvirus-1 and Preliminary Application

作　　者：黄坚 刘韵佳 杨晓农[1,2] 李妍 HUANG Jian;LIU Yunjia;YANG Xiaonong;LI Yan(College of Animal Husbandry & Veterinary Sciences, Southwest Minzu University, Chengdu 610041, China;Veterinary Teaching Hospital, Southwest Minzu University, Chengdu 610041, China)

机构地区：[1]西南民族大学畜牧兽医学院,成都610041 [2]西南民族大学教学动物医院,成都610041

出　　处：《畜牧兽医学报》2022年第5期1638-1643,共6页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：西南民族大学中央高校基本科研业务费专项(2020NQN32);西南民族大学引进人才科研启动金资助项目(RQD2021098)。

摘　　要：猫疱疹病毒1型(feline herpesvirus-1,FHV-1)为水痘病毒属的双链DNA包膜病毒,是引起猫上呼吸道感染的主要病原之一。本研究以重组聚合酶扩增(RPA)、Cas12a反式酶切反应和侧流层析试纸条(LFD)显示技术为基础,旨在建立靶向FHV-1 TK基因的RPA-Cas12a-LFD快速可视化检测方法。根据FHV-1 TK基因的保守片段序列设计RPA引物和合成crRNA的引物,并通过反应体系验证、RPA-Cas12a-LFD检测灵敏度和特异性分析,以及临床样本的符合检测,评价FHV-1 RPA-Cas12a-LFD方法的有效性。结果显示,建立的FHV-1 RPA-Cas12a-LFD方法可以特异性地检出FHV-1(与其他猫相关病原无交叉反应),灵敏度极高(最低检测限为2.35×10^(-1) copies·μL^(-1)),检测时间短,结果可视化。该方法对20份表现明显症状的患猫呼吸道样本的FHV-1检出率(35%,7/20)高于现有的TB Green qPCR方法(25%,5/20),对其中5份阳性样本的检测符合率为100%。综上表明,成功建立的FHV-1 RPA-Cas12a-LFD检测方法的特异性好和敏感性极高,不需要特殊的检测设备,可以作为FHV-1现场快速检测的有效工具。Feline herpesvirus-1(FHV-1)is double-stranded DNA enveloped virus of Varicellovirus,is one of major causative pathogen for feline upper respiratory tract infection.Based on recombinase polymerase amplification(RPA),Cas12a trans-cleavage reaction and lateral flow dipstick(LFD),a rapid visulization method for FHV-1 detection(RPA-Cas12a-LFD)was developed and validated.RPA amplified primers and crRNA synthesized primers were designed by targeting the FHV-1 TK gene,and then validation of RPA-Cas12a-LFD reaction system,sensitivity and specificity tests,and consistensy analysis of TB Green qPCR and RPA-Cas12a-LFD for FHV-1 detection were performed respectively.The results showed that the RPA-Cas12a-LFD method could specifically detect FHV-1 without cross-reaction with other associated pathogens,with advantages of high sensitivity(detection limit was 2.35×10^(-1)copies·μL^(-1)),short detection time and visual readout.The detection rate of FHV-1 in 20 clinical samples(35%,7/20)was higher than TB Green qPCR(25%,5/20)and the coincidence rate of 5 positive samples was 100%.In conclusion,RPA-Cas12a-LFD method was of good specificity and ultrasensitivity for FHV-1 detection without special equipment,and could be a promising and reliable tool for rapid on-site diagnosis.

关 键 词：猫疱疹病毒1型 RPA-Cas12a-LFD 核酸检测 应用 

分 类 号：S852.659.6[农业科学—基础兽医学]