白斑综合征病毒DETECTR检测系统的建立  

Establishment of DETECTR detection system for white spot syndrome virus

作　　者：徐博文 周傲白雪 廖秀云 蒋晓霞 邵建宏 沙才华 黄海超 赵福振 陈轩 罗宝正 XU Bowen;ZHOU Aobaixue;LIAO Xiuyun;JIANG Xiaoxia;SHAO Jianhong;SHA Caihua;HUANG Haichao;ZHAO Fuzhen;CHEN Xuan;LUO Baozheng(Technical Center of Gongbei Customs District,Zhuhai 519015,China)

机构地区：[1]拱北海关技术中心,广东珠海519015

出　　处：《中国兽医科学》2024年第8期1056-1064,共9页Chinese Veterinary Science

基　　金：广东省重点领域研发计划(2022B1111030001)。

摘　　要：将重组酶介导等温扩增技术(RAA)与成簇的规律间隔短回文重复序列及其相关蛋白12a(CRISPR-Cas12a)检测系统相结合,针对白斑综合征病毒(WSSV)建立快速、灵敏、特异的DNA内切酶靶向CRISPR反式报告(DETECTR)检测系统。针对WSSV保守基因设计RAA引物与crRNA,通过实验筛选最优组合和最佳反应条件,构建WSSV DETECTR检测系统。对该系统进行灵敏度与特异性测试,与WOAH推荐的Real-timePCR和NestedPCR检测方法比较。结果显示,WSSV DETECTR检测系统最低检出限为10copies/μL,灵敏度与Real-time PCR、Nested PCR检测方法相当(均为3.34×10^(1)copies/μL),与其他病原之间无交叉反应,与Real-time PCR、Nested PCR对临床样本的检测结果相一致。结果表明,建立的WSSV DETECTR检测系统具有快速简单、灵敏、特异等优点,为白斑综合征病毒的快速检测提供了新的方法。To establish a rapid,sensitive and specific DNA endonuclease targeted CRISPR trans reporter(DETECTR)detection system for white spot syndrome virus(WSSV)by combining recombinase-mediated amplification(RAA)with CRISPR-Cas12a.RAA primers and cr RNA were designed for the conserved genes of WSSV,and the optimal combination and best reaction condition were screened experimentally to construct the WSSV DETECTR detection system.The sensitivity and specificity of the system were tested and compared with the real-time PCR and Nested PCR methods recommended by WOAH.The Results showed that the WSSV DETECTR system has a minimum detection limit of 10 copies/μL,which is the same as real-time PCR and Nested PCR(3.34×10^(1)copies/μL),there was no cross-reactivity with other pathogens and no difference in the test results from real-time PCR and Nested PCR for clinical samples.In conclusion,WSSV DETECTR detection system has the advantages of being rapid and simple,sensitive and specific,and provides a new method for the rapid detection of white spot syndrome virus.

关 键 词：重组酶介导等温扩增技术(RAA) CRISPR-Cas12a 白斑综合征病毒 快速检测 

分 类 号：S852.659.1[农业科学—基础兽医学]