猪瘟病毒和猪圆环病毒2型二重荧光LAMP检测方法的建立  

Development of Duplex Fluorescence-based Loop-mediated Isothermal Amplification Assay for Detection of CSFV and PCV Type 2

作　　者：谢志勤 张民秀 谢芝勋 范晴 罗思思 谢丽基 黄娇玲 王盛 曾婷婷 张艳芳 李孟 李丹 邓显文 刘加波 XIE Zhi-qin;ZHANG Min-xiu;XIE Zhi-xun;FAN Qing;LUO Si-si;XIE Li-ji;HUANG Jiao-ling;WANG Sheng;ZENG Ting-ting;ZHANG Yan-fang;LI Meng;LI Dan;DENG Xian-wen;LIU Jia-bo(Guangxi Veterinary Research Institute,Guangxi Key Laboratory of Veterinary Biotechnology,Nanning,Guangxi,530001,China)

机构地区：[1]广西壮族自治区兽医研究所/广西兽医生物技术重点实验室,广西南宁530001

出　　处：《动物医学进展》2021年第3期1-6,共6页Progress In Veterinary Medicine

基　　金：广西重点研发计划项目(桂科AB16380003);国家“万人计划”领军人才专项项目(W02060083);“广西八桂学者”专项项目(2019.79)。

摘　　要：建立二重荧光LAMP方法,检测猪瘟病毒(CSFV)和猪圆环病毒2型(PCV2)。根据CSFV E2基因和PCV2 ORF2基因序列保守区域,分别设计并合成了2组针对CSFV和PCV2序列的特异性引物和2条标记不同的荧光基团探针,在CSFV探针序列5′端标记FAM荧光基团,3′端标记BHQ1淬灭基团,PCV2探针序列5′端标记CY5.5荧光基团,3′端标记BHQ2淬灭基团。以设计合成的引物、探针以及LAMP反应试剂建立二重荧光LAMP检测方法区分CSFV和PCV2,对该方法中的反应体系进行优化,并进行特异性、敏感性和干扰性测试以及临床样品验证测试。结果表明,建立的方法可鉴别CSFV和PCV2,特异性试验显示,该方法只检测出CSFV或PCV2,且与参试的对照病毒无交叉反应,特异性好。该方法最低检测CSFV或PCV2为100拷贝/μL,敏感性好。临床样品的检测结果与荧光定量PCR检测结果一致。建立的CSFV和PCV2二重荧光LAMP检测方法具有良好的特异性和敏感性,可适用于CSFV和PCV2的鉴别检测。The aim of this study was to establish a rapid nucleic acid visible detection assay for classical swine fever virus(CSFV)and porcine circovirus type 2(PCV2).The primers and probes targeting the CSFV E2 gene and PCV2 ORF2 gene were designed.The CSFV-probe was labeled with FAM fluorescence-based at 5'terminal and BHQ1 quenches-base at 3'terminal.The PCV-probe was labeled with CY5.5 fluorescence-based at 5'terminal and BHQ2 quenches-base at 3'terminal.A duplex fluorescence-based loop-mediated isothermal amplification(LAMP)assay was developed.The specificity,sensitivity and stability were evaluated using primers and probes of CSFV and PCV2,followed by clinical specimen verification.The results showed that the specificity of this assay was high,and was able to detect CSFV and PCV2 without any cross-reaction with other known non-targeted bovine pathogens.The limits of detection of assay were 100 copies.Different concentration templates of CSFV and PCV2 could be identified when mixed together without any interference.Further,the results of this LAMP were 100%agreement with real-time PCR as a contrast in detection of clinical samples.In conclusion,a duplex LAMP assay for CSFV and PCV2 was established and proved to be specific and sensitive.So this test method can be used for clinical identification for CSFV and PCV2.

关 键 词：猪瘟病毒 猪圆环病毒2型 二重荧光LAMP 检测 

分 类 号：S852.659.6[农业科学—基础兽医学] S852.659.2[农业科学—兽医学]