猫疱疹病毒ERA-LFD检测技术的建立与应用  

Establishment and application of ERA-LFD detection technology of feline herpesvirus

作　　者：刘迪 许鑫燕 郑亚婷 杨育鹏 康洪涛 姜骞[1] 杨鸣发 刘家森[1] 曲连东[1] LIU Di;XU Xin-yan;ZHENG Ya-ting;YANG Yu-peng;KANG Hong-tao;JIANG Qian;YANG Ming-fa;LIU Jia-sen;QU Lian-dong(State Key Laboratory of Veterinary Biotechrwlogy/Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Harbin 150069,China;College of Veterinary Medicine,Northeast Agricultural University,Harbin,150030,China)

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室,黑龙江哈尔滨150069 [2]东北农业大学动物医学学院,黑龙江哈尔滨150030

出　　处：《中国兽医科学》2022年第1期11-18,共8页Chinese Veterinary Science

基　　金：兽医生物技术国家重点实验室开放课题(SKLVBP202104)。

摘　　要：为建立一种快速检测猫疱疹病毒(FHV)的酶促恒温扩增-横向流动试纸条(ERA-LFD)方法,根据GenBank上FHV TK基因保守区域序列,设计多对ERA特异性引物用于筛选最佳引物对及探针,以现场快速检测FHV。灵敏性试验表明,该方法的检测限为7.86×10^(1)copies/μL,比普通PCR的检测下限高10倍,与实时荧光定量PCR方法的检测限相当。特异性试验表明,该检测方法与猫细小病毒、猫杯状病毒、猫传染性腹膜炎病毒等无交叉反应。重复性试验表明,组内的变异系数在1.15%~4.76%之间,组间变异系数在2.09%~2.66%之间,均小于5%。对30份临床样品进行检测,该方法的阳性检出数为6,包含用普通PCR方法的检出的5份阳性病料。对临床检测为阳性的ERA扩增产物进行测序,BLAST对比结果证实为FHV毒株。综上所述,建立的ERA-LFD方法可用于猫疱疹病毒的快速检测及流行病学调查。To establish an enzymatic recombinase amplification(ERA) assay combined with a lateral flow dipstick(LFD) for the rapid detection of feline herpesvirus(FHV),according to the conserved region sequence of TK gene of FHV in Gen Bank,several pairs of specific ERA primers were designed for screening the most optimal primer pair and probe for detection of FHV on the spot.The results showed that the method was highly sensitive to detect as little as 7.86×10^(1) copies/μL,and it was 10 times more sensitive than conventional PCR.There was no amplification from feline parvovirus,feline calicivirus and feline infectious peritonitis virus.The repeatability tests showed that the intra-assay coefficient of variation value was 1.15% —4.76%,and the inter-assay coefficient of variation value was2.09%—2.66%,both of which were less than 5%.Of 30 clinical samples,6 samples were tested positive for FHV by ERA-LFD,including 5 positive samples tested by conventional PCR.The ERA amplification products detected clinically positive were sequenced,and the results exhibited a high similarity with other FHVs by BLAST.In conclusion,the ERA-LFD method can provide an effective way for the rapid detection and epidemiological investigation of FHV.

关 键 词：猫疱疹病毒 ERA-LFD 快速检测 

分 类 号：S852.659.1[农业科学—基础兽医学]