检测猫杯状病毒RT-qPCR方法的优化与应用  

Optimization and application of RT-qPCR assay for detection of feline calicivirus

作　　者：刘迪 郑亚婷 许鑫燕 董丽丽 康洪涛 姜骞[1] 杨鸣发 刘家森[1] 曲连东[1] LIU Di;ZHENG Yating;XU Xinyan;DONG Lili;KANG Hongtao;JIANG Qian;YANG Mingfa;LIU Jiasen;QU Liandong(State Key Laboratory of Veterinary Biotechnology,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：《中国兽医学报》2022年第1期53-60,共8页Chinese Journal of Veterinary Science

基　　金：国家重点研发计划资助项目(2019YFC1200701);兽医生物技术国家重点实验室课题资助项目(SKLVBP202104)。

摘　　要：为建立一种通用性良好的SYBR GreenⅠ荧光定量RT-PCR方法,根据GenBank上猫杯状病毒(feline calicivirus,FCV)ORF1保守区域序列,设计合成1对特异性引物,用于FCV的快速检测。灵敏性试验结果显示,该方法的检测下限为4.93×10;拷贝/μL,比普通RT-PCR检测下限高100倍;特异性试验结果显示,该检测方法与猫疱疹病毒、猫细小病毒、猫传染性腹膜炎病毒等无交叉反应;通用性试验结果显示,针对引物靶位点区间,该方法对所有已知变异位点的人工合成质粒和本实验室保存的不同变异位点的FCV毒株,均能够良好检出;重复性试验结果显示,组内的变异系数为0.09%~0.71%,组间变异系数为0.05%~0.82%,均小于1%。对23份临床样品进行检测,该方法的阳性检出率为60.9%,检出的14份阳性病料分别接种到CRFK细胞中,对细胞病变产物进行扩增并测序,BLAST对比后结果证实为FCV毒株。结果表明,本试验建立的荧光定量RT-PCR检测方法可用于FCV的快速诊断及流行病学的调查。According to the conserved region of ORF1 of feline calicivirus(FCV)in GenBank,a pair of specific primers were designed for amplifying specific fragment from FCV.A universal SYBR GreenⅠreal-time RT-PCR was established for the rapid detection of FCV.The result of sensitivity test showed that detection limit of the assay was 4.93×10;copies/μL,and it was 100 times higher than that of conventional RT-PCR.There was no amplification from feline herpesvirus,feline parvovirus and feline infectious peritonitis virus.The universality test showed that this method could detect all the synthetic plasmids with known variation sites and FCV strains with different variation sites preserved in our laboratory.The repeatability tests showed that the intra-assay coefficient of variation value was between 0.09%and 0.71%,and the inter-assay coefficient of variation value was between 0.05%and 0.82%,both of which were less than 1%.Twenty-three clinical samples were detected by this method,and the positive rate was 60.9%.Fourteen positive samples were inoculated into CRFK cells,then gene was cloned and sequenced when cytopathic effect appeared,the results were confirmed as FCV strains after BLAST comparison.In conclusion,the real-time RT-PCR assay established in this study can provide an effective way for the rapid diagnosis and epidemiological investigation of FCV.

关 键 词：猫杯状病毒 实时荧光定量RT-PCR SYBR GreenⅠ ORF1 

分 类 号：S852.65[农业科学—基础兽医学]