猫泛白细胞减少症病毒、猫杯状病毒和猫疱疹病毒1型三重TaqMan荧光定量RT-PCR检测方法的建立与应用

Establishment and application of a triplex TaqMan fluorescence quantitative RT-PCR assay for simultaneous detection of feline panleukopenia virus,feline calicivirus and feline herpesvirus 1

为建立一种能够快速检测猫泛白细胞减少症病毒(FPV)、猫杯状病毒(FCV)和猫疱疹病毒1型(FHV-1)的三重荧光定量RT-PCR方法,本研究根据FPV VP2基因、FCV ORF2基因和FHV-1 TK基因分别设计引物与探针,采用上述引物经PCR分别扩增上述3种病原的目的基因并克隆至pMD18-T载体中,构建重组质粒标准品pMD18-T-FPV、pMD18-T-FCV和pMD18-T-FHV-1,并均经PCR和测序鉴定。将3种重组质粒标准品稀释到同一浓度1×10^(8)拷贝/μL,按体积比1∶1∶1混合后作为模板,采用方阵法优化各反应条件后,建立了检测上述病原的三重TaqMan荧光定量RT-PCR方法。以FPV、FCV、FHV-1、猫博卡病毒、猫冠状病毒、猫支原体、猫白血病病毒、猫星状病毒的基因组为模板采用本研究建立的方法检测,评估该方法的特异性,结果显示,该方法仅能检测到FPV、FCV、FHV-1,而其他病原的检测结果均为阴性。选取1×10^(0)拷贝/μL~1×10^(6)拷贝/μL的混合质粒标准品和10^(-0.5) TCID_(50)/mL~10^(5.5) TCID_(50)/mL的混合病毒液分别作为模板,采用本研究建立的方法检测,评估该方法检测重组质粒和病毒的敏感性,结果显示,该方法对重组质粒标准品pMD18-T-FPV、pMD18-T-FCV和pMD18-T-FHV-1的检测限均为1.0×10^(1)拷贝/μL,对3种病毒的检测限均约为10^(0.5) TCID_(50)/mL;以1×10^(7)拷贝/μL、1×10^(5)拷贝/μL、1×10^(3)拷贝/μL质粒标准品混合物作为模板分别进行组内和组间的重复性试验,结果显示,组内和组间重复性试验的变异系数(CV)均低于3%。利用建立的该三重荧光定量RT-PCR和常规PCR对81份临床样品(77份猫的眼、鼻、咽、肛混合拭子样品和4份组织病料样品)分别检测,结果显示三重荧光定量RT-PCR检测出47份FPV、13份FCV和13份FHV-1阳性样品,而常规PCR分别检测出39份FPV、9份FCV和6份FHV-1阳性样品,该三重荧光定量RT-PCR与FPV、FCV、FHV-1常规PCR的总符合率分别为90.12%、95.06%、92.59%。本研究建立的三重TaqMan荧光定量RT-PCR检测方法特异性强、敏感性高、重复性好,为FPV、FCV、FHV-1临床样品的快速鉴别检测提供了有力技术支持。

In order to establish a method for rapid detection of feline panleukopenia virus(FPV),feline calicivirus(FCV)and feline herpesvirus 1(FHV-1),primers and probes were designed according to the sequences of FPV VP2 gene,FCV ORF2 gene and FHV-1 TK gene,which were used to amplify the three target genes,and the recombinant plasmids pMD18-T-FPV,pMD18-T-FCV and pMD18-T-FHV-1 were constructed as standard plasmids,respectively.Three recombinant standard plasmids were diluted to the same concentration of 1×10^(8) copies/μL,mixed according to the volume ratio of 1∶1∶1 as a template,and a triplex TaqMan fluorescence quantitative RT-PCR method was established by optimizing the reaction system.The specificity of the method was evaluated with the genomes of FPV,FCV,FHV-1,FBoV,M.felis,FeLV,FCoV,and FAstV as templates.The results showed that the method was positive for detecting FPV,FCV,and FHV-1,and negative for detecting FBoV,M.felis,FeLV,FCoV and FAstV.Sensitivity tests were carried out on the mixed standard plasmids of 1×10^(0) copies/μL-1×10^(6) copies/μL and the mixed viruses of 10^(-0.5)TCID_(50)/mL-10^(5.5)TCID_(50)/mL,which showed that the detection limits of pMD18-T-FPV,pMD18-T-FCV and pMD18-T-FHV-1 were 1.0×10^(1) copies/μL.The detection limits of the three viruses were about 10^(0.5)TCID_(50)/mL.Using 1×10^(7) copies/μL,1×10^(5) copies/μL,and 1×10^(3) copies/μL mixed standard plasmids as templates,the results showed that the coefficient of variation(CV)of intra-batch and inter-batch repeatability assay were lower than 3%.The established triplex TaqMan fluorescence quantitative RT-PCR and conventional PCR detection methods were used to detect 81 clinical samples(77 eye-nasopharyngeal and anal mixed swab samples and 4 tissue samples of felin).The results showed that the triplex TaqMan fluorescence quantitative RT-PCR detected 47,13,and 13 positive FPV,FCV,and FHV-1 samples.However,conventional PCR detected only 39,9,and 6 positive FPV,FCV and FHV-1 samples.The total positive coincidence rates of FPV,FCV,and FHV-1 were 90.12%,95.06%,and 92.59%,respectively.The triplex TaqMan fluorescence quantitative RT-PCR method established in this study has strong specificity,high sensitivity,and good reproducibility,which provides powerful technical support for the rapid identification of FPV,FCV,and FHV-1.