摘要
为建立检测马病毒性动脉炎病毒(EAV)的荧光定量RT-PCR检测方法,本研究针对EAV ORF7基因序列设计引物及探针,并对其反应体系进行优化,建立标准曲线,结果显示,EAV荧光定量RT-PCR方法在退火温度为61℃、引物和探针终浓度均为0.6 μmol/L的反应条件下效果最优;质粒标准品体外转录的cRNA在1.6×107拷贝/μL～1.6×102拷贝/μL时与Ct值之间呈现良好的线性关系,标准曲线方程为y=-2.68x+32.88,R2=0.9927,初步建立了EAV荧光定量RT-PCR方法.采用该方法检测EAV、马焦虫、马疱疹病毒1型、马疱疹病毒4型、马流感病毒,结果显示,该方法仅能特异性检测到EAV,其他病原检测均为阴性,该方法特异性强;将体外转录合成的质粒标准品cRNA 10倍倍比稀释后利用该方法检测,结果显示该方法最低检测限为1.6×102拷贝/μL,灵敏性高;重复性试验结果显示,该方法组内及组间变异系数均小于2.0%,重复性好.采用本实验建立的荧光定量RT-PCR方法和文献发表的EAV荧光定量RT-PCR方法分别对234份临床样品进行检测,两者的阳性检出率均为14.1%(33/234),两种方法的符合率为100%.本研究建立的荧光定量RT-PCR检测方法为马病毒性动脉炎的防控提供了新的技术手段和思路.
Abstract
A real-time RT-PCR detection method was established for detecting equine arteritis virus.Primers and probes were designed and synthesized within highly conserved region ORF7 of the EAV genome sequence.The annealing temperature was 61℃and a final concentration of primers and probes were 0.6μmol/L after optimizing the reaction system.There was a well linear relationship between the dilution series of EAV cRNA(1.6×107 copies/μL-1.6×102 copies/μL)and Ct values(y=-2.68x+32.88,R2=0.9927).This method was specific for detecting EAV and had no cross reaction with horse Taylor wom,horse herpesvirus type 1,horse herpesvirus type 4,and horse influenza virus.The sensitivity could reach at 160 copies/μL of EAV cRNA.The inter-group and intra-group variable coefficient of this method were both within 2.0%.234 clinical samples were tested by this real-time RT-PCR method and a method from previous research.The above two methods had consistent positive rate of detection(14.1%,33/234),This method can be used for the early detection of equine viral arteritis virus infection,which provides workable solutions for the prevention and control of equine viral arteritis.
基金项目
海关总署科研项目(2022HK126)
国家自然科学基金青年科学基金(2022D01B08)
NETL
NSTL
万方数据