马动脉炎病毒Eva Green荧光定量RT-PCR检测方法的建立及初步应用  

Development and application of Eva Green qRT-PCR assay for detection of equine arteritis virus

作　　者：胡月[1,2] 戚亭[2] 胡哲[2] 关平原[1] 王晓钧[2] 

机构地区：[1]内蒙古农业大学兽医学院/农业部动物疾病临床诊疗技术重点实验室,内蒙古呼和浩特010018 [2]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/马传染病与慢病毒创新团队,黑龙江哈尔滨150001

出　　处：《中国预防兽医学报》2014年第12期943-947,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家科技支撑计划(2012BAD46B01-02;2012BAD46B03);公益性行业(农业)科研专项经费项目(201003075)

摘　　要：为建立马动脉炎病毒(EAV)的快速检测方法,本研究设计了一对针对EAV Bucyrus株ORF7基因序列的特异性引物,建立了检测EAV的Eva Green荧光定量RT-PCR(q RT-PCR)方法。结果表明,该方法在102拷贝/μL^107拷贝/μL范围内具有良好的线性关系;最低检出量为101.5TCID50/m L病毒,敏感性为常规RT-PCR的100倍,与其他马常见病毒无交叉反应;组内及组间重复性试验的变异系数均小于2%,具有较好的重复性。此外,采用该方法测定了EAV Bucyrus株及其拯救病毒ic EAV感染RK-13细胞后的复制动态,并与TCID50方法进行了比较。结果显示ic EAV与其亲本病毒接种RK-13细胞后,0 h测定病毒TCID50及拷贝数分别为103.33TCID50/m L(104.65拷贝/2μL)和103.50TCID50/m L(104.70拷贝/2μL);60 h时,细胞病变达到100%,ic EAV及其亲本病毒的TCID50及拷贝数分别为105.67TCID50/m L(107.0拷贝/2μL)和105.57TCID50/m L(106.98拷贝/2μL),两株病毒在RK-13细胞内的复制效率相似。本研究建立的Eva Green q RT-PCR方法可以为细胞培养物中EAV的检测提供有效的技术手段。In this study,an Eva Green based real-time RT-PCR （qRT-PCR） was developed for detecting equine arteritis virus （EAV） with a pair of primers designed according to the ORF7 sequence of EAV Bucyrus strain.The standard curve showed that the correlation coefficient of the assay was 0.999 with a linear relationship from 102 copies/μL to 107 copies/μL of the standard recombinant plasmid.The assay was specific for detecting EAV with a limit detection of 101.5 TCID50/mL which was 100 times to the regular RT-PCR method,and had no cross-reaction to other related equine viruses.In addition,the coefficient of variation was less than 2％ for both intra-assay and inter-assay.Moreover,the loads （in viral RNA copies） of icEAV and EAV Bucyrus strain in EAV-infected RK-13 cells were compared with the TCID50 assay.The results showed that the titers and copies of icEAV and EAV were 103.33 TCID50/mL（104.65 copies/2 μL） and 103.50 TCID50/mL（104.70 copies/ 2 μL） at 0 hour post infection （PI）,respectively.When the RK-13 cells infected icEAV and EAV developed 100％ CPE over 60 hours PI,the titers and copies of icEAV and EAV were 105.67 TCID50/mL（1070 copies/2 μL） and 105.57 TCID50/mL（106.98 copies/2 μL）,respectively,indicating that the replication kinetics of the two virus were similar in RK-13 cells.These data showed that the Eva Green qRT-PCR assay could be applied to quantitative detection of EAV infection.

关 键 词：马动脉炎病毒 QRT-PCR EVA Green 病毒含量 

分 类 号：S852.65[农业科学—基础兽医学]