牛血清中牛腺病毒3型EvaGreen qPCR检测方法的建立  

Establishment of an EvaGreen qPCR for the detection of bovine adenovirus type 3 in bovine serum

作　　者：贺倩 鱼轲 郭敏 李永佳 司璐 刘军 李佳林 魏至栋 HE Qian;YU Ke;GUO Min;LI Yong-jia;SI Lu;LIU Jun;LI Jia-lin;WEI Zhi-dong(The First Department of Vaccine in Lanzhou Institute of Biological Products Co.,Ltd.,Center for Gansu Provincial Vaccine Engineering Research,Lanzhou 730046,Gansu Province,China)

机构地区：[1]兰州生物制品研究所有限责任公司疫苗一室甘肃省疫苗工程技术研究中心,甘肃兰州730046

出　　处：《微生物学免疫学进展》2022年第4期15-20,共6页Progress In Microbiology and Immunology

摘　　要：目的建立EvaGreen qPCR检测牛腺病毒3型(bovine adenovirus 3,BAV3)的方法,用于牛血清等牛源材料的检测。方法依据GenBank公布的BAV3标准毒株WBR-1全基因序列,基于E2B区域高度保守序列设计引物,提取并制备DNA标准品,建立EvaGreen qPCR检测BAV3的方法,并对该方法的特异性、灵敏度及精密度进行验证。最后利用该方法检测牛血清中的BAV3,并与细胞培养法及荧光抗体检测对比,以验证该方法的适用性。结果建立的EvaGreen qPCR检测BAV3的方法标准曲线R^(2)>0.99,扩增效率处于90%~110%,可检测范围为0.2×10^(0)~0.2×10copies/μL。实验内Ct值的CV<2.2%,在不同实验间Ct值的CV<1.8%。设计的BAV3引物与牛腹泻病毒(bovine viral diarrhea virus,BVDV)、副流感病毒3型(parainfluenza virus 3,PI3)、呼肠孤病毒(reovirus,REO)、呼吸道合胞病毒(respiratory syncytial virus,RSV)、狂犬病毒(rabies virus,RV)、羊轮状病毒(Lanzhou lamb rotavirus,LLR)基因组均无交叉反应,特异性良好。采用EvaGreen qPCR对198份新生牛血清进行BAV3检测,阳性检出率为10.1%,检出率高于细胞培养法及荧光抗体检测。结论成功建立了EvaGreen qPCR检测BAV3的方法,该方法快速、灵敏、特异、易于标准化,可用于牛血清中BAV3的检测。Objective To develop an EvaGreen real-time quantitive polymerase chain reaction(EvaGreen qPCR)assay to detect BAV3 in bovine serum.Methods Primers were designed based on the highly conservative nucleotide sequence of E2 B region of the genome of BAV3 reference strain,DNA extracted from BAV3 culture was serially diluted as the standard DNA,and an EvaGreen qPCR method was established for detecting BAV3.Afterward,the specificity,sensitivity and precision of the method were also evaluated.Finally,BAV3 in bovine serum was detected with this method,cell culture method and fluorescence antibody method respectively,and the results were compared to verify the applicability of this method.Results The correlation coefficient of the standard curve generated with 10×10^(0)serial dilution DNA was higher than 0.99,the PCR efficiency was between 90%—110%.The intra-assay coefficient of variation(CV)of Ct values were less than 2.2%,and the inter-assay CV of the Ct values were less than 1.8%.No cross reaction to BVDV,PI3,REO,RSV,RV and LLR were detected,thus the method had a good specificity.This EvaGreen qPCR was used to detect BAV3 in 198 newborn bovine serum samples,and the positive rate was 10.1%,which was higher than the rate detected with cell culture method and immunofluorescence antibody detection method.Conclusion An EvaGreen qPCR method for detecting BAV3 was established successfully.This method was rapid,high sensitive,specific and easier to be standardized.This method can be used to detect BAV3 in raw materials derived from bovine serum.

关 键 词：外源病毒 牛腺病毒3型 实时定量PCR EvaGreen 

分 类 号：R373.9[医药卫生—病原生物学]