BVDV-1型SYBR GreenⅠ荧光定量PCR方法的建立与初步应用  

Development and Preliminary Application of a SYBR Green I Fluorescent Quantitative PCR Method for Detection of Bovine Viral Diarrhea Virus Type 1

作　　者：付兢锋 陈姗 马浩原 祝昆儒 牟碧莹 薛皓文 宋彦昊 李强 高旭[1] FU Jingfeng;CHEN Shan;MA Haoyuan;ZHU Kunru;MU Biying;XUE Haowen;SONG Yanhao;LI Qiang;GAO Xu(Department of Veterinary Medicine,Agricultural College,Yanbian University,Yanji 133000,China)

机构地区：[1]延边大学农学院动物医学系,延吉133000

出　　处：《中国动物传染病学报》2025年第1期148-154,共7页Chinese Journal of Animal Infectious Diseases

基　　金：高等学校学科创新引智计划(D20034);延边大学校企合作项目(20201271135)。

摘　　要：本研究构建了一种牛病毒性腹泻病毒1型(BVDV-1)的高效临床检测方法。利用SYBR Green I荧光定量PCR扩增BVDV-1保守基因5'UTR的片段(124 bp),并构建重组质粒作为阳性标准品,建立标准曲线,对该方法的敏感性、特异性和重复性进行验证,同时采集临床样品,评价其检测效率。结果显示:BVDV-1阳性标准品在1.169×10^(8)~1.169×10^(1)拷贝/μL范围内呈良好的线性关系,相关系数R^(2)=0.997,熔解曲线无非特异性峰产生;重复性试验组内和组间变异系数低于2%;敏感性试验最低检测拷贝数为1.169×10^(1)拷贝/μL,为普通PCR的100倍;特异性良好,与BEV、BEFV、IBRV、BPIV3无交叉反应产生。采用建立的BVDV-1 SYBR GreenⅠ荧光定量方法检测62份牛腹泻粪便样品,阳性率为46.77%,高于普通PCR^(2)0.97%。本研究成功建立了BVDV-1荧光定量方法,该方法特异性强,灵敏度高,重复性稳定,可用于养殖业BVDV-1临床的快速检测。In this study,a SYBR Green I fluorescent quantitative PCR assay was developed for detection of Bovine viral diarrhea viruses type 1(BVDV-1).A fragment(124 bp)of the 5'UTR of the conserved gene of BVDV-1 was amplified and a recombinant plasmid was constructed as a positive standard.Subsequently,a standard curve was established to validate the sensitivity,specificity and reproducibility of the method,and clinical samples were collected to evaluate its detection efficiency.The results showed that the BVDV-1 positive standards showed good linearity in the range of 1.169×10^(8)-1.169×10^(1)copies/μL with the correlation coefficient R^(2)=0.997 and no non-specific peaks were generated from the melting curve.The intra-and inter-group coefficients of variation were less than 2%in the reproducibility test.The lowest detectable copy number was 1.169×10^(1)copies/μL,indicating its sensitivity was 100 times higher than that of the conventional PCR.Moreover,this method had good specificity as it had no cross-reactivity with BEV,BEFV,IBRV and BPIV3.The BVDV-1 SYBR Green I fl uorescence quantification method developed here was used to test 62 fecal samples from diarrhea cattle and the positive rate was 46.77%,which was 20.97%higher than that of the conventional PCR.In conclusion,this study,developed a fl uorescence quantification method for detection of BVDV-1,which was specific,sensitive and stable in reproducibility and thus could be used for rapid clinical detection of BVDV-1 in the farming industry.

关 键 词：牛病毒性腹泻病毒 荧光定量 临床应用 

分 类 号：S852.65[农业科学—基础兽医学]