鹅出血性多瘤病毒SYBR GreenⅠ荧光定量PCR检测方法的建立  

Establishment of a SYBR GreenⅠ-based fluorescent quantitative PCR assay for detection of goose hemorrhagic polyomavirus

作　　者：罗灵芝 杨鹏江 丁彦彬 勾明郗 赵墩[1] 余兴龙[1] 杨磊[1] LUO Ling-zhi;YANG Peng-jiang;DING Yan-bin;GOU Ming-xi;ZHAO Dun;YU Xing-long;YANG Lei(College of Veterinary Medicine,Hunan Agricultural University,Changsha 410128,China)

机构地区：[1]湖南农业大学动物医学院,湖南长沙410128

出　　处：《中国兽医科学》2022年第3期278-284,共7页Chinese Veterinary Science

基　　金：湖南省教育厅科学研究项目(19C0923)。

摘　　要：为了建立一种能快速、灵敏且准确检测鹅出血性多瘤病毒(goose hemorrhagic polyomavirus,GHPV)的诊断方法,将GHPV的VP1基因构建到载体pClone007 Blunt Simple Vector上,将获得的重组质粒作为标准阳性模板,以建立GHPV VP1基因的SYBR GreenⅠ荧光定量PCR检测方法,并对该方法的特异性、敏感性、重复性和在临床检测中的应用进行验证。结果显示,所建立的SYBR GreenⅠ荧光定量PCR方法的标准曲线呈良好的线性关系,其线性相关系数R^(2)=0.975,其扩增效率E=86.731%,熔解曲线呈现单峰;检测鹅细小病毒、鹅圆环病毒、鹅副黏病毒、鸭坦布苏病毒、鹅星状病毒时呈阴性,具有较好的特异性;最低检出浓度为(1.16×10^(1)copies/μL),比普通PCR方法灵敏100倍;其中组内变异系数在0.10%~1.48%之间,组间变异系数在1.07%~2.24%之间,具有较好的重复性和稳定性;对20份临床组织样品检测结果进行分析发现,与普通PCR检测结果相比阳性率高出20%,其中阳性符合率为100%,总符合率为80%。上述结果表明,本研究建立的检测GHPV的SYBR GreenⅠ荧光定量PCR方法,对GHPV的快速诊断和监测具有重要意义。To establish a rapid,sensitive and accurate diagnostic method for detecting goose hemorrhagic polyomavirus(GHPV),the VP1 gene of GHPV was constructed on the p Clone007 Blunt Simple Vector,and the recombinant plasmid was obtained as a standard positive template.The SYBR GreenⅠ-based fluorescent quantitative PCR assay for detecting GHPV VP1 gene was established,and the specificity,sensitivity,reproducibility and application of the method in clinical testing were also validated.In result,the standard curve of the established SYBR GreenⅠfluorescent quantitative PCR method showed good linearity with a linear correlation coefficient R^(2)=0.975,its amplification efficiency E=86.731%,and the melting curve showed a single peak;The results were negative for detecting goose microvirus,goose circovirus,goose paramyxovirus,duck tambusovirus,and goose astrovirus,and this method had good specificity.The minimum detection concentration was(1.16×10^(1)copies/μL)100 times more sensitive than the general PCR;the within-group coefficient of variation ranged from 0.10%to 1.48%,and the between-group coefficient of variation ranged from 1.07%to 2.24%,with good reproducibility and stability;Twenty clinical tissue samples were analyzed,and the positive rate was 20%higher than that of general PCR,while the positive accordance rate was 100%and the overall accordance rate was 80%.The above-mentioned results showed that the SYBR GreenⅠ-based fluorescent quantitative PCR method for detecting GHPV in this study is important for the rapid diagnosis and monitoring of GHPV.

关 键 词：鹅出血性多瘤病毒 SYBR GreenⅠ 荧光定量PCR 

分 类 号：S852.659.6[农业科学—基础兽医学]