鸽疱疹病毒荧光定量PCR检测方法的建立和初步应用  

Establishment and preliminary application of fluorescent quantitativePCR for detection of pigeon herpesvirus

作　　者：安乐乐 杨宣叶 周海云 宋晓琛 李彦彤 罗迅 赵永清[1,3] AN Le-le;YANG Xuan-ye;ZHOU Hai-yun;SONG Xiao-chen;LI Yan-tong;LUO-Xun;ZHAO Yong-qing(Biomedical Research Center of Northwest University for Nationalities Animal Cell Technology Innovation Center of Gansu province,Lanzhou 730030,China;China-malaysia National Joint Laboratory of Biomedical Research Center,Northwest University for Nationalities,Lanzhou 730100,China;College of Life Science and Engineering,Northwest University for Nationalities,Lanzhou 730100,China;Institute of Animal Husbandry and Veterinary Medicine,Chinese Academy of Agricultural Sciences,Beijing 100080,China)

机构地区：[1]西北民族大学生物医学研究中心/甘肃省动物细胞技术创新中心,甘肃兰州730030 [2]西北民族大学生物医学研究中心中国-马来西亚国家联合实验室,甘肃兰州730100 [3]西北民族大学生命科学与工程学院,甘肃兰州730100 [4]中国农业科学院北京畜牧兽医研究所,北京100080

出　　处：《中国预防兽医学报》2025年第2期166-171,共6页Chinese Journal of Preventive Veterinary Medicine

基　　金：甘肃省自然科学基金项目(21JR1RA221);兰州市科技计划项目(2023-1-31);中央高校基本科研项目(31920220050)。

摘　　要：为建立可快速、灵敏且特异性检测鸽疱疹病毒(Pi HV)的SYBR Green I荧光定量PCR方法(qPCR),本实验根据GenBank中登录的PiHV DNA聚合酶基因(KJ995972.1)保守区域设计引物,经PCR扩增该基因后连接至pMD19-T载体,构建质粒标准品pMD19-T-PiHV。采用方阵试验对引物终浓度和退火温度优化后,建立检测PiHV的qPCR方法,并用10倍倍比稀释的质粒标准品进行qPCR扩增建立标准曲线。利用建立的该检测方法对鸽圆环病毒(PiCV)、鸽痘病毒(PPV)、鸽腺病毒(PiADV)、鸽轮状病毒(PiRV)、鸽细环病毒(PTTV)核酸和重组质粒pMD19-T-PiHV进行检测,评估该qPCR方法的特异性;利用建立的qPCR和常规PCR分别检测10~0拷贝/μL~10^(8)拷贝/μL的质粒标准品,比较二者的敏感性;以5个浓度质粒标准品作为模板进行组内和组间重复性试验,评估该qPCR方法的重复性;使用该qPCR方法和常规PCR方法对112份疑似Pi HV感染鸽临床组织样品同时检测。标准曲线结果显示,重组质粒标准品在9.47×10^(3)拷贝/μL~9.47×10^(9)拷贝/μL内与其各自的Ct值呈良好的线性关系,相关系数R^(2)=0.9988,扩增效率E=97.7%;特异性试验结果显示,该qPCR方法仅重组质粒pMD19-T-PiHV出现S型扩增曲线,与其他病原核酸均未出现交叉反应,特异性强;敏感性试验结果显示,该qPCR方法对质粒标准品的检测限为9.47拷贝/μL,敏感性是常规PCR方法的100倍;重复性试验结果显示,该qPCR方法组内和组间重复性试验的变异系数均小于1%,重复性好。临床样品检测结果显示,qPCR方法阳性检出率45.54%(51/112),高于常规PCR方法的阳性检出率35.71%(40/112),二者阳性符合率为100%,总符合率为90.18%。本实验在国内首次建立的PiHV qPCR检测方法特异性强、敏感性高和重复性好,可快速高效检测临床样品中的PiHV,为鸽疱疹病毒的临床检测及流行病学调查提供了新的技术手段。To establish a rapid,sensitive and specific SYBR GreenⅠfluorescent quantitative PCR(qPCR)method for detecting pigeon herpesvirus(PiHV).The method was established using the conservative DNA polymerase gene of PiHV to construct the plasmid standard pMD19-T-PiHV.Multiple primers were designed,screened,and optimized for concentrations and annealing temperature.The standard curve was established by qPCR amplification with 10-fold diluted plasmid standards.The specificity of the qPCR method was assessed by testing pigeon circovirus(PiCV),pigeon poxvirus(PPV),pigeon adenovirus(PiADV),pigeon rotavirus(PiRV),pigeon torquetenovirus(PTTV)and recombinant plasmid pMD19-T-PiHV.The sensitivity was compared between established qPCR and conventional PCR using plasmid standards at 100 copies/μL-10^(8)copies/μL.Intra-repeatability assay and interrepeatability assay were performed with five plasmid concentrations.To evaluate the repeatability of the qPCR method.This qPCR method was preliminary applied to clinical samples.Results showed the qPCR standard curve of the method had a good linear relationship(R^(2)=0.9988)and high amplification efficiency(E=97.7%)within 9.47×10^(3)copies/μL-9.47×10^(9)copies/μL.Specificity tests confirmed no cross-reactivity with other pathogens.Sensitivity test results showed a detection limit to plasmid standard was 9.47 copies/μL for qPCR,which was 100 times more sensitive than the conventional PCR method.The repeatability test showed coefficients of variation of the intra-group and inter-group repeatability test of the qPCR method was less than 1%.In clinical sample detection,qPCR identified 51 positive samples(45.54%)out of 112,higher than conventional PCR's 40(35.71%).All 40 PCRpositive samples were included in the qPCR-positive group,with a 100%positive coincidence rate and 90.18%total coincidence rate.The results showed that the established fluorescence quantitative PCR detection method for pigeon herpesvirus had good linear relationship,strong specificity,high sensitivity and good repeat

关 键 词：鸽疱疹病毒 DNA聚合酶基因 荧光定量PCR 临床检测 流行病学调查 

分 类 号：S855.3[农业科学—临床兽医学]