猪细小病毒TaqMan荧光定量PCR检测方法的建立及应用  

Establishment and Application of Taq Man Fluorescent Quantitative PCR Method for Detection of Porcine Parvovirus

作　　者：杨奕 吴发兴[2] 康京丽[2] 孙洪涛[2] 王志亮[2] 许信刚[1] 张琪[1] YANG Yi;WU Fa-xing;KANG Jing-li;SUN Hong-tao;WANG Zhi-liang;XU Xin-gang;ZHANG Qi(College of Veterinary Medicine,Northwest A&F University,Yangling,Shaanxi,712100,China;China Animal Health and Epidemiology Center,Qingdao,Shandong,266032,China)

机构地区：[1]西北农林科技大学动物医学院,陕西杨凌712100 [2]中国动物卫生与流行病学中心,山东青岛266032

出　　处：《动物医学进展》2024年第2期75-79,共5页Progress In Veterinary Medicine

基　　金：陕西省重点研发计划项目(2019NY-081)。

摘　　要：旨在建立一种猪细小病毒(PPV)快速准确PCR检测方法,根据PPV VP2基因的保守序列设计并合成1对特异性引物和1条特异性探针,建立了可检测PPV的TaqMan荧光定量PCR检测方法。建立的检测方法敏感性高,最低可检测8.76×10^(1)copies/μL的PPV VP2基因标准品;特异性试验结果显示,该方法检测猪流行性腹泻病毒、猪圆环病毒2型、伪狂犬病病毒、猪繁殖与呼吸综合征病毒均无交叉反应;重复性试验组内与组间变异系数均低于2%,重复性良好。用该方法检测102份临床样本,检出阳性样本16份,阳性检出率为15.69%,将检测出的阳性样本进行PCR扩增并测序,证实检测样品中确实存在PPV。该方法敏感性高、特异性强,适用于PPV的定量检测及猪细小病毒病的流行病学调查。In this study,the aim was to establish a rapid and accurate PCR detection method for porcine parvovirus(PPV).One pair of specific primers and one specific probe were designed and synthesized according to the conserved sequence of PPV VP2 gene,a Taq Man real-time PCR method for detection of PPV was established.The results showed that the established method had high sensitivity,and the lowest detectable PPV gene standard was 8.76×10^(1)copies/μL.The specificity test results showed that there was no cross-reaction in the detection of porcine epidemic diarrhea virus,porcine circovirus type 2,pseudorabies virus,porcine reproductive and respiratory disorder syndrome virus.The intra-group and inter-group coefficients of variation of the repeatability test were less than 2%,indicating that the method was reproducible.102 clinical samples were detected by this method,16 samples were positive,with a positive rate 15.69%.The PCR amplification and sequencing of the detected positive samples confirmed the presence of PPV in the test samples.This method has high sensitivity and specificity,and is suitable for the quantitative detection of PPV and the epidemiological investigation of porcine parvovirus disease.

关 键 词：猪细小病毒 VP2 TAQMAN探针 荧光定量PCR 

分 类 号：S859.659.2[农业科学—临床兽医学]