摘要
非洲猪瘟在临床上出现多种毒株,主要分为野毒株和基因变异毒株。因其在临床上造成不同程度的危害,有必要建立一种方法进行正确诊断和健康监测。为实现野毒株和基因变异毒株的鉴别诊断,研究根据GenBank中的ASFV中国流行株(登录号:MK333180.1)的P72、CD2v、MGF基因保守序列设计引物,建立一种可以同时检测上述三种基因的三重荧光定量PCR方法。三重荧光定量PCR方法对猪细小病毒(PPV)、猪伪狂犬病病毒2型(PRV)和猪圆环病毒(PCV2)检测均无交叉反应,表现出较强的特异性。敏感性检测显示针对P72、CD2v和MGF重组质粒标准品的最低检测下限为103~102 copies/mL。在重复性试验中,大部分组间变异系数均小于2%或接近2%,具有较好的重复性。试验表明,三重荧光定量PCR方法可用来鉴别野毒株和基因变异毒株。
African swine fever(ASF)has a variety of clinical strains,mainly divided into wild strains and gene variant strains.It is necessary to establish a method for correct diagnosis and health monitoring because it causes different degrees of harm in clinical practice.In order to realize the differential diagnosis between wild and genovariation strains,primers were designed according to the conserved sequences of P72,CD2v and MGF of ASFV in China(entry number:MK333180.1)in GenBank,and a triple fluorescence quantitative PCR method was established to detect the above three genes simultaneously.There was no cross-reaction in the detection of porcine parvovirus(PPV),porcine pseudorabies virus(PRV)and porcine circovirusⅡ(PCV2)by triple fluorescence quantitative PCR method,showing strong specificity.Sensitivity tests showed that the lower limit of detection for P72,CD2v and MGF recombinant plasmid standards was 103~102 copies/mL.In the repeatabitity test,the coefficient of variation between most groups was less than 2%or close to 2%,showing good repeatabitity.The results showed that the triple fluorescence quantitative PCR method could be used to identify wild strains and gene variant strains.
作者
张红云
陈秋英
严斯刚
杜泓明
陆志翔
郑敏
罗廷荣
Zhang Hong-yun;Chen Qiu-ying;Yan Si-gang;Du Hong-ming;Lu Zhi-xiang;Zheng Min;Luo Ting-rong(College of Life Science and Technology,Guangxi University,Nanning,Guangxi 530004,China;Guangxi Yuemu Biotechnology Co.,Ltd.,Nanning,Guangxi 530006,China;Guangxi Pudi Enwei Biotechnology Co.,Ltd.,Nanning,Guangxi 530006,China;Liuzhou Center for Animal Disease Prevention and Control,Liuzhou,Guangxi 545026,China;College of Animal Science and Technology,Guangxi University,Nanning,Guangxi 530004,China;Guangxi Center for Animal Disease Prevention and Control,Nanning,Guangxi 530004,China)
出处
《广西农学报》
2023年第1期32-39,共8页
Journal of Guangxi Agriculture
基金
柳州市科学研究与科技开发项目(2020NACB0801):非洲猪瘟快速预警关键技术开发与应用。
关键词
非洲猪瘟
野毒株
基因缺失毒株
三重荧光定量PCR
检测方法
African swine fever
wild strain
gene deletion strain
triple fluorescence quantitative PCR
detection method
