摘要
传染性法氏囊病病毒新型变异株(Novel variant Infectious bursal disease virus,nVarIBDV)给我国养禽业防控法氏囊带来更大的挑战。目前缺乏快速、灵敏的针对nVarIBDV的检测方法。为解决这一问题,该试验根据已发表的nVarIBD的VP2基因序列,设计了一对引物和一条特异性Taq Man探针,建立了一种nVarIBD的Taq Man荧光定量RT-PCR检测方法,该方法可通过特异性扩增nVarIBD VP2基因来确定样品是否为IBDV新型变异株。通过构建重组质粒pMD18-T-VP2对引物和探针进行灵敏性和重复性试验,以及特异性检测,最后使用该检测方法对临床样品进行验证。荧光定量RT-PCR结果显示灵敏性可达1.32×10^(1)copies/μL,高于常规RT-PCR的100倍。批内和批间重复试验变异系数(CV)均小于0.5%,结果表明该检测方法有良好的重复性与稳定性。该方法与常见禽病毒核酸无交叉反应,说明检测方法具有较好的特异性。综合本研究结果表明,该研究建立的nVarIBD荧光定量RT-PCR检测方法特异性强、灵敏性高、重复性好,可用于临床样品中IBDV新型变异株的监测。
The Novel variant Infectious bursal disease virus(nVarIBDV)has brought greater challenges to the prevention and control of bursal bursal disease in the poultry industry in China.At present,there is a lack of rapid and sensitive detection methods for nVarIBDV.In order to solve this problem,based on the published VP2 gene sequence of nVarIBD,we designed a pair of primers and a specific Taq Man probe,and established a real-time Taq Man real time RT-PCR detection method for nVarIBD.This method can determine whether the sample is a new variant of IBDV by specifically amplifying the nVarIBD VP2 gene.The sensitivity,repeatability and specificity of the primers and probes were tested by constructing the recombinant plasmid pMD18-T-VP2,and finally the detection method was used to verify the clinical samples.The sensitivity of real-time RT-PCR was 1.32×10^(1)copies/μL,which was 100 times higher than that of conventional RT-PCR.The coefficient of variation(CV)of intra-assay and inter-assay was less than 0.5%.The results showed that the method had good repeatability and stability.There was no cross reaction between the method and the common avian virus nucleic acid,indicating that the detection method had good specificity.The nVarIBD real-time RT-PCR assay established in this study has high specificity,sensitivity and repeatability,and can be used for the detection of new IBDV variants in clinical samples.
作者
曹雪珍
周庆丰
王连想
严专强
林丽苗
李薇
李群辉
CAO Xuezhen;ZHOU Qingfeng;WANG lianxiang;YAN Zhuanqiang;LIN Limiao;LI Wei;LI Qunhui(College of Animal Science and Technology,Zhongkai University of Agriculture and Engineering,Guangzhou Guangdong 510225;Guangdong Provincial Key Laboratory of Livestock and Poultry Health Breeding and Environmental Control,Wens Food Group Co.,Ltd.,Yunfu Guangdong 527400)
出处
《广东畜牧兽医科技》
2024年第3期54-59,82,共7页
Guangdong Journal of Animal and Veterinary Science
基金
重大动物疫病新型综合防控技术资助(2022SDZG02)。
