牛结节性皮肤病病毒GPCR基因荧光定量PCR检测方法的建立

Establishment of fluorescence quantitative PCR detection method for GPCR gene of lumpy skin disease virus

为了更好地进行牛结节性皮肤病的临床检测,根据GenBank登录的牛结节性皮肤病病毒(LSDV)的GPCR基因序列,设计并合成1对特异性引物,建立一种基于染料法的实时荧光定量PCR(qPCR)方法。本研究进一步对此检测方法进行了敏感性试验、特异性试验、重复性试验,随后用模拟临床样品进行了验证。结果显示,该方法至少可以检测出的病毒序列含量为7.11×102拷贝/μL;仅对LSDV有特异性扩增;批内重复和批间重复的Cq值变异系数均小于1.5%。模拟临床样品检测结果显示,建立的qPCR方法在临床采集的牛皮肤结节、肛拭子、泪拭子中,能特异性检测出LSDV的序列片段。结果表明,本研究建立的LSDV的qPCR检测方法敏感性高、特异性强、重复性好,能应用于临床诊断。

In August 2019,lumpy skin disease(LSD)broke out in Yili,Xinjiang Province of China,followed by outbreaks in many provinces in southern China.To better detect the LSD in field,a pair of specific primers was designed and synthesized based on the GPCR gene sequence of lumpy skin disease virus(LSDV)published in GenBank and the real-time fluorescence quantitative PCR method(qPCR)was established.Then the sensitivity test,specificity test and repeatability test for this detection method were carried out.Subsequently,simulated clinical samples were used for verification.The results showed that the method could detect the virus content as low as 7.11×102 copies/μL;the variation coefficient of Cq value of in-batch repetition and inter-batch repetition was less than 1.5%;only LSDV sequence could be amplified.The detection results of the simulated clinical samples showed that the qPCR method could specifically detect LSDV in lumpy skin,anal swabs and lacrimal swabs collected in the field,indicating that the established qPCR method was sensitive,specific and reproducible,and could be used in diagnosis.