罗非鱼湖病毒微滴式逆转录数字PCR检测方法的建立

Establishment of reverse transcription droplet digital PCR assay for detection of Tilapia Lake Virus

建立一种敏感性高、特异性强、重复性好的罗非鱼湖病毒反转录微滴式数字PCR (RT-ddPCR)检测方法,可为罗非鱼湖病毒的定量检测提供技术支持。参照NCBI中GenBank登陆的TiLV第3段全基因序列,选择hypothetical protein gene基因作为靶位基因设计合成了1对引物和探针,以TiLV-cDNA为模板,摸索、优化反应方法,建立与实时荧光RTPCR检测方法的线性关系,分析方法的敏感性、特异性、重复性,最后进行临床样品检测。结果显示,当引物、探针浓度分别为500、300 nmol·L^(-1)且退火温度为54.2℃时,建立的TiLV RT-ddPCR扩增反应效率最高、阴阳性微滴分布界限最明显、平均拷贝数较高;敏感性强,检测限低至2拷贝·μL^(-1),且在1~90 000拷贝·μL^(-1)范围内与实时荧光RT-PCR检测的线性关系较好(R2=0.995 8);检测变异系数低(4.86%);与其他5种常见的水生动物疫病病毒[鲤浮肿病毒(Carp edema virus, CEV)、锦鲤疱疹病毒(Koi herpesvirus, KHV)、草鱼出血病毒(Grass carp reovirus, GCRV)、鲫造血器官坏死病毒(Cyprinid herpesvirus 2, CyHV-2)、细胞肿大虹彩病毒(Red sea bream iridovirus, RSIV)]阳性样品未发生交叉反应;在临床样品的检测中,48份罗非鱼样品结果均为阴性,5份能力验证样品中3份为阳性,与能力验证满意结果一致。

To establish an assay of reverse transcription droplet digital PCR(RT-ddPCR) for Tilapia Lake Virus(TiLV), we designed a pair of specific primers and probe based on the conserved region of TiLV segment 3 and evaluated the specificity, sensitivity and repeatability of this method. The structured standard curve was evaluated by using TiLV-cDNA as a template. Finally,the samples were tested. When the concentrations of primers and probes were 500 and 300 nmol·L^(-1)and the annealing temperature was 54.2 ℃, the established TiLV RT-ddPCR amplification reaction efficiency was the highest, the distribution boundary of the positive and negative droplets was the most obvious, and the average copy number was higher. The RT-ddPCR of TiLV had a lower limit of detection with 2 copies·μL^(-1)and showed a good linear relationship between 1–90 000 copies·μL^(-1)(Correlation coefficient R^(2)=0.995 8). There was no amplification reaction to other viruses in aquatic animals. The CV of ddPCR for TiLVcDNA was 4.86%. There was no cross reaction with the positive samples of other five common aquatic animal disease viruses [Carp edema virus(CEV), Koi herpesvirus(KHV), Grass carp reovirus(GCV), Cyprinid herpesvirus 2(CyHV-2), Red sea bream iridovirus(RSIV)]. Among the 53 detected samples, 48 were negative, three of five proficiency testing samples were positive, consistent with satisfactory previous proficiency testing results.