李敏, 李永福, 黄育浩, 陈灼均, 莫钻兰, 钟群芳, 李本旺, 张险朋. 罗非鱼湖病毒微滴式逆转录数字PCR检测方法的建立[J]. 南方水产科学, 2023, 19(1): 75-85. DOI: 10.12131/20220184
引用本文: 李敏, 李永福, 黄育浩, 陈灼均, 莫钻兰, 钟群芳, 李本旺, 张险朋. 罗非鱼湖病毒微滴式逆转录数字PCR检测方法的建立[J]. 南方水产科学, 2023, 19(1): 75-85. DOI: 10.12131/20220184
LI Min, LI Yongfu, HUANG Yuhao, CHEN Zhuojun, MO Zuanlan, ZHONG Qunfang, LI Benwang, ZHANG Xianpeng. Establishment of reverse transcription droplet digital PCR assay for detection of Tilapia Lake Virus[J]. South China Fisheries Science, 2023, 19(1): 75-85. DOI: 10.12131/20220184
Citation: LI Min, LI Yongfu, HUANG Yuhao, CHEN Zhuojun, MO Zuanlan, ZHONG Qunfang, LI Benwang, ZHANG Xianpeng. Establishment of reverse transcription droplet digital PCR assay for detection of Tilapia Lake Virus[J]. South China Fisheries Science, 2023, 19(1): 75-85. DOI: 10.12131/20220184

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

  • 摘要: 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RT-PCR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。

     

    Abstract: 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 R2=0.995 8). There was no amplification reaction to other viruses in aquatic animals. The CV of ddPCR for TiLV-cDNA 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.

     

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