一种用于检测中国稻田鳗病毒的液滴数字 PCR 检测方法。

A droplet digital PCR assay to detect Chinese rice-field eels rhabdovirus.

机构信息

Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Wuhan, China.

College of Bioscience and Bioengineering, Jiangxi Agricultural University, Nanchang, China.

出版信息

J Fish Dis. 2024 Dec;47(12):e14020. doi: 10.1111/jfd.14020. Epub 2024 Sep 16.

DOI:10.1111/jfd.14020

PMID:39282798

Abstract

Chinese rice-field eels rhabdovirus (CrERV) causes haemorrhagic disease in Chinese rice-field eels (Monopterus albus), leading to significant mortality and economic losses. Sensitive detection of CrERV nucleic acids is essential to control the spread of this pathogen and to treat infected individuals. Herein, we developed an efficient and sensitive droplet digital PCR (ddPCR) method to rapidly detect and quantify CrERV. The ddPCR assay optimal conditions were an annealing temperature of 53°C, and primer and probe concentrations of 0.5 and 0.25 μM, respectively. The assay had a diagnostic sensitivity of 0.23 copies/μL, and was highly specific, showing no cross reactivity with other viruses (infectious haematopoietic necrosis virus, grass carp reovirus, spring viremia of carp virus, largemouth bass ranavirus, carp edema virus, Chinese giant salamander iridovirus, and white spot syndrome virus). Real-time quantitative PCR testing of 30 Chinese rice-field eels samples detected CrERV in 7 samples (23.3%), whereas ddPCR detected CrERV in 12 samples (40%), demonstrating its higher sensitivity. Thus, ddPCR represents an advanced method to absolutely quantify CrERV in infected fish with low virus concentrations, providing a valuable tool to manage the spread and impact of CrERV.

摘要

中国稻田鳗病毒（CrERV）可引起中国稻田鳗（Monopterus albus）出血性疾病，导致严重的死亡率和经济损失。对 CrERV 核酸的敏感检测对于控制该病原体的传播和治疗感染个体至关重要。本文开发了一种高效、敏感的微滴式数字 PCR（ddPCR）方法，用于快速检测和定量 CrERV。ddPCR 检测的最佳条件为退火温度 53°C，引物和探针浓度分别为 0.5 和 0.25 μM。该检测方法的诊断灵敏度为 0.23 拷贝/μL，具有高度特异性，与其他病毒（传染性造血坏死病毒、草鱼出血病病毒、鲤春病毒血症病毒、大口黑鲈虹彩病毒、草鱼水肿病毒、中国大鲵虹彩病毒和白斑综合征病毒）无交叉反应。对 30 份中国稻田鳗样本进行实时定量 PCR 检测，在 7 份样本（23.3%）中检测到 CrERV，而 ddPCR 在 12 份样本（40%）中检测到 CrERV，表明其具有更高的灵敏度。因此，ddPCR 是一种先进的方法，可对低病毒浓度感染鱼类中的 CrERV 进行绝对定量，为管理 CrERV 的传播和影响提供了有价值的工具。