开发一种用于灵敏检测和定量 largemouth bass ranavirus 的液滴数字 PCR 方法。

Development of a droplet digital PCR method for the sensitive detection and quantification of largemouth bass ranavirus.

机构信息

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

Agriculture Ministry Key Laboratory of Healthy Freshwater Aquaculture, Key Laboratory of Fish Health and Nutrition of Zhejiang Province, Zhejiang Institute of Freshwater Fisheries, Huzhou, China.

出版信息

J Fish Dis. 2023 Feb;46(2):91-98. doi: 10.1111/jfd.13721. Epub 2022 Oct 9.

DOI:10.1111/jfd.13721

PMID:36209477

Abstract

Largemouth bass ranavirus (LMBRaV), also known as largemouth bass virus (LMBV), is a high mortality pathogen in largemouth bass. A rapid, sensitive, specific and convenient diagnosis method is an urgent requirement for the prevention of virus transmission. In the present study, a droplet digital PCR (ddPCR) method based on the major capsid protein (mcp) gene was established to detect and quantify the virus genome copy number. Oligonucleotide primers were designed based on the LMBRaV mcp gene sequence. The specificity and sensitivity of ddPCR assay were analysed. The other aquatic virus including Chinese giant salamander iridovirus (GSIV), Cyprinid herpesvirus II (CyHV-2) and infectious spleen and kidney necrosis virus could not be detected by LMBRaV ddPCR assay. The detection limit of ddPCR assay was 2 ± 0.37 copies/μl DNA sample. And this ddPCR assay had great repeatability and reproducibility. In clinical diagnosis of 50 largemouth bass, 43 positive samples were detected by ddPCR, whereas only 34 positive samples were detected by quantitative PCR (qPCR). This LMBRaV detection assay provided a specific and sensitive method for the rapid diagnosis of LMBRaV infection in largemouth bass as well as quantification of the virus load.

摘要

大口黑鲈虹彩病毒（LMBRaV），又称大口黑鲈病毒（LMBV），是大口黑鲈的高致死性病原体。一种快速、敏感、特异和方便的诊断方法是预防病毒传播的迫切要求。本研究基于主要衣壳蛋白（mcp）基因建立了一种用于检测和定量病毒基因组拷贝数的液滴数字 PCR（ddPCR）方法。根据 LMBRaV mcp 基因序列设计了寡核苷酸引物。分析了 ddPCR 检测的特异性和灵敏度。其他水生病毒，包括中国大鲵虹彩病毒（GSIV）、鲤鱼疱疹病毒 II（CyHV-2）和传染性脾肾坏死病毒，不能通过 LMBRaV ddPCR 检测到。ddPCR 检测的检测限为 2±0.37 拷贝/μl DNA 样本。该 ddPCR 检测具有很好的重复性和再现性。在对 50 尾大口黑鲈的临床诊断中，ddPCR 检测到 43 个阳性样本，而 qPCR 仅检测到 34 个阳性样本。该 LMBRaV 检测方法为大口黑鲈 LMBRaV 感染的快速诊断以及病毒载量的定量提供了一种特异和敏感的方法。