蛙病毒PCR检测方法的建立与比较分析

为了进一步丰富蛙虹彩病毒检测方法,实验针对蛙病毒3型(frog virus 3,FV3)核衣壳蛋白(major capsid protein,MCP)基因保守区设计一对特异性引物,并选取另外2种蛙病毒PCR检测方法进行对比实验,通过反应体系的优化、反应特异性和敏感性实验,建立了一种针对FV3的PCR检测方法。结果显示,该方法最低检测限可达1.2个拷贝数的病毒粒子,与神经坏死病毒、虾血细胞虹彩病毒、大口黑鲈虹彩病毒、锦鲤疱疹病毒、鲤浮肿病毒、传染性脾肾坏死病毒、加州鲈弹状病毒等常见水产动物致病毒株无交叉反应。临床样品检测表明,该方法所获得的检测结果与另外2种方法一致,结果可靠。本研究所建立的FV3 PCR检测方法,具有简便、快速、敏感度好、特异性高、低成本等特点,可用于FV3蛙病毒的快速诊断和分子流行病学调研。

Establishment and comparative analysis of PCR detection methods for frog virus

Ranavirus has a wide range of hosts, can infect reptiles, fish and amphibians, and lead to a high mortality. In order to further enrich its detection methods, in this study, a pair of specific primers was designed for the frog virus type 3 (FV3) based on its major capsid protein gene, and two other frog virus PCR detection methods were selected for comparison experiments. After the optimization of PCR reaction system, specificity and sensitivity test, a new PCR method for the detection of FV3 strain was established. This method has a minimum detection limit of 1.2 copies, has no cross-reaction with nervous necrosis virus, shrimp hemocyte iridovirus, largemouth bass Ranavirus, koi herpesvirus, carp edema virus, infectious spleen and kidney necrosis virus and Micropterus salmoides rhabdovirus. The present new method was used to detect the clinical samples, obtaining consistent results with the other two methods. The FV3 PCR detection method established in this research has the characteristics of simplicity, rapidity, good sensitivity, high specificity, and low cost. It can be used for rapid diagnosis and molecular epidemiological investigation of FV3 frog virus.