鲤疱疹病毒2型与3型双重PCR检测方法的建立与应用

Dual PCR Detection of Type 2 and 3 Cyprinus Herpesvirus

【目的】鲤疱疹病毒2型和3型对鲤科鱼类危害严重,本研究旨在建立快捷、高效且能同时检测2种鲤疱疹病毒的检测技术。【方法】根据鲤疱疹病毒2型和3型的DNA聚合酶基因保守序列,设计2对特异性引物,通过对双重PCR反应条件的优化,建立检测鲤疱疹病毒2型和3型的双重PCR方法;使用该方法对试验室保存的鲫和鲤病鱼组织样品进行检测。【结果】该方法能够分别对鲤疱疹病毒2型和鲤疱疹病毒3型各扩增出715 bp和456bp的特异性条带,表现出良好的特异性;敏感性试验结果显示,该方法的检测极限值为100 copies·μL-1,具有较高的灵敏性。使用该方法对本试验室保存的18份临床病料进行PCR检测并对PCR产物进行测序验证,结果显示CyHV-2和CyHV-3阳性的各有3份,其阳性率都为33.3%;分别对2份CyHV-2和CyHV-3阳性样品混合后进行检测,结果混合样品均为CyHV-2和CyHV-3双阳性,与常规检测方法得到的结果相同。【结论】本研究建立的双重PCR检测方法特异性强、灵敏度高,可用于CyHV-2和CyHV-3的快速检测和鉴别诊断。

【Objective】A PCR method for simultaneously detecting Type 2 and Type 3 Cyprinid herpesvirus that cause serious diseases on Cyprinidae was developed and tested for clinical diagnosis.【Methods】Two pairs of specific primers were designed according to the conserved sequences of the DNA polymerase gene of the two types of virus,CyHV-2 and CyHV-3.PCR reaction conditions of the method were optimized.The assay was applied on the stored tissue samples of diseased Carassius auratus and Cyprinus carpio to verify validity of the methodology.【Result】The newly developed Dual PCR Assay amplified specific bands with the base numbers of 715 bp for CyHV-2 and 456 bp for CyHV-3.It exhibited high sensitivity with a detection limit of 100 copies·μL−1.On the 18 clinical samples,3 were found to be CyHV-2 and 3 CyHV-3 with a positive detection rate of 33.3%.Furthermore,the assay successfully identified the two type viruses in a mixed sample of CyHV-2 and CyHV-3 in a challenge test.【Conclusion】The Dual PCR Assay demonstrated high specificity and sensitivity in simultaneously detecting CyHV-2 and CyHV-3.It could be adequately applied for rapid diagnosis of the viral diseases on carps.