鲤疱疹病毒2型和3型三重PCR检测方法的建立与应用

Establishment and application of the triplex PCR method for simultaneous detection of Cyprinid herpesvirus 2 and Cyprinid herpesvirus 3

关键词:  鲤疱疹病毒  三重PCR  鲫(金)鱼  鲤鱼

Key words:  Cyprinid herpesvirus  Triplex PCR  Carassius auratus  Cyprinus carpio

摘要:

鲤疱疹病毒2型和3型主要感染鲫(金)鱼(Carassius auratus)、鲤鱼(Cyprinus carpio)和锦鲤(Cyprinus carpio haematopterus)及其杂交种，具有高度的传染性和致病性，对养殖鲤科鱼类危害严重。为了建立快捷、高效且能同时检测这2种类型鲤疱疹病毒的检测技术，本研究根据鲤疱疹病毒的DNA聚合酶基因(2、3型)、解旋酶基因(2型)和胸苷激酶基因(3型)的保守序列，设计了3对特异性引物，通过对三重PCR的反应条件进行优化，建立起鲤疱疹病毒2型和3型三重PCR检测方法，并运用该方法对实验室保存的鲫鱼和鲤鱼组织样品进行检测。结果显示，该三重PCR检测方法仅在鲤疱疹病毒阳性样品中扩增出3条特异性条带，表现出良好的特异性；以克隆的目的基因质粒为模板，进行10倍梯度稀释，该检测方法能检出的极限值为2.85×102 copies/μL，表现出较高的灵敏性；运用该方法检测122份鲫鱼和60份鲤鱼样品，其结果与运用国家标准(GB/T 36194-2018)和行业标准(SC/T 7212.1-2011)方法检测的结果一致。本研究表明，构建的三重PCR检测方法不仅具有较高的准确性和灵敏度，还能同时检测2种类型的鲤疱疹病毒，有效提高检测效率。

Abstract:

Cyprinid herpesvirus 2 (CyHV-2) and Cyprinid herpesvirus 3 (CyHV-3) mainly infect crucian carp (goldfish), common carp, koi, and their hybrids. They are highly infectious and pathogenic viruses and can seriously damage the culture of Cyprinid fishes. In order to establish a rapid, and efficient method for simultaneously detecting the two viruses, three pairs of specific primers were designed based on the conserved sequences of the genes encoding DNA polymerase (CyHV-2, 3), helicase (CyHV-2), and thymidine kinase (CyHV-3). By optimizing the reaction conditions and system for the triplex PCR, a detection method for identifying different types of carp herpesviruses was established and validated using the Carassius auratus and Cyprinus carpio tissue samples preserved in the laboratory. The results showed that the triplex PCR method amplified only three specific bands from the herpes virus positive carp samples, showing good specificity. Using the cloned plasmid as a template with 10-fold dilution, the detection limit of the method was 2.85×102 copies/μL, showing high sensitivity. A total of 122 samples of crucian carp and 60 samples of common carp were analyzed by this method and the detection results were consistent with those of the national standard (GB/T 36194-2018) and industry standard (SC/T 7212.1-2011). In conclusion, the newly established triplex PCR method is not only highly accurate and sensitive, but can also detect two types of carp herpesvirus simultaneously, thereby effectively improve the detection efficiency.