实时荧光重组酶聚合酶扩增技术(RPA)快速检测Ⅱ型鲤疱疹病毒

A real-time recombinase polymerase amplification assay for the rapid detection of CyHV2

摘要:

【背景】Ⅱ型鲤疱疹病毒(Cyprinid Herpesvirus 2，CyHV2)感染鲫引起的疱疹病毒性造血器官坏死病(Herpes Viral Haematopoietic Necrosis，HVHN)是鲫养殖业的主要病害，造成严重的经济损失。目前尚无治疗HVHN的有效药物。对CyHV2进行早期监测和有效防控是阻止该病暴发的有效手段。【目的】建立一种针对CyHV2的orf72基因的实时荧光重组酶聚合酶扩增技术(Recombinase Polymerase Ampli?cation，RPA)检测方法，并评价其特异性和灵敏度。【方法】通过比较CyHV2五株毒株间orf72核苷酸序列，在保守区设计特异性引物和探针。设置5个反应温度，优化实时荧光RPA反应的条件。在最优的条件下验证实时荧光RPA检测方法在不同水产动物病毒间的特异性。以梯度稀释的CyHV2阳性DNA为模板比较实时荧光RPA与qPCR的灵敏度。【结果】实时荧光RPA能在37.8 °C条件下20 min内快速准确地检测CyHV2病毒，而且种间特异性高，与其他病毒无交叉反应，反应灵敏度与qPCR相同。【结论】研究建立的实时荧光RPA可用于CyHV2的现场快速检测，具有一定应用价值。

Abstract:

[Background] Herpes viral haematopoietic necrosis (HVHN) caused by cyprinid herpesvirus 2 (CyHV2) is one of the main diseases in crucian carp breeding, causing serious economic losses. The disease has no effective therapeutic treatment, therefore early detection of the virus and efficient control is an effective way to prevent its outbreak. [Objective] A real-time recombinase polymerase ampli?cation (RPA) assay for CyHV2 orf72 gene was established, and its specificity and sensitivity were evaluated. [Methods] Specific primers and probes were designed in the conserved region by comparing the orf72 nucleotide sequences between the five CyHV2 strains. Five reaction temperatures were set to optimize the conditions of real-time RPA assay. Under the optimal conditions, the specificity of the real-time RPA assay was verified among different species. The sensitivity of real-time RPA and qPCR was compared using gradient diluted CyHV2 positive DNA as template. [Results] Real-time RPA assay can quickly and accurately detect CyHV2 virus within 20 minutes at 37.8 °C, with high specificity, no cross-reaction with other viruses, and the same sensitivity as qPCR. [Conclusion] The real-time RPA assay developed in this study can be used for rapid on-site detection of CyHV2.