HRCA技术检测中华鳖虹彩病毒

Detection of soft-shelled turtle iridovirus with HRCA

摘要:

【目的】中华鳖虹彩病毒(STIV)是甲鱼重要的病毒性病原之一, 建立特异性好、灵敏度高的中华鳖虹彩病毒超分支滚环扩增体系(HRCA), 对STIV进行快速、准确地检测。【方法】根据中华鳖虹彩病毒(STIV)独有的基因序列和锁式探针公共连接序列分别设计特异性的锁式探针及其扩增引物, 对HRCA的系列反应条件进行优化, 验证该方法的特异性和灵敏度, 并用该方法对感染STIV显症和未显症的甲鱼组织进行检测。【结果】10 nmol/L探针经T4 DNA连接酶作用20 min环化, Bst DNA聚合酶大片段扩增20 min即可获得很好的检测效果。特异性试验表明, 在多种待检病毒样中, HRCA能特异地检测出STIV, 同时HRCA具有极高的灵敏度, 能检测出的最低模板量为101拷贝, 而常规PCR的检测下限为103拷贝, 对显症和尚未显症的感染早期甲鱼组织的检测结果均为阳性。【结论】建立的中华鳖虹彩病毒HRCA检测体系具有灵敏、快速、简便等特点, 能从组织样品中准确地检测出STIV, 可以用于该类疾病的早期诊断, 具有较好的推广前景。

Abstract:

[Objective] To establish a detection system of soft-shelled turtle iridovirus (STIV, a causative agent of severe diseases in cultured Trionyx sinensis) with hyper-branched rolling cycle amplification (HRCA) assay, which provides a stable, reliable and novel technique for early diagnosis of the disease and identification of the pathogen. [Methods] The padlock probe consists of a universal linking sequence and the two target complementary regions at 5′ and 3′ ends, which was designed based on the unique fragment sequence of STIV, was synthesized. Detection system of HRCA was established and optimized. The specificity and sensitivity of HRCA was determined and compared with conventional PCR. Sick turtles with or without obvious symptom were detected applying HRCA. [Results] HRCA is capable of amplifying STIV DNA, while can not detect related virus and other aquatic animal virus. The detection sensitivity of HRCA is as low as 101 copy which is 100-fold higher than that of conventional PCR. All the detections of the infected soft-shell turtles with or yet without evident symptoms at early stage of infection showed positive results. [Conclusion] The detection system of HRCA for STIV was successfully established. It is sensitive, specific, rapid and simple. It can be used for early diagnosis with good prospects for generalizing in the turtle culture.