论文部分内容阅读
【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。
【Objective】 STI is one of the most important virulent pathogens in soft-shelled turtle. To establish HRCA with high specificity and high sensitivity, and to detect STIV rapidly and accurately Detection. 【Method】 A series of specific probes and their primers were designed according to the unique sequence of STIV and the common sequence of the probe. The series of reaction conditions of HRCA were optimized and verified Method specificity and sensitivity, and the method used to detect STIV symptoms and symptoms of soft-shelled turtle tissue was detected. 【Results】 The 10 nmol / L probe was circularized by T4 DNA ligase for 20 min. The amplification of Bst DNA polymerase fragment for 20 min gave good results. Specificity tests showed that HRCA could detect STIV specifically in a wide range of virus samples to be tested, HRAC also had the highest sensitivity, the lowest detectable template amount was 101 copies, and the lower limit of detection by conventional PCR was 103 copies , The symptoms and symptoms of early symptoms of turtles were positive for the test. 【Conclusion】 The established HRCA detection system of iridescent virus of soft-shelled turtle is sensitive, rapid and simple. STIV can be accurately detected from tissue samples, which can be used for the early diagnosis of such diseases and has a good promotion prospect.