重组酶聚合酶扩增技术结合侧流层析试纸条快速检测新加坡石斑鱼虹彩病毒

[目的]为建立一种快速灵敏、可视化的适用于临床样品检测新加坡石斑鱼虹彩病毒（Singapore grouper iridovirus，SGIV）的方法。[方法]本研究针对SGIV特异基因ORF014序列设计特异性引物及探针，建立重组酶聚合酶扩增（recombinase polymerase amplification, RPA）技术及结合侧流层析试纸条（lateral flow dipstick, LFD）（RPA-LFD）的SGIV检测技术。[结果]RPA反应使用10 μmol/L的引物浓度，在40.1℃恒温反应20 min即可完成特异性病毒的检测，最低检测限为100个／μl。RPA-LFD反应在42℃恒温反应8 min可将检测结果通过试纸条可视化呈现，最低检测限为10个／μl，且不与其他常见水生动物病原发生交叉反应，临床样品检测结果也与PCR检测结果一致。[结论]RPA、RPA-LFD均能特异性检测SGIV，两者的检测限均比常规PCR灵敏。[意义]RPA-LFD法具有快捷简单、结果可视化的特点，在临床应用具有较好的应用前景。

Rapid detection of Singapore grouper iridovirus by a recombinase polymerase amplification combined with lateral flow dipstick

[Background] Singapore grouper iridovirus (SGIV), a novel species of Ranavirus, caused more than 90% mortality in larval and juvenile of grouper. Up to now, there is still a lack of effective prevention and control strategies for SGIV. Therefore, it is essential to develop convenient diagnostic methods for filed detection of SGIV without special equipment. [Methods] In this study, to establish a rapid, sensitive and visualized method for the detection of SGIV in clinical samples, specific primers and probes was designed by targeting the SGIV ORF014, and a recombinase polymerase amplification (RPA) technique combined with Lateral flow dipstick (LFD) (RPA-LFD) was developed for the detection of SGIV. [Results] The results showed that the RPA reaction specially detect target fragment of SGIV within 20min at 40.1 ℃ with the lowest detection limit of 100 copies/μl. The RPA-LFD reaction at constant temperature of 42 ℃ for 8 min was able to visualized the results on the test strips with the lowest detection limit of 10 copies/μl, and showed no cross-reaction with other common aquatic pathogens. The coincidence rate of positive test of clinical samples was consistent with between RPA-LFD and PCR methods. [Conclusion] Both RPA and RPA-LFD could specifically detect SGIV with lower limit than conventional PCR assay. [Significance] Taken together, RPA-LFD assay developed in present study provided a convenient, specific, sensitive and visualized method for on-site rapid detection of SGIV without special equipment.