中国海洋大学学报（自然科学版）2025，Vol.55Issue(7)：19-27,9.DOI:10.16441/j.cnki.hdxb.20240178
基于RPA和CRISPR/Cas12a技术构建的VHSV快速检测方法
Development of Rapid Detection Methods for VHSV Based on RPA and CRISPR/Cas12a
摘要
Abstract
Viral hemorrhagic septicemia virus(VHSV)is an important fish Novirhabdovirus virus that can cause high lethality in cold-water fish and is widely prevalent worldwide,seriously threatening global food security and aquatic biodiversity.To achieve the convenient,rapid,and sensitive diagnosis of VHSV,a rapid detection system was developed through the screening of RPA primers and crRNA basing on RPA and CRISPR/Cas technologies and the optimization of the detection system by combining with the application of lateral flow chromatography and fluorescence colorimetry in the present study.The experimental results showed that RT-RPA reaction at 39℃ for 30 min can obtain the best amplification effect,and the detection of the virus samples can be completed in 50 min by combining CRISPR/Cas12a with lateral flow chromatography.The results can be judged by the naked eyes.The system was highly sensitive and specific;the sensitivity of the assay was 9 copies/μL,and there was no cross-reactivity with infectious hematopoietic necrosis virus(IHNV)and hirame rhabdovirus(HIRRV).The clinical sample test showed that the results obtained with RPA-CRISPR/Cas12a method was in consistent with those obtained with RT-PCR method,demonstrating that it is highly accurate and stable.In this study,we have developed an RPA-CRISPR/Cas12a detection method for VHSV,and provided an important technical support for the convenient and rapid diagnosis for VHSV.关键词
病毒性出血性败血症病毒/重组酶聚合酶扩增/CRISPR/Cas12a/便捷诊断/快速检测/牙鲆Key words
viral hemorrhagic septicemia virus/recombinase polymerase amplification/CRISPR/Cas12a/convenient diagnosis/rapid detection/Paralichthys olivaceus分类
水产学引用本文复制引用
王洪升,唐小千,绳秀珍,邢婧,迟恒,战文斌..基于RPA和CRISPR/Cas12a技术构建的VHSV快速检测方法[J].中国海洋大学学报（自然科学版）,2025,55(7):19-27,9.基金项目
国家重点研究发展计划项目(2023YFD2400704)资助Supported by the National Key Research and Development Program of China(2023YFD2400704) （2023YFD2400704）
