摘要
应用逆转录-重组酶介导等温扩增技术(Reverse transcription recombinase-aided isothermal amplification,RT-RAA)建立一种乙型流感病毒(Influenza B virus,FLU B)的快速检测方法。在NCBI检索乙型流感病毒的基因序列,利用DNAstar软件进行序列比对,根据比对结果选择较为保守的HA基因进行引物和探针的设计并验证,同时评价该方法特异性、灵敏性、重复性并进行临床样本检测。成功建立了乙型流感病毒荧光RT-RAA的快速检测方法,最佳反应温度为42℃,最佳引物浓度为10μmol/L,最佳探针浓度为25μmol/L,20min内即可完成检测,极大的缩减了检测时间,最低可检测出浓度为3.16×100 copies/μL的重组质粒,并且与其他呼吸道病原体等无交叉反应,具有较好的特异性,应用本研究所建立的荧光RT-RAA方法对20份FLU B样本进行检测。检测结果表明,本研究建立的荧光RT-RAA方法对20份阳性样本均可检出,证明所建立方法可用于FLU B的临床检测,本研究成功建立了快速检测FLU B的荧光RT-RAA方法,具有良好的特异性、灵敏性、重复性,为FLU B的检测提供了有力工具。
This study aimed to develop a rapid detection method for Influenza B virus(FLU B)using reverse transcription recombinase-aided isothermal amplification(RT-RAA).The hemagglutinin(HA)gene,identified as a conserved region through sequence alignment of FLU B genomes retrieved from NCBI and analyzed using DNAStar software,was selected for primer and probe design.The established fluorescent RT-RAA method was systematically evaluated for specificity,sensitivity,repeatability,and clinical applicability.Optimal reaction conditions were determined as follows:reaction temperature at 42℃,primer concentration of 10μmol/L,and probe concentration of 25μmol/L.The assay achieved rapid detection within 20 minutes,significantly reducing testing time,and demonstrated a limit of detection(LOD)of 3.16×100 copies/μL for recombinant plasmids.Cross-reactivity tests confirmed high specificity with no interference from other common respiratory pathogens.Clinical validation using 20 FLU B-positive samples showed 100%detection accuracy.In conclusion,the fluorescent RT-RAA method devel oped in this study exhibits excellent specificity,sensitivity,and reproducibility,providing a robust tool for rapid clinical diagnosis of Influenza B virus.
作者
杨森
张瑾
冯茹荔
刘迪
滕新栋
YANG Sen;ZHANG Jin;FENG Ruli;LIU Di;TENG Xindong(College of Veterinary Medicine,Qingdao Agricultural University,Qingdao Shandong 266109,China;Qingdao International Travel Health Center,Qingdao Shandong 266071,China)
基金
2023年度海关总署科研项目(2023HK020)
山东省重点研发计划(重大科技创新工程)(2021CXG011306)。
关键词
等温扩增技术
乙型流感病毒检测
isothermal amplification technology
influenza B virus
detection
