摘要
为建立一种特异性强、灵敏度高且简单方便的诺如病毒GⅡ.6核酸快速检测方法,下载诺如病毒GⅡ.6基因组序列,用Mega7.0对基因组进行比对分析获得高度保守序列;基于保守序列设计诺如病毒GⅡ.6的反转录重组酶和聚合酶等温检测(RT-RAPID)技术扩增引物、荧光探针、crRNA和报告分子;优化RT-RAPID的反应引物、反应温度和反应时间及Cas12a检测用的crRNA,并分析RT-RAPID-Cas12a方法的特异性和灵敏度。基于RT-RAPID荧光法和RT-RAPID-Cas12a荧光法的诺如病毒GⅡ.6亚型核酸快速检测方法能在1 h内完成检测并得出结果,灵敏度分别为10 copies/μL和0.5 copies/μL,且两个方法均与诺如病毒GⅡ.17型、鼻病毒、偏肺病毒和博卡病毒无交叉反应。2个检测方法与RT-qPCR阳性样本一致率均为90%,阴性样本一致率均为100%。论文建立的基于RT-RAPID和Cas12a的诺如病毒GⅡ.6核酸快速检测方法均可高效快速地检测出诺如病毒GⅡ.6,具有灵敏度高、特异性强、操作便捷且快速等特点。
To establish a specific,sensitive,simple and convenient method for rapid detection of norovirus GⅡ.6,norovirus GⅡ6 genome sequence was downloaded and evaluated using Mega7.0 to obtain highly conservative sequence for norovirus GⅡ.6.The primer pairs and fluorescent probe for reverse transcriptase polymerase(RT-RAPID)and crRNA were designed based on conservative sequences.Then,the optimal primer pairs and reaction temperature of RT-RAPID and the crRNA for Cas12a detection were screened.And the specificity and sensitivity of RT-RAPID-based fluorescence and RT-RAPID-Cas12a-based fluorescence methods were analyzed.Results showed that RT-RAPID-based fluorescence and RT-RAPID-Cas12a-based fluorescence methods for detecting norovirus GⅡ.6 can be completed within 1 h,and their sensitivities were 10 copies/μL and 0.5 copies/μL,respectively.And there were no cross reaction with norovirus GⅡ.17,rhinovirus,metapneumovirus and bocavirus.The consistency rate between the two detection methods and RT-qPCR for positive samples was 90%,and the consistency rate for negative samples was 100%.The detection method of norovirus GⅡ.6 nucleic acid based on RT-RAPID and Cas12a established in this study can be applied to efficiently and rapidly detect norovirus GⅡ.6 with the characteristics of high-sensitivity,high-specificity,convenience and fast-operation.
作者
冉红志
樊成
王雪飞
刘静
康婕
钱卫东
王婷
RAN Hong-zhi;FAN Cheng;WANG Xue-fei;LIU Jing;KANG Jie;QIAN Wei-dong;WANG Ting(Shaanxi Animal Disease Prevention and Control Center,Xi′an,Shaanxi,710016,China;Shaanxi Institute of Supervision and Testing on Product Quality,Xi′an,Shaanxi,710021,China;Shaanxi University of Science and Technology,Xi′an,Shaanxi,710021,China)
出处
《动物医学进展》
北大核心
2023年第9期44-50,共7页
Progress In Veterinary Medicine
基金
国家市场监管总局科技计划项目(2021MK107)
西安市科技计划项目(22GXFW0007)
西安市未央区科技计划项目(202208)。
