NoV GⅡ.2亚型RT-RPA-CRISPR/Cas12a检测方法的建立

Establishment of RT-RPA-CRISPR/Cas12a Assay for Norovirus G II.2

近年来,一种新的诺如病毒GⅡ.2亚型(norovirus GⅡ.2,NoV GⅡ.2)通过牡蛎等生食海鲜感染人群,引发急性胃肠炎,并在全球广泛传播。为实现NoV感染的现场诊断和快速疫情响应,亟需建立快速便捷的NoV GⅡ.2亚型核酸检测方法。本研究通过设计引物、crRNA,经优化反应条件后,建立了NoV GⅡ.2亚型RTRPA-CRISPR/Cas12a检测方法。结果显示:利用引物NoV-GⅡ.2-F1B和NoV-GⅡ.2-R1对NoV GⅡ.2标准RNA进行RT-RPA扩增,结合crRNA1介导的CRISPR/Cas12a报告体系,能在40 min内完成NoV GⅡ.2核酸检测;该方法检测灵敏度为10 copies/μL,且与轮状病毒、腺病毒、星形病毒、人副肠孤病毒、博卡病毒和札如病毒无交叉反应;应用该方法和qRT-PCR法,分别对30份临床模拟样本进行检测,发现总符合率达96.67%。结果表明,本研究建立的NoV GⅡ.2亚型RT-RPA-CRISPR/Cas12a检测方法无需依赖昂贵设备,具有灵敏度高、特异性强、操作便捷快速等特点,为NoV GⅡ.2感染的现场检测和防控提供了新方法。

In recent years,a novel G II.2 subtype of Norovirus(NoV G II.2)has appeared,which causes human acute gastroenteritis through raw seafood such as oysters,is widely distributed in the world.In order to achieve onsite inspection of NoV infection and rapidly response to an outbreak,it was urgent to establish a fast and convenient method for detection of nucleic acids of NoV G II.2.In the study,a RT-RPA-CRISPR/Cas12a assay for NoV G II.2 was established through designing primers and crRNA and optimizing reaction conditions.The results showed that RT-RPA amplification was conducted for NoV G II.2 standard RNA using primers of NoV-G II.2-F1B and NoV-G II.2-R1,and the detection could be completed within 40 min in combination with crRNA1-mediated CRISPR/Cas12a reporting system;the method,with a detection limit of 10 copies/μL,failed to interact with rotavirus,adenovirus,astrovirus,human parvovirus,bocavirus and zarovirus;30 clinical simulated samples were tested separately by the established method and qRT-PCR,it was found that the overall compliance rate was 96.67%.In conclusion,the established method was characterized by strong sensitivity,specificity and accessibility without any expensive equipment,contributing to the on-site detection,prevention and control of NoV G II.2 infection.