新型冠状病毒亚基因组RNA荧光重组酶介导等温扩增检测方法的建立及评价  

Establishment and evaluation of fluorescent RT-RAA assay for SARS-CoV-2 sgRNAs detection

作　　者：乔乔 吴涛[1] 朱小娟[1] 葛以跃[1] 陈银[1] 崔仑标[1] QIAO Qiao;WU Tao;ZHU Xiao-juan;GE Yi-yue;CHEN Yin;CUI Lun-biao(Jiangsu Provincial Center for Disease Control and Prevention,Institute of Pathogenic Microbiology,NHC Key Laboratory of Enteric Pathogenic Microbiology,Nanjing,Jiangsu 210009,China;不详)

机构地区：[1]江苏省疾病预防控制中心病原微生物研究所、国家卫生健康委员会肠道病原微生物重点实验室,江苏南京210009

出　　处：《中国病毒病杂志》2022年第6期444-447,共4页Chinese Journal of Viral Diseases

基　　金：江苏省自然科学基金(BK20191489,BK20211373);江苏省六大人才高峰项目(WSN-024);江苏省卫健委重点科研项目(ZD2021060)。

摘　　要：目的 建立新型冠状病毒(SARS-CoV-2)亚基因组RNA(sgRNAs)荧光重组酶介导等温扩增(reverse-transcription recombinase-aided amplification,RT-RAA)检测方法。方法 根据SARS-CoV-2的5′-leader和7a、N基因序列设计特异性等温扩增引物和探针,在39℃等温条件下,30 min内对SARS-CoV-2的sgRNAs进行快速检测,并对该方法的灵敏度、特异度及与实时荧光定量PCR(real-time fluorescence quantitative PCR,real time PCR)法的一致性进行评价。结果 该方法检测限可达到20拷贝/μl,且与其他呼吸道病原体均无交叉反应,具有良好的灵敏度和特异度;与荧光定量PCR法相比,2种检测方法一致性较好(κ=0.762,P<0.001)。结论 本文建立的荧光RT-RAA法可用于SARS-CoV-2 sgRNAs的快速检测,对于临床样本感染性的快速诊断有重要意义。Objective To establish a reverse-transcription recombinase-aided amplification assay(RT-RAA) to rapidly detect SARS-CoV-2 sub-genomic RNAs(sgRNAs). Methods The primers and probe for isothermal nucleic acid amplification were designed based on the 5′-leader and 7a and N gene sequence of SARS-CoV-2, and the sgRNAs of SARS-COV-2 were rapidly detected within 30 min at 39 ℃.The sensitivity, specificity and consistency of the assay were evaluated. Results The detection limit of the method was 20 copies/μl and there were no cross-reactions with other respiratory pathogens, showing decent sensitivity and specificity.The results of the assay were concordant with that of real-time PCR, indicating a better consistency of two methods(κ=0.762,P<0.001). Conclusions The fluorescence RT-RAA assay established in the study can be used for the rapid detection of SARS-CoV-2 sgRNAs, which is of great significance for the rapid diagnosis of COVID 19.

关 键 词：新型冠状病毒 亚基因组RNA 荧光重组酶介导等温扩增 

分 类 号：R183.3[医药卫生—流行病学]