建立新型冠状病毒实时荧光逆转录环介导等温扩增快速检测方法

Establishment of SARS-CoV-2 rapid detection method based on Real-time fluorescence reverse transcription loop mediated isothermal amplification

目的建立实时荧光逆转录环介导等温扩增方法(RT-LAMP),对新型冠状病毒(SARS-CoV-2)进行现场快速检测。方法在充分比对SARS-CoV-2与其他相关病毒基因组序列后,针对其核衣壳蛋白N基因序列设计了RT-LAMP引物组,经优化反应条件后,进行特异性、灵敏度与添加回收试验。结果建立的RT-LAMP方法对SARS-CoV-2的检测特异性良好,优化条件后在102~107体外转录RNA拷贝/反应的范围内,RT-LAMP反应荧光信号达到最大值的扩增时间(peak time)与每个反应内模板RNA初始拷贝数的对数值的线性方程为y=-1.486x+19.63,R2=0.9921,线性关系良好。检测可在30 min内完成,检出限以体外转录RNA为模板达到200拷贝/μl。结论自主研发建立了可以特异性检测SARS-CoV-2的实时荧光逆转录环介导等温扩增快速检测方法,可用于新冠肺炎病例确诊和人群筛查。

Objective To establish Real-time fluorescence reverse transcription loop mediated isothermal amplification(RT-LAMP) assay for the rapid detection of SARS-CoV-2. Methods The RT-LAMP primers for SARS-CoV-2 were designed according to the sequence of nucleocapsid protein N gene,after fully comparing the genome sequences among SARS-CoV-2 and other related viruses. Based on the optimized reaction conditions,the RT-LAMP specificity,sensitivity and adding recovery experiments were carried out. Results The RT-LAMP method expressed excellent specificity for SARS-CoV-2 detection. After optimization of reaction conditions,the linear equation between peak time and log(copies/reaction) was y=-1.486 x+19.63,R2=0.9921 within the range of 102-107 copies/reaction in vitro transcription RNA,exhibiting a fantastic linear relationship. The detection could be completed within 30 min,and the detection limit was 200 copies/μl in vitro transcription RNA as the template. Conclusion This study independently developed a Real-time fluorescence reverse transcription loop mediated isothermal amplification rapid detection method that can specifically detect SARS-CoV-2. We hope that this method will be contributed to the diagnosis and popula-tion screening in SARS-CoV-2 epidemic.