新型冠状病毒核酸荧光型RT-RAA检测方法的建立及其评价  

Establishment and evaluation of fluorescence RT-RAA detection method for SARS-CoV-2 nucleic acid

作　　者：高越 刘伯玉 任翠平 高勇[2] 朱禹 杨莉 温萌 钱振 芦宝静 柳燕 Gao Yue;Liu Boyu;Ren Cuiping(Dept of Microbiology,Anhui Medical University,Hefei 230032)

机构地区：[1]安徽医科大学基础医学院微生物学教研室,合肥230032 [2]阜阳市第二人民医院,阜阳236015

出　　处：《安徽医科大学学报》2021年第6期980-985,共6页Acta Universitatis Medicinalis Anhui

基　　金：国家自然科学基金(编号:81772203);“十三五”国家科技重大专项(编号:2018ZX10711001);安徽省科技厅新型冠状病毒应急攻关项目(编号:202004a07020010);安徽医科大学应急攻关项目(编号:YGJJ202007)。

摘　　要：目的建立新型冠状病毒(SARS-CoV-2)荧光型RT-RAA快速检测方法。方法以体外转录SARS-CoV-2的RNA为模板,利用单链DNA结合蛋白、重组酶和DNA聚合酶,在40.5℃恒温下,快速完成orf1ab基因和S基因序列片段的扩增,并用已知新冠核酸阳性和其他呼吸道病毒感染患者咽拭子标本进行初步评价。结果该研究建立的方法分别检测SARS-CoV-2两种基因的所需时长均在20 min以内,检测2种基因的灵敏度均为2拷贝数/反应管,特异性为100%,2种引物的最低检出限3次重复实验扩增反应起峰时间一致,曲线形态接近。结论该研究建立的方法灵敏性高,特异性强,重复性良好,临床标本检测结果符合率高,且不需要昂贵的仪器,适用于现场快速检测。Objective To establish a rapid detection method of fluorescent RT-RAA for novel coronavirus(SARS-CoV-2). Methods Using SARS-CoV-2 RNA transcribed in vitro as template, orf1 ab gene and S gene were amplified rapidly by single strand DNA binding protein, recombinant enzyme and DNA polymerase at constant temperature of 40.5 ℃. Pharyngeal swabs from patients with known COVID-19 nucleic acid positive and other respiratory virus infections were used for preliminary evaluation. Results The method established in this research took less than 20 minutes to detect the two SARS-CoV-2 genes separately. The sensitivity of the two genes was 2 copies per reaction tube, and the specificity was 100%. The lowest detection limit of two primers was 100%. The peak time of the amplification reaction of the three repeated experiments was the same, and the shape of the curve was similar. Conclusion The method established in this study has high sensitivity, specificity and good reproducibility. The test results of clinical specimens have a high coincidence rate and do not require expensive equipment, so this method is suitable for rapid detection on site.

关 键 词：SARS-CoV-2 RT-RAA 核酸 快速检测 

分 类 号：R373.1[医药卫生—病原生物学]