新型冠状病毒胶体金抗原快速检测试剂的研制及性能评价*

目的:建立新型冠状病毒(severe acute respiratory syndrome coronavirus 2,SARS-CoV-2)胶体金抗原快速检测试剂的制备方法,并对检测试剂的性能指标进行评价。方法:采用柠檬酸三钠还原法制备胶体金溶液,用鼠抗核衣壳蛋白(nucleocapsid protein, NP)单克隆抗体及二硝基苯酚-牛血清白蛋白(DNP-BSA)作为标记抗体,硝酸纤维素膜上分别包被鼠抗核衣壳蛋白单克隆抗体和兔抗DNP多抗作为检测线和质控线制备免疫胶体金试纸条;对试剂最低检出限、交叉反应性、加速稳定性及临床诊断特异性和灵敏度进行性能评价。结果:检测热灭活培养物的最低检出限为2.0×10² TCID₅₀/mL;测试16种常见呼吸道病原体高浓度样本均无交叉反应;试剂盒50℃加速破坏8周稳定。临床及健康人群鼻咽拭子样本测试,诊断灵敏度为96.67%(29/30),特异性为99.23%(129/130),总符合率为98.75%(158/160);一致性检验Kappa值为0.959 0,P<0.05。结论:SARS-CoV-2胶体金抗原快速检测试剂检测灵敏度和特异性高,检测速度快,操作便携,无需设备,肉眼观察,可作为现有核酸检测法的补充手段,用于新型冠状病毒的早期筛查。

Development and Performance Evaluation of a Rapid Antigen Test for SARS-CoV-2

Objective: To establish a colloidal gold technique assay for the rapid detection of antigen against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and to evaluate its clinical performance. Methods: The colloidal gold was prepared by trisodium citrate reduction. The monoclonal antibody of mouse anti-SARS-CoV-2 nucleocapsid protein (NP) and dinitrophenol-bovine serum albumin (DNP-BSA) were labeled with colloidal gold nanoparticles. The monoclonal antibody of mouse anti-nucleocapsid protein and rabbit anti DNP polyclonal antibody were coated on the nitrocellulose membrane as detection line and quality control line to prepare immunofluorescence test strip. The performance of the limit of detection, cross-reactivity, accelerated stability, sensitivity and specificity of clinical diagnosis were evaluated. Results: The limit of detection for heat inactivated SARS-CoV-2 was 2.0×10² TCID₅₀/mL. There were no cross reaction with high concentration samples or cultured virus of 16 common pathogens. The kit was stable after 8 weeks accelerated at 50℃. Nasopharyngeal swab samples of clinical and healthy people were tested, the sensitivity was 96.67% (29/30), the specificity was 99.23% (129/130), the total coincidence rate was 98.75% (158/160), and the Kappa consistency test had a Kappa value of 0.959 0 (P<0.05). Conclusion: The SARS-CoV-2 antigen detection reagent (colloidal gold method) has the advantages of high sensitivity and specificity, fast detection speed, portable operation, no need for equipment and naked eye observation, which can be used as a supplementary method for the existing SARS-CoV-2 nucleic acid detection method.