新型冠状病毒和甲/乙型流感病毒抗原联合快速检测试剂的研制及性能评价

Development and performance evaluation of a new rapid detection reagent for novel coronavirus and influenza A/B virus antigen

目的建立新型冠状病毒(2019-nCoV)、甲型流感病毒(Flu A)和乙型流感病毒(Flu B)胶体金抗原联合快速检测试剂的制备工艺,并系统评价试剂的性能指标。方法采用柠檬酸三钠两步还原法制备胶体金溶液,用抗2019-nCoV核衣壳蛋白(NP)单抗、抗Flu A NP单抗、抗Flu B NP单抗及多肽作为标记抗体,硝酸纤维素膜上分别包被抗2019-nCoV NP单抗和抗多肽单抗制备新型冠状病毒抗原试纸条,另一硝酸纤维素膜上分别包被抗Flu A NP单抗、抗Flu B NP单抗和抗多肽单抗制备甲/乙型流感病毒抗原试纸条。对试剂盒最低检出限、重复性、交叉反应及临床诊断符合率进行性能评价。结果检测国家参考品,试剂盒最低检出限和重复性均符合要求;分析特异性研究显示,试剂盒与16种呼吸道相关病原体毒株均无交叉反应性。临床研究显示,检测新型冠状病毒总符合率为99.11%(111/112),甲型流感病毒总符合率为98.78%(81/82),乙型流感病毒诊断总符合率为97.56%(80/82)。结论新型冠状病毒和甲/乙型流感病毒抗原联合快速检测试剂检测灵敏度和特异性高,出结果快,操作便携,可用于新型冠状病毒和甲/乙型流感病毒的快速筛查及鉴别诊断。

Objective To establish a immunoassay for the combo rapid detection of antigen against novel coronavirus(2019-nCoV),influenza A(Flu A)and influenza B(Flu B),and to evaluate its clinical performance.Methods The colloidal gold was synthesized by two-step reduction of trisodium citrate.The monoclonal antibodies against 2019-nCoV nucleocapsid protein(NP),Flu A NP,Flu B NP and peptides were labeled with colloidal gold nanoparticles.The monoclonal antibodies of anti-2019-nCoV NP and anti-peptides were immobilized on the nitrocellulose membrane to prepare 2019-nCoV test strip.The other nitrocellulose membrane was immobilized with the monoclonal antibodies of anti-Flu A NP,anti-Flu B NP and anti-peptides to prepare influenza A/B test strip.The performance metrics of the lowest detect limit,repeatability,cross-reactivity,total coincidence rate of clinical diagnosis were evaluated.Results The lowest detect limit and repeatability of the kit for testing national reference materials met the requirements.The analysis specificity study showed that the kit had no cross reactivity with 16 strains of respiratory related pathogens.Clinical research showed that the diagnostic total coincidence rate of 2019-nCoV,influenza A and influenza B were 99.11%(111/112),98.78%(81/82)and 97.56%(80/82),respectively.Conclusion The combined rapid detection reagent of 2019-nCoV and influenza A/B has high sensitivity and specificity,portable operation,fast speed,which can be used for rapid screening and differential diagnosis of 2019-nCoV and influenza A/B virus.