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<sup>*</sup>

doi:10.13523/j.cb.2102025

中国生物工程杂志

2021, Vol. 41

Issue (5): 27-34 DOI: 10.13523/j.cb.2102025

技术与方法

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^*

张赛¹,王刚¹,刘仲明²,李辉军³,汪大明⁴,钱纯亘^5,^**(

)

1 深圳市亚辉龙生物科技股份有限公司深圳 518116
2 中国人民解放军南部战区总医院广州 510010
3 华中科技大学同济医学院附属同济医院武汉 430030
4 中国科学院苏州生物医学工程技术研究所苏州 215163
5 华中科技大学生命科学与技术学院武汉 430074

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

ZHANG Sai¹,WANG Gang¹,LIU Zhong-ming²,LI Hui-jun³,WANG Da-ming⁴,QIAN Chun-gen^5,^**(

)

1 Shenzhen YHLO Biotech Co., Ltd., Shenzhen 518116, China
2 General Hospital of Southern Theater Command, Guangzhou 510010, China
3 Tongji Hospital affiliated to Huazhong University of Science and Technology, Wuhan 430030, China
4 Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences, Suzhou 215163, China
5 College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China

全文: PDF(14688 KB) HTML

摘要：

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。

关键词： 新型冠状病毒; 抗原; 胶体金; 免疫层析; 性能评价

Abstract:

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.

Key words: SARS-CoV-2 Antigen Colloidal gold Immunochromatography Performance evaluation

收稿日期: 2021-02-22 出版日期: 2021-06-01

ZTFLH:

Q816

基金资助: * 深圳市科技创新委员会“新型冠状病毒肺炎疫情应急防治”悬赏项目(2020254008);深圳市龙岗区科技发展资金“新型冠状病毒感染应急防治”科技专项(LGKCXGZX2020012)

通讯作者: 钱纯亘 E-mail: chungen_qian@hust.edu.cn

	服务
	把本文推荐给朋友
	加入引用管理器
	E-mail Alert
	RSS
	作者相关文章
	张赛
	王刚
	刘仲明
	李辉军
	汪大明
	钱纯亘

引用本文:

张赛,王刚,刘仲明,李辉军,汪大明,钱纯亘. 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^*[J]. 中国生物工程杂志, 2021, 41(5): 27-34.

ZHANG Sai,WANG Gang,LIU Zhong-ming,LI Hui-jun,WANG Da-ming,QIAN Chun-gen. Development and Performance Evaluation of a Rapid Antigen Test for SARS-CoV-2. China Biotechnology, 2021, 41(5): 27-34.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2102025 或 https://manu60.magtech.com.cn/biotech/CN/Y2021/V41/I5/27

图1 抗原检测试纸条结构图

表1 胶体金及胶体金偶合物粒径和Zeta电位

图2 胶体金及胶体金偶合物粒径分布图

图3 不同浓度灭活培养物的显色强度

表2 加速稳定性结果

表3 交叉反应结果

表4 临床性能评价

[1]	Chen Y, Liu Q Y, Guo D Y. Emerging coronaviruses: genome structure, replication, and pathogenesis. Journal of Medical Virology, 2020,92(4):418-423. doi: 10.1002/jmv.25681 pmid: 31967327
[2]	Chu D K W, Pan Y, Cheng S M S, et al. Molecular diagnosis of a novel coronavirus (2019-nCoV) causing an outbreak of pneumonia. Clinical Chemistry, 2020,66(4):549-555. doi: 10.1093/clinchem/hvaa029
[3]	Corman V M, Landt O, Kaiser M, et al. Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR. Eurosurveillance, 2020,25(3):1-8.
[4]	Lu R J, Zhao X, Li J, et al. Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding. Lancet (London, England), 2020,395(10224):565-574. doi: 10.1016/S0140-6736(20)30251-8
[5]	Paraskevis D, Kostaki E G, Magiorkinis G, et al. Full-genome evolutionary analysis of the novel Corona virus (2019-nCoV) rejects the hypothesis of emergence as a result of a recent recombination event. Infection, Genetics and Evolution, 2020,79:104212. doi: 10.1016/j.meegid.2020.104212
[6]	An J H, Liao X J, Xiao T Y, et al. Clinical characteristics of recovered COVID-19 patients with re-detectable positive RNA test. Annals of Translational Medicine, 2020,8(17):1084. doi: 10.21037/atm
[7]	Broughton J P, Deng X D, Yu G X, et al. CRISPR-Cas12-based detection of SARS-CoV-2. Nature Biotechnology, 2020,38(7):870-874. doi: 10.1038/s41587-020-0513-4 pmid: 32300245
[8]	Li Z T, Yi Y X, Luo X M, et al. Development and clinical application of a rapid IgM-IgG combined antibody test for SARS-CoV-2 infection diagnosis. Journal of Medical Virology, 2020,92(9):1518-1524. doi: 10.1002/jmv.v92.9
[9]	Porte L, Legarraga P, Vollrath V, et al. Evaluation of a novel antigen-based rapid detection test for the diagnosis of SARS-CoV-2 in respiratory samples. International Journal of Infectious Diseases, 2020,99:328-333. doi: 10.1016/j.ijid.2020.05.098
[10]	Long Q X, Liu B Z, Deng H J, et al. Antibody responses to SARS-CoV-2 in patients with COVID-19. Nature Medicine, 2020,26(6):845-848. doi: 10.1038/s41591-020-0897-1
[11]	Qian C G, Zhou M, Cheng F M, et al. Development and multicenter performance evaluation of fully automated SARS-CoV-2 IgM and IgG immunoassays. Clinical Chemistry and Laboratory Medicine, 2020,58(9):1601-1607. doi: 10.1515/cclm-2020-0548
[12]	Okba N M A, Müller M A, Li W T, et al. Severe acute respiratory syndrome coronavirus 2-specific antibody responses in coronavirus disease patients. Emerging Infectious Diseases, 2020,26(7):1478-1488. doi: 10.3201/eid2607.200841
[13]	Ai T, Yang Z L, Hou H Y, et al. Correlation of chest CT and RT-PCR testing for coronavirus disease 2019 (COVID-19) in China: a report of 1014 cases. Radiology, 2020,296(2):E32-E40. doi: 10.1148/radiol.2020200642
[14]	Zhou P, Yang X L, Wang X G, et al. A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature, 2020,579(7798):270-273. doi: 10.1038/s41586-020-2012-7
[15]	Ji T X, Liu Z W, Wang G Q, et al. Detection of COVID-19: a review of the current literature and future perspectives. Biosensors and Bioelectronics, 2020,166:112455. doi: 10.1016/j.bios.2020.112455
[16]	Shibata S, Ishiguro T, Kobayashi Y, et al. High incidence of false-positive results of IgG antibody against SARS-CoV-2 with rapid immunochromatographic antibody test due to human common cold coronavirus infection. Respiratory Medicine Case Reports, 2020,31:101180. doi: 10.1016/j.rmcr.2020.101180 pmid: 32766111
[17]	Cerutti F, Burdino E, Milia M G, et al. Urgent need of rapid tests for SARS CoV-2 antigen detection: Evaluation of the SD-Biosensor antigen test for SARS-CoV-2. Journal of Clinical Virology, 2020,132:104654. doi: 10.1016/j.jcv.2020.104654
[18]	Nalumansi A, Lutalo T, Kayiwa J, et al. Field evaluation of the performance of a SARS-CoV-2 antigen rapid diagnostic test in Uganda using nasopharyngeal samples. International Journal of Infectious Diseases, 2021,104:282-286. doi: 10.1016/j.ijid.2020.10.073
[19]	Dinnes J, Deeks J J, Adriano A, et al. Rapid, point-of-care antigen and molecular-based tests for diagnosis of SARS-CoV-2 infection. The Cochrane Database of Systematic Reviews, 2020, 8: CD013705.
[20]	Scohy A, Anantharajah A, Bodéus M, et al. Low performance of rapid antigen detection test as frontline testing for COVID-19 diagnosis. Journal of Clinical Virology, 2020,129:104455. doi: 10.1016/j.jcv.2020.104455
[21]	Jans H, Liu X, Austin L, et al. Dynamic light scattering as a powerful tool for gold nanoparticle bioconjugation and biomolecular binding studies. Analytical Chemistry, 2009,81(22):9425-9432. doi: 10.1021/ac901822w
[22]	Hunter R J. Zeta potential in colloid science: principles and applications. New York and London: Academic Press, 1981: 59-121.
[23]	James F. EP25-A evaluation of stability of in vitro diagnostic reagents; Approved Guideline. [2021-03-31]. https://clsi.org/standards/products/method-evaluation/documents/ep25/.
[24]	Lambert-Niclot S, Cuffel A, Le Pape S, et al. Evaluation of a rapid diagnostic assay for detection of SARS-CoV-2 antigen in nasopharyngeal swabs. Journal of Clinical Microbiology, 2020,58(8):e00977-e00920.
[25]	Kashiwagi K, Ishii Y, Aoki K, et al. Immunochromatographic test for the detection of SARS-CoV-2 in saliva. Journal of Infection and Chemotherapy, 2021,27(2):384-386. doi: 10.1016/j.jiac.2020.11.016
[26]	Diao B, Wen K, Chen J, et al. Diagnosis of acute respiratory syndrome coronavirus 2 infection by detection of nucleocapsid protein. [2020-03-13]. https://www.researchgate.net/publication/339846095_Diagnosis_of_Acute_Respiratory_Syndrome_Coronavirus_2_Infection_by_Detection_of_Nucleocapsid_Protein.

[1]	贠涛,巩玥,谷芃,徐冰冰,李瑾,赵洗尘. 中国与“一带一路”参与国家抗击新冠肺炎疫情的国际科技合作现状与展望[J]. 中国生物工程杂志, 2021, 41(7): 110-121.
[2]	康可人,袁强,梁飞敏,伍丽贤. 苄非他明人工抗原合成[J]. 中国生物工程杂志, 2021, 41(7): 58-65.
[3]	史瑞,严景华. 抗新型冠状病毒单克隆中和抗体药物研发进展^*[J]. 中国生物工程杂志, 2021, 41(6): 129-135.
[4]	陈晨,胡劲超,曹姗姗,门冬. 新型冠状病毒抗原快速检测研发现状及展望^*[J]. 中国生物工程杂志, 2021, 41(6): 119-128.
[5]	范月蕾,王跃,王恒哲,李丹丹,毛开云. 新型冠状病毒体外诊断技术研发现状与展望 ^*[J]. 中国生物工程杂志, 2021, 41(2/3): 150-161.
[6]	张赛,向乐,李林海,李辉军,王刚,钱纯亘. 新型冠状病毒(2019-nCoV)IgM /IgG抗体检测试剂的研制及性能评价[J]. 中国生物工程杂志, 2020, 40(8): 1-9.
[7]	王猛,宋慧茹,程雨洁,王毅,杨波,胡征. 以核糖体蛋白L7/L12为分子标志物精准检测肺炎链球菌的研究 ^*[J]. 中国生物工程杂志, 2020, 40(4): 34-41.
[8]	程永庆,刘金毅,林福玉,童梅. 重组人干扰素α1b与新型冠状病毒肺炎防治[J]. 中国生物工程杂志, 2020, 40(1-2): 71-77.
[9]	王国强,于茵茵,曾华辉,王旭东,吴玉彬,尚立芝,李玉林,张怡青,张西西,张振强,王云龙. 基于MS2噬菌体病毒样颗粒的RT-PCR检测新型冠状病毒(SARS-CoV-2)质控品制备^*[J]. 中国生物工程杂志, 2020, 40(12): 31-40.
[10]	徐应永. 基因治疗产品的开发现状与挑战[J]. 中国生物工程杂志, 2020, 40(12): 95-103.
[11]	林福玉,刘金毅,程永庆. 重组人干扰素α1b抗新型冠状病毒的基础和临床研究进展[J]. 中国生物工程杂志, 2020, 40(12): 1-7.
[12]	陈利军,屈晶晶,项春生. 间充质干细胞在2019新型冠状病毒肺炎(COVID-19)中的治疗潜能、临床研究与应用前景^*[J]. 中国生物工程杂志, 2020, 40(11): 43-55.
[13]	刀凤亭,杨璐,王亚哲,常艳,袁晓英,李玲娣,陈文敏,龙玲玉,刘艳荣,秦亚溱. 成人t(8;21)急性髓系白血病患者初诊Ki-67的表达特征及预后意义 ^*[J]. 中国生物工程杂志, 2019, 39(9): 11-18.
[14]	陈曼,王爱先,傅旻婧,吴雪英,甄军毅,宫美维,郭亚,王卉. CAR细胞疗法在T细胞-急性淋巴细胞白血病应用的新进展[J]. 中国生物工程杂志, 2019, 39(9): 103-107.
[15]	区裕升,郑红俊,钟时,李懿. TAEST16001:TCR亲和力增强型特异性T细胞免疫治疗[J]. 中国生物工程杂志, 2019, 39(2): 49-61.

Viewed

Full text

Abstract

Cited

Shared

Discussed