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山东大学学报 (医学版) ›› 2020, Vol. 58 ›› Issue (10): 127-133.doi: 10.6040/j.issn.1671-7554.0.2020.0923

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2019新型冠状病毒核酸检测试剂优化、验证及分析

周云英1,张通2,钊倩倩1,王海岩2,汪运山3   

  1. 1. 山东大学附属济南市中心医院医学实验诊断中心, 山东 济南 250013;2. 山东艾克韦生物技术有限公司, 山东 济南 250110;3. 山东第一医科大学附属中心医院临床基础研究中心, 山东 济南 250013
  • 发布日期:2020-10-08
  • 通讯作者: 汪运山. E-mail:sdjnwys@163.com
  • 基金资助:
    国家自然科学基金(81802761);山东省2019重点研发计划(2019GSF107014);山东第一医科大学“学术提升计划”(2019QL024);新型冠状病毒肺炎防控应急科技公关计划(202001005-3)

Optimization, validation and analysis of a 2019-nCoV nucleic acid detection kit

ZHOU Yunying1, ZHANG Tong2, ZHAO Qianqian1, WANG Haiyan2, WANG Yunshan3   

  1. 1. Medical Research &
    Laboratory Diagnostic Center, Jinan Central Hospital, Cheeloo College of Medicine, Shandong University, Jinan 250013, Shandong, China;
    2. Shandong ACV Biotech Limited Company, Jinan 250110, Shandong, China;
    3. Research Center of Basic Medicine, Jinan Central Hospital Affiliated to Shandong First Medical University, Jinan 250013, Shandong, China
  • Published:2020-10-08

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摘要： 目的 优化敏感性及特异性更高的新型冠状病毒(2019-nCoV)核酸检测试剂盒,提高阳性检出率。并与市售试剂盒进行对比,对临床使用提出指导。 方法 收集2020年1月至3月在山东大学附属济南市中心医院等6家2019-nCoV核酸检测单位的确诊患者88例及阴性患者572例,共计660例。通过对2019-nCoV的全基因组进行序列分析,设计更加灵敏的引物、探针,优化buffer配比及扩增过程。采用交叉实验及内源性物质干扰实验评价其特异性。另外选择3家市售核酸检测试剂盒,对梯度稀释的阳性核酸样本进行对比验证实验。 结果 优化后的开放读码框1a/b(ORF1ab)、核衣壳蛋白(N)对于2019-nCoV的RNA靶点,比市售试剂盒具有更高的灵敏度。当核酸样本中1个病毒浓度为1个拷贝时即可检出,同时扩增曲线更加优越。相比较与世界卫生组织(WHO)或自行设计引物的试剂盒,ORF1ab的灵敏度提高了2倍(1∶10 vs 1∶5);而N基因的灵敏度比中国疾病预防控制中心(CDC)和自行设计引物的试剂盒提高了8倍(1∶80 vs 1∶10),比WHO试剂盒提高了2倍(1∶80 vs 1∶40)。临床验证实验显示,鼻咽拭子检测准确度均为100%,与其他感染部位相同或感染症状相似的其他病原体无交叉反应,试剂盒的分析特异性为100%,阳性符合率和阴性符合率均为100%。 结论 优化后的2019-nCoV核酸检测试剂盒具有更高的检测灵敏度和特异性,有助于解决核酸检测假阴性,提高阳性率,将对低病毒载量患者及出院患者的判定具有重要的意义。

关键词: 新型冠状病毒, 低病毒载量, 假阴性, 灵敏度, 特异性

Abstract: Objective To optimize the sensitivity and specificity of a 2019-nCoV nucleic acid detection kit, so as to improve the positive detection rate and provide guidance for clinical use by comparison with different kits. Methods From January to March 2020, 88 confirmed and 572 negative specimens of 2019-nCoV were recruited from 6 2019-nCOV nucleic acid testing centers including Jinan Central Hospital Affiliated to Shandong University. By sequence analysis of the whole genome of 2019-nCoV, a more sensitive primer and probe were designed to optimize the buffer ratio and amplification process. The sensitivity was evaluated with cross-reactive experiment and endogenous substances interference test. The optimized kit and 3 other commercial kits were compared with gradient dilution tests. Results The optimized ORF1ab and N gene had higher sensitivity to the RNA targets of 2019-nCoV. When the concentration of virus was 1, the virus could be detected, and the amplification curve was better than the commercial kits. The sensitivity of ORF1ab was increased by 2 times(1∶10 vs 1∶5)compared with WHO or other self-designed kits, and the sensitivity of N gene was increased by 8 times(1∶80 vs 1∶10)and 2 times(1∶80 vs 1∶40)compared with CDC and WHO kits respectively. The results of cross-reactive test and endogenous substances interference test showed that the accuracy of nasopharyngeal swabs was 100%, there was no cross-reaction with other pathogens with similar infection symptoms, and the accuracy and specificity of the kit were 100%. Conclusion The optimized 2019-nCoV nucleic acid detection kit has higher sensitivity and specificity, which is helpful to solve the false negative nucleic acid detection and increase the positive rate, and is significant to formulate discharge criteria of low viral load patients.

Key words: Novel coronavirus, Low viral load, False negative, Sensitivity, Specificity

中图分类号: 

  • R574
[1] Wang C, Horby PW, Hayden FG, et al. A novel coronavirus outbreak of global health concern[J]. Lancet, 2020, 395(10223):470-473.
[2] Munster VJ, Koopmans M, van Doremalen N, et al. A novel coronavirus emerging in China-Key questions for impact assessment[J]. N Engl J Med, 2020, 382(8):692-694.
[3] Zhu N, Zhang D, Wang W, et al. A novel coronavirus from patients with pneumonia in China, 2019[J]. N Engl J Med, 2020, 382(8):727-733.
[4] Wang D, Hu B, Hu C, et al. Clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in Wuhan, China[J]. JAMA, 2020, 323(11):1061-1069.
[5] 李士雪, 单莹. 新型冠状病毒肺炎研究进展述评[J].山东大学学报(医学版), 2020, 58(3):19-25. LI Shixue, SHAN Ying. Latest research advances on novel coronavirus pneumonia[J]. Journal of Shandong University(Health Sciences), 2020, 58(3):19-25.
[6] Yan R, Zhang Y, Li Y, et al. Structural basis for the recognition of SARS-CoV-2 by full-length human ACE2[J]. Science, 2020, 367(6485):1444-1448.
[7] Zhang L, Lin D, Sun X, et al. Crystal structure of SARS-CoV-2 main protease provides a basis for design of improved alpha-ketoamide inhibitors[J]. Science, 2020, 368(6489):409-412.
[8] National Genomics Data Center Members and Partners. Database Resources of the National Genomics Data Center in 2020[J]. Nucleic Acids Res, 2020, 48(1): 24-33.
[9] Wu F, Zhao S, Yu B, et al. A new coronavirus associated with human respiratory disease in China[J]. Nature, 2020, 579(7798):265-269.
[10] Angeletti S, Benvenuto D, Bianchi M, et al. COVID-2019: The role of the nsp2 and nsp3 in its pathogenesis[J]. J Med Virol, 2020, 92(6):584-588.
[11] 国家药品监督管理局.国家药监局应急审批新型冠状病毒检测产品[EB/OL].(2020-03-16)[2020-06-05].http://www.nmpa.gov.cn/WS04/CL2578/375802.html.
[12] 郭雅琼,张笠,鲁彦.新型冠状病毒引起的临床相关疾病及其分子生物学检测[J/OL].检验医学与临床,2020.http://kns.cnki.net/kcms/detail/50.1167.R.20200316.1810.002.html.
[13] 郭元元, 王昆, 张宇, 等. 6 种国产新型冠状病毒核酸检测试剂检测性能比较与分析[J]. 重庆医学, 2020, 49(15):2435-2439. GUO Yuanyuan, WANG Kun, ZHANG Yu, et al. Comparison and analysis of the detection performance of six SARS-CoV-2 nucleic acid detection reagents[J]. Chongqing Medicine, 2020, 49(15):2435-2439.
[14] Zou L, Ruan F, Huang M, et al. SARS-CoV-2 Viral Load in Upper Respiratory Specimens of Infected Patients[J]. N Engl J Med, 2020, 382(12):1177-1179.
[15] Su YC, Anderson DE, Young BE, et al. Discovery of a 382-nt deletion during the early evolution of SARS-CoV-2[J]. bioRxiv, 2020. doi:10.1101/2020.03.11.987222.
[16] Corman VM, Landt O, Kaiser M, et al. Detection of 2019 novel coronavirus(2019-nCoV)by real-time RT-PCR[J]. Euro Surveill, 2020, 25(3): 2000045.
[17] 国家卫生健康委员会办公厅.新型冠状病毒肺炎实验室检测技术指南[EB/OL].(2020-03-09)[2020-06-05]. http://www.chinacdc.cn/jkzt/crb/zl/szkb11803/jszl11815/202003/t20200309214241.html.
[18] Barra GB, Santa Rita TH, Mesquita PG, et al. Analytical sensibility and specificity of two RT-qPCR protocols for SARS-CoV-2 detection performed in an automated workflow[J]. medRxiv, 2020. doi:10.1101/2020.03.07.20032326.
[19] Diao B, Wen K, Chen J, et al. Diagnosis of Acute Respiratory Syndrome Coronavirus 2 Infection by Detection of Nucleocapsid Protein[J]. medRxiv, 2020. doi: 10.1101/2020.03.07.20032524.
[20] Broughton JP, Deng X, Yu G, et al. Rapid Detection of 2019 Novel Coronavirus SARS-CoV-2 Using a CRISPR-based DETECTR Lateral Flow Assay[J]. medRxiv, 2020. doi:10.1101/2020.03.06.20032334.
[21] 钟慧钰, 赵珍珍, 宋兴勃, 等. 新型冠状病毒临床检测要点及经验[J].国际检验医学杂志,2020,41(5):523-526. ZHONG Huiyu, ZHAO Zhenzhen, SONG Xingbo, et al. Clinal points and experience in nucleic acid testing of SARS-Cov-2[J]. International Journal of Laboratory Medicine, 2020, 41(5):523-526.
[22] Lan L, Xu D, Ye G, et al. Positive RT-PCR test results in patients recovered from COVID-19[J]. JAMA, 2020, 323(15):1502-1503.
[23] Zhang W, Du RH, Li B, et al. Molecular and serological investigation of 2019-nCoV infected patients: implication of multiple shedding routes[J]. Emerg Microbes Infect, 2020, 9(1):386-389.
[24] 赵怀龙, 吕燕, 赵红. 济南市47例新型冠状病毒肺炎患者取样部位对核酸检测结果的影响[J].山东大学学报(医学版), 2020, 58(4):28-31. ZHAO Huailong, LU Yan, ZHAO Hong. Effect of sampling in mucleic acid test on the test result: a report from 47 confirmed COVID-19 cases in Jinan City[J]. Journal of Shandong University(Health Sciences), 2020, 58(4):28-31.
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