您的位置:山东大学 -> 科技期刊社 -> 《山东大学学报(医学版)》

山东大学学报 (医学版) ›› 2021, Vol. 59 ›› Issue (1): 1-7.doi: 10.6040/j.issn.1671-7554.0.2020.1069

• 基础医学 •    下一篇

新型冠状病毒核酸检测过程中灵敏度损失的定量分析

董宏杰1,张俊梅1,2,王宏伟1,王茂凤1,张坤迪1,张冯瑜1,王帅1,胡玮1,谢时灵2,谷立川1   

  1. 1.山东大学微生物技术国家重点实验室, 山东 青岛 266000;2.山东仕达思生物产业有限公司, 山东 济南 250000
  • 发布日期:2021-01-09
  • 通讯作者: 谷立川. E-mail:lcgu@sdu.edu.cn谢时灵. E-mail:xieshiling@shidasi.net
  • 基金资助:
    国家自然科学基金(31970043)

Quantitative analysis of the loss of sensitivity in the nucleic acid detection of SARS-CoV-2

DONG Hongjie1, ZHANG Junmei1,2, WANG Hongwei1, WANG Maofeng1, ZHANG Kundi1, ZHANG Fengyu1, WANG Shuai1, HU Wei1, XIE Shiling2, GU Lichuan1   

  1. 1. State Key Laboratory of Microbial Technology, Shandong University, Qingdao 266000, Shandong, China;
    2. Shandong Shtars Biological Industry Co., Ltd., Jinan 250000, Shandong, China
  • Published:2021-01-09

摘要: 目的 定量分析新型冠状病毒核酸检测过程中的灵敏度损失,为改进检测流程、解决假阴性问题提供支持。 方法 以N基因假病毒为样本,对核酸提取方法、保存液pH、保存液体积、提取核酸所用的样本保存液体积中可能影响诊断灵敏度的各种因素进行系统的定量分析和优化,并对优化的检测方法与目前常规检测法进行比较。 结果 用柱法提取核酸,使用酸性样本保存液可提高检测灵敏度;随着保存液体积增大,假病毒释放得更充分,当保存液体积达到3 mL时假病毒接近完全洗脱;分别取200 μL和3 mL保存液样本提取核酸,检测灵敏度相差10倍以上;全部保存液样本用离心柱法提取核酸且PCR体系中加入12 μL模板时,检测灵敏度达70 copies/mL;取200 μL样本提取核酸且PCR体系中加入4 μL模板时,灵敏度为700 copies/mL。 结论 将新型冠状病毒标本存放于偏酸性样本保存液中,利用尽量多的样本保存液,用离心柱法提取核酸,并在PCR体系中加入最大量的模板,该操作法可较目前常规检测法将最终检测灵敏度提高10倍以上。

关键词: 新型冠状病毒, 核酸检测, 逆转录-聚合酶链反应, 假阴性

Abstract: Objective To conduct the quantitative analysis of SARS-CoV-2 nucleic acid detection sensitivity loss, and to provide support for improving the detection process and solving the false negative problem. Methods The N gene pseudovirus was taken as the sample, the factors that might affect the diagnostic sensitivity in nucleic acid extraction method, pH of preservation solution, volume of preservation solution used for nucleic acid extraction were analyzed and optimized systematically, and the optimized detection method was compared with the current conventional detection method. Results The detection sensitivity could be improved by extraction of nucleic acid by centrifugal column method and using acid sample preservation solution. With the increase of the volume of the preservation solution, the pseudovirus was released more fully. When the volume of the preservation solution reached 3 mL, the pseudovirus was almost completely eluted. The detection sensitivity exhibited more than ten times the difference when 200 μL and 3 mL preservation solution were used to extract nucleic acid, respectively. When the whole preservation samples was extracted by centrifugal column method, and 12 μL template was added into the PCR system, the detection sensitivity could reach 70 copies/mL; when 200 μL preservation sample was taken to extract the nucleic acid, and 4 μL template was added into the PCR system, the detection sensitivity was 700 copies/mL. Conclusion The novel coronavirus specimens are stored in the slightly acidic sample, and the nucleic acid is extracted by centrifugation column method using as many preservation solution samples as possible, and the largest number of nucleic acid templates are added into the PCR system. The sensitivity of the nucleic acid detection method is 10 times higher than that of the conventional detection method.

Key words: SARS-CoV-2, Nucleic acid detection, Reverse transcription-polymerase chain reaction, False negative

中图分类号: 

  • R574
[1] 国际病毒分类委员会将新型冠状病毒命名为SARS-CoV-2[EB/OL].(2020-02-12)[2020-07-22]. http://www.chinanews.com/gj/2020/02-12/9088314.shtml.
[2] 疫情实时大数据报告[EB/OL].(2020-12-09)[2020-07-22]. https://voice.baidu.com/act/newpneumonia/newpneumonia/?from=osari_aladin_banner#tab4.
[3] 中国疾病预防控制中心. 新型冠状病毒核酸检测引物和探针序列(Specific primers and probes for detection 2019 novel coronavirus)[EB/OL].(2020-01-21)[2020-07-22]. http://ivdc.chinacdc.cn/kyjz/202001/t20200121_211337.html.
[4] Jiang SB, Shi ZL. The first disease X is caused by a highly transmissible acute respiratory syndrome coronavirus[J]. Virol Sin, 2020, 35(3): 263-265.
[5] Li XG, Zai JJ, Zhao Q, et al. Evolutionary history, potential intermediate animal host, and cross-species analyses of SARS-CoV-2[J]. J Med Virol, 2020, 92(6): 602-611.
[6] Li C, Yang YL, Ren LZ. Genetic evolution analysis of 2019 novel coronavirus and coronavirus from other species[J]. Infect Genet Evol, 2020, 82: 104285. doi:10.1016/j.meegid.2020.104285.
[7] Li Q, Guan XH, Wu P, et al. Early transmission dynamics in Wuhan, China, of novel coronavirus-infected pneumonia[J]. N Engl J Med, 2020, 382(13): 1199-1207.
[8] Kahn JS, McIntosh K. History and recent advances in coronavirus discovery[J]. Pediatr Infect Dis J, 2005, 24(11 Suppl): S223-S227, discussion S226.
[9] Guo YR, Cao QD, Hong ZS, et al. The origin, transmission and clinical therapies on coronavirus disease 2019(COVID-19)outbreak—an update on the status[J]. Mil Med Res, 2020, 7(1): 11. doi:10.1186/s40779-020-00240-0.
[10] Xie MX, Chen Q. Insight into 2019 novel coronavirus-an updated interim review and lessons from SARS-CoV and MERS-CoV[J]. Int J Infect Dis, 2020, 94: 119-124. doi:10.1016/j.ijid.2020.03.071.
[11] Chan JF, Kok KH, Zhu Z, et al. Genomic characterization of the 2019 novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting Wuhan[J]. Emerg Microbes Infect, 2020, 9(1): 221-236.
[12] 国家卫生健康委办公厅. 新型冠状病毒肺炎诊疗方案(试行第七版)国卫办医函〔2020〕184号[EB/OL].(2020-03-04)[2020-07-22]. http://www.nhc.gov.cn/yzygj/s7653p/202003/46c9294a7dfe4cef80dc7f5912eb1989.shtml.
[13] Wu JT, Leung K, Leung GM. Nowcasting and forecasting the potential domestic and international spread of the 2019-nCoV outbreak originating in Wuhan, China: a modelling study [J]. Lancet, 2020, 395(10225): 689-697.
[14] 袁易, 王铭杰, 张欣欣. 第三代测序技术的主要特点及其在病毒基因组研究中的应用[J]. 微生物与感染, 2016, 11(6): 380-384. YUAN Yi, WANG Mingjie, ZHANG Xinxin. Characteristics of the third generation sequencing technology and its application in researching of viral genomes[J]. Journal of Microbes and Infections, 2016, 11(6): 380-384.
[15] Binnicker MJ. Emergence of a novel coronavirus disease(COVID-19)and the importance of diagnostic testing: why partnership between clinical laboratories, public health agencies, and industry is essential to control the outbreak[J]. Clin Chem, 2020, 66(5): 664-666.
[16] Chu DKW, Pan Y, Cheng SMS, et al. Molecular diagnosis of a novel coronavirus(2019-nCoV)causing an outbreak of pneumonia[J]. Clin Chem, 2020, 66(4): 549-555.
[17] Corman VM, Landt O, Kaiser M, et al. Detection of 2019 novel coronavirus(2019-nCoV)by real-time RT-PCR[J]. Eur Commun Dis Bull, 2020, 25(3): 2000045. doi:10.2807/1560-7917.ES.2020.25.3.2000045.
[18] 中国医疗. 新冠核酸检测治疗评价报告出炉,对于各地检验都有参考意义[EB/OL].(2020-04-13)[2020-07-22]. http://med.china.com.cn/content/pid/170006/tid/1026.
[19] 李彩玉, 陈梦媛, 张师音, 等. 新型冠状病毒核酸检测假阴性原因分析及控制要点[J]. 厦门大学学报(自然科学版), 2020, 59(3): 310-316. LI Caiyu, CHEN Mengyuan, ZHANG Shiyin, et al. Cause analysis and control points of false negative nucleic acid test for severe acute respiratory syndrome coronavirus 2[J]. Journal of Xiamen University(Natural Science), 2020, 59(3): 310-316.
[20] 里进, 叶光明, 陈良君, 等. 新型冠状病毒核酸检测假阴性结果原因分析及对策[J]. 中华检验医学杂志, 2020, 43(3): 221-225. LI Jin, YE Guangming, CHEN Liangjun, et al. Causes and countermeasures of false-negative results for 2019 novel coronavirus nucleic acid test[J]. Chinese Journal of Laboratory Medicine, 2020, 43(3): 221-225.
[21] 冯毅, 袁连方, 郑冲霄, 等. CT与核酸检测在新型冠状病毒肺炎诊断中的应用[J]. 广东医学, 2020, 41(5): 440-443. FENG Yi, YUAN Lianfang, ZHENG Chongxiao, et al. Practical application of CT and nucleic acid detection in the diagnosis of COVID-19[J]. Guangdong Medical Journal, 2020, 41(5): 440-443.
[22] Bai Y, Yao LS, Wei T, et al. Presumed asymptomatic carrier transmission of COVID-19[J]. JAMA, 2020, 323(14): 1406-1407.
[23] Yao HP, Lu XY, Chen Q, et al. Patient-derived mutations impact pathogenicity of SARS-CoV-2[J]. SSRN Journal, 2020. doi:10.2139/ssrn.3578153.
[24] 钟慧钰, 赵珍珍, 宋兴勃, 等. 新型冠状病毒核酸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. Clinical points and experience in nucleic acid testing of SARS-CoV-2[J]. International Journal of Laboratory Medicine, 2020, 41(5): 523-526.
[25] 许朋, 郑春辉, 孙智勇, 等. 磁珠法快速提取基因组DNA的实验研究[J]. 生物信息学, 2018, 16(3): 190-195. XU Peng, ZHENG Chunhui, SUN Zhiyong, et al. Rapid extraction of genomic DNA by magnetic beads method[J]. Chinese Journal of Bioinformatics, 2018, 16(3): 190-195.
[1] 曹义海. 血管生成在疾病治疗中的应用与展望[J]. 山东大学学报 (医学版), 2021, 59(9): 9-14.
[2] 杨璇,李岩志,马伟,贾崇奇. 基于两样本孟德尔随机化的肺功能与新型冠状病毒肺炎病死风险的因果关系[J]. 山东大学学报 (医学版), 2021, 59(7): 104-111.
[3] 周溪,黄霂晗,任玉洁,邱洋. 新型冠状病毒感染与天然免疫及炎症反应[J]. 山东大学学报 (医学版), 2021, 59(5): 15-21.
[4] 董宏杰,张俊梅,王帅,王宏伟,张坤迪,胡玮,谢晓鸿,谢时灵,谷立川. 新型冠状病毒混合样品检测研究[J]. 山东大学学报 (医学版), 2021, 59(4): 1-5.
[5] 于莹,张功,刘晶,颜世童,韩涛,黄海量. 基于网络药理学和分子对接方法探析黄芪预防新型冠状病毒肺炎的潜在作用机制[J]. 山东大学学报 (医学版), 2021, 59(4): 6-16.
[6] 任敏敏,王广梅,张丽,杨瑶瑶,封丹珺. 335名抗疫一线护理人员心理弹性对共情疲劳的影响[J]. 山东大学学报 (医学版), 2021, 59(2): 88-94.
[7] 曹爱华,段春红,邱丙平,路苓,李可亮, 刘学工,周志远,孙建华,鞠秀丽. 山东省儿童新型冠状病毒感染者流行病学及临床特征[J]. 山东大学学报 (医学版), 2020, 58(6): 34-40.
[8] 李晓非,刘贵明,李婉澜,吕松琴,聂磊,张娟,徐丽萍,陆霓虹. 化学发光法检测新型冠状病毒抗体在临床的应用价值[J]. 山东大学学报 (医学版), 2020, 58(5): 46-50.
[9] 程召平,段艳华,姚金坤,李岩,顾慧,袁宪顺,刘斌,毕万利,宋照亮,聂佩,陈月芹,孙占国,刘善平,王鲁光,唐忠仁,魏相磊,董亮,王春亭,王锡明. 105例新型冠状病毒肺炎胸部CT影像学特征——山东省多中心回顾性分析[J]. 山东大学学报 (医学版), 2020, 58(5): 38-45.
[10] 王蔚茹,李新蕊,王芳,赵小冬,于秋燕,杨国樑,李战,常彩云,赵梦娇,刘晓雪,李传彬,孙湛,耿兴义. 济南市一起新型冠状病毒肺炎家庭聚集性疫情分析[J]. 山东大学学报 (医学版), 2020, 58(4): 54-57.
[11] 杨柳,李战,许华茹,常彩云,刘仲,李传彬,孙湛,景睿,刘铁诚,耿兴义,周敬文. 济南市10例儿童新型冠状病毒肺炎确诊病例流行病学和临床特征[J]. 山东大学学报 (医学版), 2020, 58(4): 36-39.
[12] 赵怀龙,吕燕,赵红,赵宝添,韩莹,杨国樑,王春荣,关恒云,刘辉,刘岚铮. 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.
[13] 杨丽,李战,刘晓雪,焦海涛,周林,刘庆皆,刘铁诚,耿兴义. 济南市新型冠状病毒肺炎密切接触者隔离医学观察情况分析与评价[J]. 山东大学学报 (医学版), 2020, 58(4): 12-16.
[14] 常彩云,于秋燕,赵小冬,王芳,李伟,阮师漫,耿兴义. 济南市首例新型冠状病毒肺炎病例及其相关家庭聚集性疫情分析[J]. 山东大学学报 (医学版), 2020, 58(4): 7-11.
[15] 马艳,张孝国,张忠法. 济南市某定点医疗机构26例新型冠状病毒感染患者流行病学分析[J]. 山东大学学报 (医学版), 2020, 58(4): 62-64.
Viewed
Full text


Abstract

Cited

  Shared   
  Discussed   
[1] 王焕亮,孙宝柱,杜洪玫,周长青,张丽. 不同麻醉监测指标调控异丙酚麻醉的比较[J]. 山东大学学报(医学版), 2006, 44(5): 471 -474 .
[2] 于慧1,2 ,陈少华1 ,赵家军2 ,高聆3
. 乙醇对人肝L02细胞糖原和GSK3β、PAMPK的影响[J]. 山东大学学报(医学版), 2009, 47(04): 75 -78 .
[3] 赵正斌1,薛双林2,张立婷1,李俊峰1,赵荣荣1,周海莲3,陈红1. 原花青素对瘦素诱导肝星状细胞增殖和TIMP-1产生的影响[J]. 山东大学学报(医学版), 2012, 50(7): 46 .
[4] 宋永红,马春红,吕红娟,朱传福,聂向民,王玫,刘艳,张萍 . 中国北方汉族人群HLA基因多态性研究[J]. 山东大学学报(医学版), 2007, 45(6): 546 -553 .
[5] 孟静1,刘晓立2,郭春3,马春红3 . 小鼠Tim-3真核表达载体pTARGET-Tim-3的构建[J]. 山东大学学报(医学版), 2010, 48(11): 37 -40 .
[6] 黄庆,田辉,李林,梁飞,刘贤锡 . 老年人肺癌组织中鸟氨酸脱羧酶基因表达及其临床意义[J]. 山东大学学报(医学版), 2006, 44(6): 556 -559 .
[7] 朱梅佳,韩巨,王新怡,鹿伟,王爱华,关心华,曹霞,曹秉振. 伴有皮层下梗死和白质脑病的常染色体显性遗传性脑动脉病临床病理研究[J]. 山东大学学报(医学版), 2006, 44(8): 834 -839 .
[8] 侯晓阳,卜培莉,张运,冯进波,刘春喜,李传保,郝明秀. 过氧化酶体增殖物激活受体α抑制血管紧张素Ⅱ促心肌纤维化作用的试验研究[J]. 山东大学学报(医学版), 2007, 45(7): 665 -668 .
[9] 孟涛1,张心雨2,李兴华2,于金贵1. 吗啡和芬太尼对心内直视手术患者全身炎性反应和心脏功能影响的比较[J]. 山东大学学报(医学版), 2011, 49(2): 102 -105 .
[10] 曾季平,王丽娜,王立祥,任晓辉,张孟业,夏文,崔行. 氯化锰致PC12细胞损伤的研究[J]. 山东大学学报(医学版), 2006, 44(5): 467 -470 .