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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2022, Vol. 42 Issue (8): 63-73    DOI: 10.13523/j.cb.2204054
综述     
新型冠状病毒(SARS-CoV-2)SNV分型检测技术
王桃雪1,2,3,刘倩1,2,3,齐浩1,2,3,*()
1.天津大学化工学院 天津 300072
2.系统生物工程教育部重点实验室 天津 300072
3.天津化学化工协同创新中心合成生物学平台 天津 300072
SARS-CoV-2 SNV Genotyping Test Technology
WANG Tao-xue1,2,3,LIU Qian1,2,3,QI Hao1,2,3,*()
1. School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China
2. Key Laboratory of Systems Bioengineering of Ministry of Education, Tianjin University, Tianjin 300072, China
3. Collaborative Innovation Center of Chemical Science and Engineering, Syn Bio Research Platform, Tianjin University, Tianjin 300072, China
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摘要：

新型冠状病毒肺炎(COVID-19)的全球大流行对整个人类社会造成了重大影响,人类面临着财政刺激、金融压力、债务重整等挑战。在特效治疗药物与方法出现之前,大规模的人群筛查隔离成为现在疫情治理的最有效方法。然而,这一次的新冠病毒(SARS-CoV-2)展示出了极高的遗传变异性,截至2022年3月31日统计突变率超过了2.3‰,迄今为止高传染性的新病毒株不断出现,被世界卫生组织正式警告的变异株就达到了7个。因此,在接下来的病毒防控与研究中,不但需要检测SARS-CoV-2,更需要精准、实用的单核苷酸变异(single nucleotide variation, SNV)基因分型技术,特别针对大规模人群筛查中,不仅需要获得SRAS-CoV-2的信息,还需要精准快速区分具有更高传染性与毒性的变异株感染。对病毒的感染和突变机制进行了简要介绍,并着重对现有主要的SARS-CoV-2 SNV分型技术进行了分类综述,希望为新型检测技术的开发提供参考。
关键词： 新型冠状病毒肺炎;  SARS-CoV-2 检测;  单核苷酸变异    
Abstract:

The global pandemic of the COVID-19 has had a major impact on the entire human society, and human beings are facing challenges such as fiscal stimulus, financial stress, and debt restructuring. Before the emergence of specific therapeutic drugs and methods, large-scale population screening and isolation has become the most effective method for epidemic management. However, the new strain of coronavirus this time has shown a very high genetic variability, with a statistical mutation rate of more than 2.3‰ as of March 31st, 2022. So far, new highly infectious virus strains have been emerging, and the number of mutant strains officially warned by the World Health Organization has reached 7. Therefore, in the next virus prevention and control and research, we not only need to detect SARS-CoV-2, but also need to explore accurate and practical single nucleotide variation (SNV) genotyping techniques, especially for large-scale population screening. It is not only necessary to obtain information on the SRAS-CoV-2, but also to accurately and quickly distinguish variant strains with higher infectivity and virulence. This paper briefly introduces the infection and mutation mechanism of the virus, and focuses on the classification and review of the existing main SARS-CoV-2 SNV genotyping techniques, hoping to provide insight into the development of new detection technologies.
Key words: COVID-19    SARS-CoV-2 test    Single nucleotide varication (SNV)
收稿日期: 2022-04-21 出版日期: 2022-09-07
ZTFLH:  Q934.9  
通讯作者: 齐浩     E-mail: haoq@tju.edu.cn
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王桃雪,刘倩,齐浩. 新型冠状病毒(SARS-CoV-2)SNV分型检测技术[J]. 中国生物工程杂志, 2022, 42(8): 63-73.

WANG Tao-xue,LIU Qian,QI Hao. SARS-CoV-2 SNV Genotyping Test Technology. China Biotechnology, 2022, 42(8): 63-73.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2204054    或    https://manu60.magtech.com.cn/biotech/CN/Y2022/V42/I8/63

图1  SARS-CoV-2基因组示意图
VOC (variant of concern) VOI (variant of intention) Former VOI (former variant of intention)
Alpha B.1.1.7 Lambda C.37 Epsilon B.1.429
Beta B.1.351 Mu B.1.621 Zeta P.2
Gamma P.1 Eta B.1.525
Delta B.1.617.2 Theta P.3
Omicron B.1.1.529 Iota B.1.526
Omicron BA.2 Kappa B.1.617.1
表1  现有的新型冠状病毒变体种类
图2  SARS-CoV-2 SNV 物化检测技术示意图
检测平台 检测位点 分型变体 分型时间 分型特异性 文献
TSP-PCR C251T S84L S/L 3 h 100% NGS [37]
RT-qPCR&TaqMan N501Y 无 46 min 99.7% NGS [38]
RT-ddPCR Δ69-70 N501Y Δ145 S982A Alpha 2.5 h 100% NGS [39]
RT-LAMP-BART L452R 无 1 h 95%NGS [40]
RT-PCR&CRISPR-Cas12a K417N∕T L452R∕Q T478K E484K∕Q
N501Y D614G		 Alpha Beta
Gamma Delta		 30 min 100%NGS [41]
MT-PCR P681R K417T K417N E484K Alpha Beta Gamma
Delta Kappa		 4 h 100%NGS [42]
表2  SARS-CoV-2的分型检测平台汇总表
ORF 1ab N E
靶标长度/nt 119 99 113
GC含量/% 48 52 41
荧光探针基团 FAM/BHQ1 FAM/TRMRA FAM/BHQ1
保守程度 较为保守 一般保守 保守
高频突变 Y32Y R203K/G204R A32A
检测特异性 较高 较高 较高
检测灵敏度 较高 一般 较高
表3  RT-qPCR检测靶点对比
检测平台 蛋白质 检测基因 时间 灵敏度 预扩增 文献
DETECTR Cas12a E gene and N gene 30 min 10~100拷贝/μL RT-LAMP [49]
CAS detec Cas12b RNA dependent polymerase 40 min 5拷贝/μL RT-RPA [50]
FELUDA FnCas9 N gene 1 h 110 fmol/L RT-PCR/RPA [51]
AIOD-CRISPR Cas12a N gene 40 min 4.6拷贝/μL RT-RPA [52]
SHERLOCK Cas13 S gene and ORF1ab gene 1 h 10~100拷贝/μL RT-qPCR [53]
CREST Cas13 N gene 1~2 h 10拷贝/μL RT-qPCR [54]
表4  SARS-CoV-2的CRISPR/Cas检测平台汇总表
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