Advance in the technologies of molecular assays for SARS-CoV-2

doi:10.3969/j.issn.1673-8640.2021.04.023

Abstract

Abstract:

The ongoing outbreak of severe acute respiratory syndrome coronavirus 2（SARS-CoV-2） infection has posed a challenge for worldwide public health. Molecular assays for SARS-CoV-2,a gold standard for the diagnosis of pathogen infection,play roles in the diagnosis,therapy,prevention and control of corona virus disease 2019（COVID-19）. This review focuses on the advance in the technologies of molecular assays and patterns of sample pooling of SARS-CoV-2,so as to provide a reference for selecting molecular assays and screening strategies for SARS-CoV-2 in clinical laboratories.

Key words: Severe acute respiratory syndrome coronavirus 2, Molecular assay, Next generation sequencing, Reverse transcription-polymerase chain reaction, Isothermal amplification, Sample pooling

CLC Number:

R446.1

HUANG Fei, ZHANG Chunyan, PAN Baishen, WANG Beili, GUO Wei. Advance in the technologies of molecular assays for SARS-CoV-2[J]. Laboratory Medicine, 2021, 36(4): 462-466.

Figures/Tables 2

References 30

[1]	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)[EB/OL].(2020-03-03)[2020-03-11]. http://www.nhc.gov.cn/yzygj/s7653p/202003/46c9294a7dfe4cef80dc7f5912eb1989.shtml.
[2]	陶悦, 傅启华, 莫茜. 病原宏基因组测序在新型冠状病毒检测中的应用与挑战[J]. 中华检验医学杂志, 2020,43(3):217-220.
[3]	XIAO M, LIU X, JI J, et al. Multiple approaches for massively parallel sequencing of SARS-CoV-2 genomes directly from clinical samples[J]. Genome Med, 2020,12(1):57. DOI URL PMID
[4]	DI L, FU Y, SUN Y, et al. RNA sequencing by direct tagmentation of RNA/DNA hybrids[J]. Proc Natl Acad Sci U S A, 2020,117(6):2886-2893. URL PMID
[5]	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. DOI URL PMID
[6]	REN L L, WANG Y M, WU Z Q, et al. Identification of a novel coronavirus causing severe pneumonia in human:a descriptive study[J]. Chin Med J(Engl), 2020,133(9):1015-1024.
[7]	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. DOI URL PMID
[8]	WANG M, FU A, HU B, et al. Nanopore targeted sequencing for accurate and comprehensive detection of SARS-CoV-2 and other respiratory viruses[J]. Small, 2020,16(32):e2002169. DOI URL PMID
[9]	CORMAN V M, 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.
[10]	阚丽娟, 陈大洋, 莫红梅, 等. 新型冠状病毒核酸检测的主要影响因素及控制措施[J]. 临床检验杂志, 2020,38(3):223-225.
[11]	NGUYEN T, BANG D D, WOLFF A. 2019 novel coronavirus disease(COVID-19):paving the road for rapid detection and point-of-care diagnostics[J]. Micromachines(Basel), 2020,11(3):306.
[12]	NOTOMI T, OKAYAMA H, MASUBUCHI H, et al. Loop-mediated isothermal amplification of DNA[J]. Nucleic Acids Res, 2000,28(12):E63. URL PMID
[13]	YU L, WU S, HAO X, et al. Rapid detection of COVID-19 coronavirus using a reverse transcriptional loop-mediated isothermal amplification(RT-LAMP) diagnostic platform[J]. Clin Chem, 2020,66(7):975-977. DOI URL PMID
[14]	YAN C, CUI J, HUANG L, et al. Rapid and visual detection of 2019 novel coronavirus(SARS-CoV-2) by a reverse transcription loop-mediated isothermal amplification assay[J]. Clin Microbiol Infect, 2020,26(6):773-779. DOI URL PMID
[15]	LU R, WU X, WAN Z, et al. A novel reverse transcription loop-mediated isothermal amplification method for rapid detection of SARS-CoV-2[J]. Int J Mol Sci, 2020,21(8):2826.
[16]	HARRINGTON A, COX B, SNOWDON J, et al. Comparison of Abbott ID now and Abbott m2000 methods for the detection of SARS-CoV-2 from nasopharyngeal and nasal swabs from symptomatic patients[J]. J Clin Microbiol, 2020,58(8):e00798-20. DOI URL PMID
[17]	KEIGHTLEY M C, SILLEKENS P, SCHIPPERS W, et al. Real-time NASBA detection of SARS-associated coronavirus and comparison with real-time reverse transcription-PCR[J]. J Med Virol, 2005,77(4):602-608. DOI URL PMID
[18]	高闪电, 常惠芸, 丛国正, 等. NASBA(依赖核酸序列的扩增)技术及其在病毒检测中的应用[J]. 中国生物工程杂志, 2009,29(1):80-85.
[19]	WANG W K, FANG C T, CHEN H L, et al. Detection of severe acute respiratory syndrome coronavirus RNA in plasma during the course of infection[J]. J Clin Microbiol, 2005,43(2):962-965. URL PMID
[20]	WANG B, POTTER S J, LIN Y, et al. Rapid and sensitive detection of severe acute respiratory syndrome coronavirus by rolling circle amplification[J]. J Clin Microbiol, 2005,43(5):2339-2344. DOI URL PMID
[21]	ABUDAYYEH O O, GOOTENBERG J S, ESSLETZBICHLER P, et al. RNA targeting with CRISPR-Cas13[J]. Nature, 2017,550(7675):280-284. DOI URL PMID
[22]	KELLNER M J, KOOB J G, GOOTENBERG J S, et al. SHERLOCK:nucleic acid detection with CRISPR nucleases[J]. Nat Protoc, 2019,14(10):2986-3012. DOI URL PMID
[23]	董召刚, 邹明瑾, 张义. 新型冠状病毒肺炎核酸检测现状及研究进展[J]. 中华检验医学杂志, 2020,43(4):386-390.
[24]	CHEN Q, LI J, DENG Z, et al. Comprehensive detection and identification of seven animal coronaviruses and human respiratory coronavirus 229E with a microarray hybridization assay[J]. Intervirology, 2010,53(2):95-104. DOI URL PMID
[25]	HOGAN C A, SAHOO M K, PINSKY B A. Sample pooling as a strategy to detect community transmission of SARS-CoV-2[J]. JAMA, 2020,323(19):1967-1969. DOI URL PMID
[26]	REGEN F, EREN N, HEUSER I, et al. A simple approach to optimum pool size for pooled SARS-CoV-2 testing[J]. Int J Infect Dis, 2020,100:324-326.
[27]	TORRES I, ALBERT E, NAVARRO D. Pooling of nasopharyngeal swab specimens for SARS-CoV-2 detection by RT-PCR[J]. J Med Virol, 2020,92(11):2306-2307. DOI URL PMID
[28]	YELIN I, AHARONY N, TAMAR E S, et al. Evaluation of COVID-19 RT-qPCR test in multi-sample pools[J]. Clin Infect Dis, 2020,71(16):2073-2078. DOI URL PMID
[29]	国家卫生健康委办公厅. 关于印发《新冠病毒核酸筛查稀释混样检测技术指引》的通知[EB/OL].(2020-07-23)[2020-09-24]. http://www.gov.cn/xinwen/2020-07/23/content_5529372.htm.
[30]	国家卫生健康委办公厅. 关于印发《新冠病毒核酸10合1混采检测技术规范》的通知[EB/OL].(2020-08-19)[2020-09-24]. http://www.gov.cn/xinwen/2020-08/19/content_5535756.htm.

方法	原理及特点	适用场景
宏基因组测序	直接对病原体核酸进行NGS,通过与数据库比对获得病原体信息,能检测出所有潜在病原体,无偏好性	混合感染（鉴别病毒是否发生变异）
靶向基因测序	使用捕获法或扩增子法对特定病原体核酸进行NGS, 能检测出特定病原体,敏感性较高	常规感染

方法	原理及特点	适用场景
宏基因组测序	直接对病原体核酸进行NGS,通过与数据库比对获得病原体信息,能检测出所有潜在病原体,无偏好性	混合感染（鉴别病毒是否发生变异）
靶向基因测序	使用捕获法或扩增子法对特定病原体核酸进行NGS, 能检测出特定病原体,敏感性较高	常规感染

检测技术	检测步骤	样本制备	扩增特点	检测仪器	诊断灵敏度/%	抑制剂	应用
实时荧光RT-PCR	繁琐,耗时长,需专业检测人员	多步,复杂	温度循环	仪器多,且体积大	95	阻碍反应	技术成熟,有商品化试剂盒
RT-LAMP	简便,耗时短,无需专业检测人员	1步,简便	等温连续	便携	>95	耐受,稳定	处于研发中

检测技术	检测步骤	样本制备	扩增特点	检测仪器	诊断灵敏度/%	抑制剂	应用
实时荧光RT-PCR	繁琐,耗时长,需专业检测人员	多步,复杂	温度循环	仪器多,且体积大	95	阻碍反应	技术成熟,有商品化试剂盒
RT-LAMP	简便,耗时短,无需专业检测人员	1步,简便	等温连续	便携	>95	耐受,稳定	处于研发中