新型冠状病毒亚基因组RNA检测方法的建立及性能评估

doi:10.3969/j.issn.1006-5725.2024.12.021

Abstract

Abstract:

Objective To establish a method for detecting the 2019 novel coronavirus subgenomic RNA （sgRNA）， and evaluate the performance of the established method. Methods Primers and probes were designed according to the subgenomic sequence of the 2019 novel coronavirus， and a reverse transcription PCR method for sgRNA detection was established. The established method was optimized， including the concentration and proportion of primers and probes， extension temperature， reaction volume and template amount. The performance of the method was evaluated， including the limit of detection， sensitivity， specificity and repeatability. sgRNA and genomic RNA （gRNA） were detected in clinical samples， and the results were analyzed. Results In this study， an RT-PCR method for the detection of sgRNA of the 2019 novel coronavirus was established. The limit of detection of this method was 100 copies/mL， and the detection results of common pathogens were negative. The CV of sgRNA in high， medium and low concentration samples were less than 5%. sgRNA was positive in 115 suspected 2019 novel coronavirus infected patients （115/330， 34.85%）. When the Ct value of gRNA-N was less than 30， the positive rate of sgRNA was 100.00%. When the Ct value of gRNA-N was in the range of 30-32， the positive rate of sgRNA was 68.75%. When the Ct value of gRNA-N was in the range of 32-35， the positive rate of sgRNA was 44.44%. When the Ct value of gRNA-N was greater than 35， the sgRNA was negative. Conclusion The RT-PCR method for the detection of sgRNA of the 2019 novel coronavirus was established， and the detection method was sensitive， specific and reproducible.

Key words: 2019 novel coronavirus, subgenomic RNA, polymerase chain reaction, reverse transcription

CLC Number:

R181.3

Zhiwei ZHAO,Cha CHEN,Bin HUANG. Establishment and performance evaluation of subgenomic RNA detection methods for the 2019 novel coronavirus[J]. The Journal of Practical Medicine, 2024, 40(12): 1737-1743.

Figures/Tables 9

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References 34

1	MARKOV P V， GHAFARI M， BEER M， et al. The evolution of SARS-CoV-2 ［J］. Nat Rev Microbiol， 2023， 21（6）： 361-379. doi:10.1038/s41579-023-00878-2 doi: 10.1038/s41579-023-00878-2
2	ZHOU P， YANG X L， WANG X G， et al. A pneumonia outbreak associated with a new coronavirus of probable bat origin ［J］. Nature， 2020， 579（7798）： 270-273. doi:10.1038/s41586-020-2012-7 doi: 10.1038/s41586-020-2012-7
3	BRANTL S. Regulatory mechanisms employed by cis-encoded antisense RNAs. ［J］. Curr Opin Microbiol， 2007， 10（2）： 102-109. doi:10.1016/j.mib.2007.03.012 doi: 10.1016/j.mib.2007.03.012
4	CHEN N， ZHOU M， DONG X， et al. Epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in Wuhan， China： a descriptive study ［J］. Lancet， 2020， 395（10223）： 507-513. doi:10.1016/s0140-6736(20)30211-7 doi: 10.1016/s0140-6736(20)30211-7
5	CARABELLI A M， PEACOCK T P， THORNE L G， et al. SARS-CoV-2 variant biology： immune escape， transmission and fitness ［J］. Nat Rev Microbiol， 2023， 21（3）： 162-177.
6	YÜCE M， FILIZTEKIN E， ÖZKAYA K G. COVID-19 diagnosis -A review of current methods ［J］. Biosens Bioelectron， 2021， 172： 112752. doi:10.1016/j.bios.2020.112752 doi: 10.1016/j.bios.2020.112752
7	许玉玲，王海霞，李金月，等. 新型冠状病毒抗原检测胶体金法与核酸检测实时荧光RT-PCR方法比较［J］. 实用医学杂志， 2022， 38（7）： 791-794. doi:10.3969/j.issn.1006-5725.2022.07.003 doi: 10.3969/j.issn.1006-5725.2022.07.003
8	KWAN L Y， JOO K O， RYOUN K H， et al. Clinical and epidemiologic characteristics of inconclusive results in SARS-CoV-2 RT-PCR assays ［J］. BMC Infect Dis， 2021， 21（1）：851. doi:10.1186/s12879-021-06534-5 doi: 10.1186/s12879-021-06534-5
9	WÖLFEL R， CORMAN V M， GUGGEMOS W， et al. Virological assessment of hospitalized patients with COVID-2019 ［J］. Nature， 2020， 581（7809）： 465-469. doi:10.1038/s41586-020-2196-x doi: 10.1038/s41586-020-2196-x
10	WANG D， JIANG A， FENG J， et al. The SARS-CoV-2 subgenome landscape and its novel regulatory features ［J］. Mol Cell， 2021， 81（10）： 2135-2147. e5. doi:10.1016/j.molcel.2021.02.036 doi: 10.1016/j.molcel.2021.02.036
11	BRANT A C， TIAN W， MAJERCIAK V， et al. SARS-CoV-2： from its discovery to genome structure， transcription， and replication ［J］. Cell Biosci， 2021， 11（1）： 136. doi:10.1186/s13578-021-00643-z doi: 10.1186/s13578-021-00643-z
12	CHANG J J Y， RAWLINSON D， PITT M E， et al. Transcriptional and epi-transcriptional dynamics of SARS-CoV-2 during cellular infection ［J］. Cell Rep， 2021，35（6）：10910. doi:10.1016/j.celrep.2021.109108 doi: 10.1016/j.celrep.2021.109108
13	WANG M， FU A， HU B， et al. Nanopore Targeted Sequencing for the Accurate and Comprehensive Detection of SARS-CoV-2 and Other Respiratory Viruses ［J］. Small， 2021， 17（32）： e2104078. doi:10.1002/smll.202104078 doi: 10.1002/smll.202104078
14	NOMBURG J， MEYERSON M， DECAPRIO J A. Pervasive generation of non-canonical subgenomic RNAs by SARS-CoV-2 ［J］. Genome Med， 2020， 12（1）： 108. doi:10.1186/s13073-020-00802-w doi: 10.1186/s13073-020-00802-w
15	ZIV O， PRICE J， SHALAMOVA L， et al. The Short- and Long-Range RNA-RNA Interactome of SARS-CoV-2 ［J］. Mol Cell， 2020， 80（6）： 1067-1077. doi:10.1016/j.molcel.2020.11.004 doi: 10.1016/j.molcel.2020.11.004
16	FORD L， LEE C， PRAY I W， et al. Epidemiologic Characteristics Associated With Severe Acute Respiratory Syndrome Coronavirus 2 （SARS-CoV-2） Antigen-Based Test Results， Real-Time Reverse Transcription Polymerase Chain Reaction （rRT-PCR） Cycle Threshold Values， Subgenomic RNA， and Viral Culture Results From University Testing ［J］. Clin Infect Dis， 2021， 73（6）： e1348-e1355. doi:10.1093/cid/ciab303 doi: 10.1093/cid/ciab303
17	KIM J Y， BAE J Y， BAE S， et al. Diagnostic usefulness of subgenomic RNA detection of viable SARS-CoV-2 in patients with COVID-19 ［J］. Clin Microbiol Infect， 2022， 28（1）： 101-106. doi:10.1016/j.cmi.2021.08.009 doi: 10.1016/j.cmi.2021.08.009
18	THORNE L G， BOUHADDOU M， A-K REUSCHL， et al. Evolution of enhanced innate immune evasion by SARS-CoV-2 ［J］. Nature， 2022， 602（7897）： 487-495. doi:10.1038/s41586-021-04352-y doi: 10.1038/s41586-021-04352-y
19	CARROLL T， FOX D， VAN DOREMALEN N， et al. The B.1.427/1.429 （epsilon） SARS-CoV-2 variants are more virulent than ancestral B.1 （614G） in Syrian hamsters ［J］. PLoS Pathog， 2022， 18（2）：e1009914. doi:10.1371/journal.ppat.1009914 doi: 10.1371/journal.ppat.1009914
20	TELWATTE S， MARTIN H A， MARCZAK R， et al. Novel RT-ddPCR assays for measuring the levels of subgenomic and genomic SARS-CoV-2 transcripts ［J］. Methods， 2022， 201： 15-25. doi:10.1016/j.ymeth.2021.04.011 doi: 10.1016/j.ymeth.2021.04.011
21	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. doi:10.1001/jama.2020.1585 doi: 10.1001/jama.2020.1585
22	SHRESTHA L B， FOSTER C， RAWLINSON W， et al. Evolution of the SARS-CoV-2 omicron variants BA.1 to BA.5： Implications for immune escape and transmission ［J］. Rev Med Virol， 2022， 32（5）： e2381. doi:10.1002/rmv.2381 doi: 10.1002/rmv.2381
23	UGOLINI C， MULRONEY L， LEGER A， et al. Nanopore ReCappable sequencing maps SARS-CoV-2 5' capping sites and provides new insights into the structure of sgRNAs ［J］. Nucleic Acids Res， 2022， 50（6）： 3475-3489. doi:10.1093/nar/gkac144 doi: 10.1093/nar/gkac144
24	KIM D， LEE J Y， YANG J S， et al. The Architecture of SARS-CoV-2 Transcriptome ［J］. Cell， 2020， 181（4）：914-921.e10. doi:10.1016/j.cell.2020.04.011 doi: 10.1016/j.cell.2020.04.011
25	TELWATTE S， KUMAR N， VALLEJO-GRACIA A， et al. Novel RT-ddPCR assays for simultaneous quantification of multiple noncoding and coding regions of SARS-CoV-2 RNA ［J］. J Virol Methods， 2021， 292： 114115. doi:10.1016/j.jviromet.2021.114115 doi: 10.1016/j.jviromet.2021.114115
26	乔乔，吴涛，朱小娟，等. 新型冠状病毒亚基因组RNA荧光重组酶介导等温扩增检测方法的建立及评价［J］. 中国病毒病杂志， 2022， 12（6）： 444-447.
27	UGOLINI C， MULRONEY L， LEGER A， et al. Nanopore ReCappable sequencing maps SARS-CoV-2 5' capping sites and provides new insights into the structure of sgRNAs ［J］. Nucleic Acids Res， 2022， 50（6）： 3475-3489. doi:10.1093/nar/gkac144 doi: 10.1093/nar/gkac144
28	DAGOTTO G， MERCADO N B， MARTINEZ D R， et al. Comparison of Subgenomic and Total RNA in SARS-CoV-2 Challenged Rhesus Macaques ［J］. J Virol， 2021， 95（8）：e02370-20. doi:10.1128/jvi.02370-20 doi: 10.1128/jvi.02370-20
29	ZHANG C， CUI H， GUO Z， et al. SARS-CoV-2 Virus Culture， Genomic and Subgenomic RNA Load， and Rapid Antigen Test in Experimentally Infected Syrian Hamsters ［J］. J Virol， 2022， 96（18）： e0103422. doi:10.1128/jvi.01034-22 doi: 10.1128/jvi.01034-22
30	BRAVO M S， BERENGUA C， MARÍN P， et al. Viral Culture Confirmed SARS-CoV-2 Subgenomic RNA Value as a Good Surrogate Marker of Infectivity ［J］. J Clin Microbiol， 2022， 60（1）：e0160921. doi:10.1128/jcm.01609-21 doi: 10.1128/jcm.01609-21
31	THORNE L G， BOUHADDOU M， REUSCHL A K， et al. Evolution of enhanced innate immune evasion by SARS-CoV-2 ［J］. Nature， 2022， 602（7897）： 487-495. doi:10.1038/s41586-021-04352-y doi: 10.1038/s41586-021-04352-y
32	AVANZATO V A， MATSON M J， SEIFERT S N， et al. Case Study： Prolonged Infectious SARS-CoV-2 Shedding from an Asymptomatic Immunocompromised Individual with Cancer ［J］. Cell， 2020， 183（7）： 1901-1912.e9. doi:10.1016/j.cell.2020.10.049 doi: 10.1016/j.cell.2020.10.049
33	栾涛，杨罡，王帅颖，等. “长新冠”综合征研究最新进展［J］. 实用医学杂志， 2023，39（10）：1195-1200.
34	李娜，白彝华，蒋红樱，等. 长期维持性透析患者的衰弱现况及其影响因素［J］. 实用医学杂志， 2024，40（3）：330-335.

引物名称	引物序列（5′?3′）	纯化方式	5'修饰	3'修饰
sgN_F	CCAACCAACTTTCGATCTCT	PAG
sgN_R	GGGTCCACCAAACGTAAT	PAG
sgN_P	TGTCTGATAATGGACCCCAAAATCA	HPLC	FAM	MGB

试剂	反应终浓度	加入量（μL）
2X One-Step RT-PCR Buffer Ⅲ	1 x	10/25
TaKaRa Ex Taq HS	5 U/μL	0.4/1.0
PrimeScript RT Enzyme Mix Ⅱ		0.4/1.0
sgN_F（50 μmol/L）	X μmol/L	X
N_R（50 μmol/L）	X μmol/L	X
N_P（50 μmol/L）	X μmol/L	X
ROX		0.4/1.0
Template		X
RNase free water		X
总体积（μL）		20/50

	温度（℃）	时间（s）	循环数
逆转录	42	300	1
预变性	95	10	1
变性	95	5	45
延伸（采集荧光）	X	31	45

浓度（copies/mL）	阳性样本数	sgRNA-N
浓度（copies/mL）	阳性样本数	Ct（x ± s）
500	20/20	32.37 ± 0.95
250	20/20	33.07 ± 0.91
100	20/20	34.99 ± 0.96
50	16/20	36.47 ± 0.98

Establishment and performance evaluation of subgenomic RNA detection methods for the 2019 novel coronavirus

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Ct（gRNA-N）	sgRNA
Ct（gRNA-N）	样本数	阳性	阴性	阳性率（%）
总计	132	115	17	87.12
< 30	86	86	0	100.00
30 ~ 32	16	11	5	68.75
32 ~ 35	18	8	10	44.44
≥ 35	2	0	2	0.00