A Novel Strategy for the Detection of SARS-CoV-2 Variants Based on Multiplex PCR-Mass Spectrometry Minisequencing Technology
ABSTRACT The objective of this study was to construct a novel strategy for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants using multiplex PCR-mass spectrometry minisequencing technique (mPCR-MS minisequencing). Using the nucleic acid sequence of a SARS-CoV-2 n...
| Main Authors: | , , , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
American Society for Microbiology
2021-12-01
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| Series: | Microbiology Spectrum |
| Subjects: | |
| Online Access: | https://journals.asm.org/doi/10.1128/Spectrum.01267-21 |
| _version_ | 1798015804554346496 |
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| author | Fei Zhao Jinxing Lu Bing Lu Tian Qin Xuemei Wang Xuexin Hou Fanliang Meng Xiaona Xu Tianyi Li Haijian Zhou Jianzhong Zhang Biao Kan Ying Huang Zehui Zhang Di Xiao |
| author_facet | Fei Zhao Jinxing Lu Bing Lu Tian Qin Xuemei Wang Xuexin Hou Fanliang Meng Xiaona Xu Tianyi Li Haijian Zhou Jianzhong Zhang Biao Kan Ying Huang Zehui Zhang Di Xiao |
| author_sort | Fei Zhao |
| collection | DOAJ |
| description | ABSTRACT The objective of this study was to construct a novel strategy for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants using multiplex PCR-mass spectrometry minisequencing technique (mPCR-MS minisequencing). Using the nucleic acid sequence of a SARS-CoV-2 nonvariant and a synthetic SARS-CoV-2 variant-carrying plasmid, a matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) method based on the single-base mass probe extension of multiplex PCR amplification products was established to detect 9 mutation types in 7 mutated sites (HV6970del, N501Y, K417N, P681H, D614G, E484K, L452R, E484Q, and P681R) in the receptor-binding domain of the spike protein of SARS-CoV-2 variants. Twenty-one respiratory tract pathogens (9 bacteria and 12 respiratory viruses) and nucleic acid samples from non-COVID-19 patients were selected for specific validation. Twenty samples from COVID-19 patients were used to verify the accuracy of this method. The 9 mutation types could be detected simultaneously by triple PCR amplification coupled with MALDI-TOF MS. SARS-CoV-2 and six variants, B.1.1.7 (Alpha), B.1.351 (Beta), B.1.429 (Epsilon), B.1.526 (Iota), P.1 (Gamma) and B.1.617.2 (Delta), could be identified. The detection limit for all 9 sites was 1.5 × 103 copies. The specificity of this method was 100%, and the accuracy of real-time PCR cycle threshold (CT) values less than 27 among positive samples was 100%. This method is open and extensible, and can be used in a high-throughput manner, easily allowing the addition of new mutation sites as needed to identify and track new SARS-CoV-2 variants as they emerge. mPCR-MS minisequencing provides a new detection option with practical application value for SARS-CoV-2 and its variant infection. IMPORTANCE The emergence of SARS-CoV-2 variants is the key factor in the second wave of the COVID-19 pandemic. An all-in-one SARS-CoV-2 variant identification method based on a multiplex PCR-mass spectrometry minisequencing system was developed in this study. Six SARS-CoV-2 variants (Alpha, Beta, Epsilon, Iota, Gamma, and Delta) can be identified simultaneously. This method can not only achieve the multisite simultaneous detection that cannot be realized by PCR coupled with first-generation sequencing technology and quantitative PCR (qPCR) technology but also avoid the shortcomings of time-consuming, high-cost, and high technical requirements of whole-genome sequencing technology. As a simple screening assay for monitoring the emergence and spread of SARS-CoV-2 and variants, mPCR-MS minisequencing is expected to play an important role in the detection and monitoring of SARS-CoV-2 infection as a supplementary technology. |
| first_indexed | 2024-04-11T15:39:32Z |
| format | Article |
| id | doaj.art-aa7ebb7c05344d24bf8a47d876e6a87a |
| institution | Directory Open Access Journal |
| issn | 2165-0497 |
| language | English |
| last_indexed | 2024-04-11T15:39:32Z |
| publishDate | 2021-12-01 |
| publisher | American Society for Microbiology |
| record_format | Article |
| series | Microbiology Spectrum |
| spelling | doaj.art-aa7ebb7c05344d24bf8a47d876e6a87a2022-12-22T04:15:51ZengAmerican Society for MicrobiologyMicrobiology Spectrum2165-04972021-12-019310.1128/Spectrum.01267-21A Novel Strategy for the Detection of SARS-CoV-2 Variants Based on Multiplex PCR-Mass Spectrometry Minisequencing TechnologyFei Zhao0Jinxing Lu1Bing Lu2Tian Qin3Xuemei Wang4Xuexin Hou5Fanliang Meng6Xiaona Xu7Tianyi Li8Haijian Zhou9Jianzhong Zhang10Biao Kan11Ying Huang12Zehui Zhang13Di Xiao14National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, State Key Laboratory of Infectious Disease Prevention and Control, Beijing, ChinaNational Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, State Key Laboratory of Infectious Disease Prevention and Control, Beijing, ChinaInstitute for Infectious Disease and Endemic Disease Control, Beijing Center for Disease Prevention and Control, Beijing Research Center for Preventive Medicine, Beijing Key Laboratory of Diagnostic and Traceability Technologies for Food Poisoning, Beijing, ChinaNational Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, State Key Laboratory of Infectious Disease Prevention and Control, Beijing, ChinaIntelligene Biosystems (Qingdao) Co., Ltd., Qingdao, ChinaNational Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, State Key Laboratory of Infectious Disease Prevention and Control, Beijing, ChinaNational Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, State Key Laboratory of Infectious Disease Prevention and Control, Beijing, ChinaNational Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, State Key Laboratory of Infectious Disease Prevention and Control, Beijing, ChinaNational Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, State Key Laboratory of Infectious Disease Prevention and Control, Beijing, ChinaNational Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, State Key Laboratory of Infectious Disease Prevention and Control, Beijing, ChinaNational Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, State Key Laboratory of Infectious Disease Prevention and Control, Beijing, ChinaNational Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, State Key Laboratory of Infectious Disease Prevention and Control, Beijing, ChinaInstitute for Infectious Disease and Endemic Disease Control, Beijing Center for Disease Prevention and Control, Beijing Research Center for Preventive Medicine, Beijing Key Laboratory of Diagnostic and Traceability Technologies for Food Poisoning, Beijing, ChinaNational Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, State Key Laboratory of Infectious Disease Prevention and Control, Beijing, ChinaNational Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, State Key Laboratory of Infectious Disease Prevention and Control, Beijing, ChinaABSTRACT The objective of this study was to construct a novel strategy for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants using multiplex PCR-mass spectrometry minisequencing technique (mPCR-MS minisequencing). Using the nucleic acid sequence of a SARS-CoV-2 nonvariant and a synthetic SARS-CoV-2 variant-carrying plasmid, a matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) method based on the single-base mass probe extension of multiplex PCR amplification products was established to detect 9 mutation types in 7 mutated sites (HV6970del, N501Y, K417N, P681H, D614G, E484K, L452R, E484Q, and P681R) in the receptor-binding domain of the spike protein of SARS-CoV-2 variants. Twenty-one respiratory tract pathogens (9 bacteria and 12 respiratory viruses) and nucleic acid samples from non-COVID-19 patients were selected for specific validation. Twenty samples from COVID-19 patients were used to verify the accuracy of this method. The 9 mutation types could be detected simultaneously by triple PCR amplification coupled with MALDI-TOF MS. SARS-CoV-2 and six variants, B.1.1.7 (Alpha), B.1.351 (Beta), B.1.429 (Epsilon), B.1.526 (Iota), P.1 (Gamma) and B.1.617.2 (Delta), could be identified. The detection limit for all 9 sites was 1.5 × 103 copies. The specificity of this method was 100%, and the accuracy of real-time PCR cycle threshold (CT) values less than 27 among positive samples was 100%. This method is open and extensible, and can be used in a high-throughput manner, easily allowing the addition of new mutation sites as needed to identify and track new SARS-CoV-2 variants as they emerge. mPCR-MS minisequencing provides a new detection option with practical application value for SARS-CoV-2 and its variant infection. IMPORTANCE The emergence of SARS-CoV-2 variants is the key factor in the second wave of the COVID-19 pandemic. An all-in-one SARS-CoV-2 variant identification method based on a multiplex PCR-mass spectrometry minisequencing system was developed in this study. Six SARS-CoV-2 variants (Alpha, Beta, Epsilon, Iota, Gamma, and Delta) can be identified simultaneously. This method can not only achieve the multisite simultaneous detection that cannot be realized by PCR coupled with first-generation sequencing technology and quantitative PCR (qPCR) technology but also avoid the shortcomings of time-consuming, high-cost, and high technical requirements of whole-genome sequencing technology. As a simple screening assay for monitoring the emergence and spread of SARS-CoV-2 and variants, mPCR-MS minisequencing is expected to play an important role in the detection and monitoring of SARS-CoV-2 infection as a supplementary technology.https://journals.asm.org/doi/10.1128/Spectrum.01267-21SARS-CoV-2 variantsSNPsscreening and identificationmPCR-MS minisequencing |
| spellingShingle | Fei Zhao Jinxing Lu Bing Lu Tian Qin Xuemei Wang Xuexin Hou Fanliang Meng Xiaona Xu Tianyi Li Haijian Zhou Jianzhong Zhang Biao Kan Ying Huang Zehui Zhang Di Xiao A Novel Strategy for the Detection of SARS-CoV-2 Variants Based on Multiplex PCR-Mass Spectrometry Minisequencing Technology Microbiology Spectrum SARS-CoV-2 variants SNPs screening and identification mPCR-MS minisequencing |
| title | A Novel Strategy for the Detection of SARS-CoV-2 Variants Based on Multiplex PCR-Mass Spectrometry Minisequencing Technology |
| title_full | A Novel Strategy for the Detection of SARS-CoV-2 Variants Based on Multiplex PCR-Mass Spectrometry Minisequencing Technology |
| title_fullStr | A Novel Strategy for the Detection of SARS-CoV-2 Variants Based on Multiplex PCR-Mass Spectrometry Minisequencing Technology |
| title_full_unstemmed | A Novel Strategy for the Detection of SARS-CoV-2 Variants Based on Multiplex PCR-Mass Spectrometry Minisequencing Technology |
| title_short | A Novel Strategy for the Detection of SARS-CoV-2 Variants Based on Multiplex PCR-Mass Spectrometry Minisequencing Technology |
| title_sort | novel strategy for the detection of sars cov 2 variants based on multiplex pcr mass spectrometry minisequencing technology |
| topic | SARS-CoV-2 variants SNPs screening and identification mPCR-MS minisequencing |
| url | https://journals.asm.org/doi/10.1128/Spectrum.01267-21 |
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