Accessible and Adaptable Multiplexed Real-Time PCR Approaches to Identify SARS-CoV-2 Variants of Concern
ABSTRACT Rapid identification and continuous surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants are critical for guiding the response to the COVID-19 pandemic. Whole-genome sequencing (WGS) is a preferred tool for this aim, but many laboratories suffer from a lack...
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| Format: | Article |
| Language: | English |
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American Society for Microbiology
2022-10-01
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| Series: | Microbiology Spectrum |
| Subjects: | |
| Online Access: | https://journals.asm.org/doi/10.1128/spectrum.03222-22 |
| _version_ | 1811198176914309120 |
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| author | Ting Yan Ye Xu Rongrong Zheng Xiaohong Zeng Zehui Chen Su Lin Zihan Xia Yiqun Liao Yongyou Zhang Qingge Li |
| author_facet | Ting Yan Ye Xu Rongrong Zheng Xiaohong Zeng Zehui Chen Su Lin Zihan Xia Yiqun Liao Yongyou Zhang Qingge Li |
| author_sort | Ting Yan |
| collection | DOAJ |
| description | ABSTRACT Rapid identification and continuous surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants are critical for guiding the response to the COVID-19 pandemic. Whole-genome sequencing (WGS) is a preferred tool for this aim, but many laboratories suffer from a lack of resources to support population-level sequencing. Here, we describe two PCR strategies targeting spike protein mutations to identify the Alpha, Delta, and Omicron variants. Signature mutations were selected using a dedicated bioinformatic program. The selected mutations in Alpha and Delta variants were detected using multicolor melting curve analysis (MMCA). Thirty-two mutations of the Omicron variant were targeted using the MeltArray approach in one reaction, which was able to detect the Omicron subvariants BA.1, BA.2, BA.3, and BA.4/5. The limits of detection varied from five to 50 copies of RNA templates/reactions. No cross-reactivity was observed with nine other respiratory viruses, including other coronaviruses. We validated the MMCA and MeltArray assays using 309 SARS-CoV-2 positive samples collected at different time points. These assays exhibited 98.3% to 100% sensitivity and 100% specificity compared with WGS. Multiplexed real-time PCR strategies represent an alternative tool capable of identifying current SARS-CoV-2 VOCs, adaptable for emerging variants and accessible for laboratories using existing equipment and personnel. IMPORTANCE Rapid detection and mutation surveillance of SARS-CoV-2 VOCs is crucial for COVID-19 control, management, and prevention. We developed two rapid molecular assays based on the real-time PCR platform to identify important variants of concern, including the Omicron variant with a large number of mutations. Signature mutations were selected by an R program. Then, MMCA assays were established for Alpha and Delta variants, and a MeltArray assay targeting 32 mutations was developed for Omicron variant. These multiplexed PCR assays could be performed in a 96-well real-time PCR instrument within 2.5 h, offering a high-throughput choice for dynamic monitoring of SARS-CoV-2 VOCs in a standard microbiology laboratory. |
| first_indexed | 2024-04-12T01:26:25Z |
| format | Article |
| id | doaj.art-7f22049172604fc4a26f5e340651f482 |
| institution | Directory Open Access Journal |
| issn | 2165-0497 |
| language | English |
| last_indexed | 2024-04-12T01:26:25Z |
| publishDate | 2022-10-01 |
| publisher | American Society for Microbiology |
| record_format | Article |
| series | Microbiology Spectrum |
| spelling | doaj.art-7f22049172604fc4a26f5e340651f4822022-12-22T03:53:38ZengAmerican Society for MicrobiologyMicrobiology Spectrum2165-04972022-10-0110510.1128/spectrum.03222-22Accessible and Adaptable Multiplexed Real-Time PCR Approaches to Identify SARS-CoV-2 Variants of ConcernTing Yan0Ye Xu1Rongrong Zheng2Xiaohong Zeng3Zehui Chen4Su Lin5Zihan Xia6Yiqun Liao7Yongyou Zhang8Qingge Li9Engineering Research Centre of Molecular Diagnostics of the Ministry of Education, State Key Laboratory of Cellular Stress Biology, State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, School of Life Sciences, Faculty of Medicine and Life Sciences, Xiamen University, Xiamen, Fujian, ChinaEngineering Research Centre of Molecular Diagnostics of the Ministry of Education, State Key Laboratory of Cellular Stress Biology, State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, School of Life Sciences, Faculty of Medicine and Life Sciences, Xiamen University, Xiamen, Fujian, ChinaXiamen Centre for Disease Control and Prevention, Xiamen, Fujian, ChinaXiamen Centre for Disease Control and Prevention, Xiamen, Fujian, ChinaXiamen Centre for Disease Control and Prevention, Xiamen, Fujian, ChinaEngineering Research Centre of Molecular Diagnostics of the Ministry of Education, State Key Laboratory of Cellular Stress Biology, State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, School of Life Sciences, Faculty of Medicine and Life Sciences, Xiamen University, Xiamen, Fujian, ChinaEngineering Research Centre of Molecular Diagnostics of the Ministry of Education, State Key Laboratory of Cellular Stress Biology, State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, School of Life Sciences, Faculty of Medicine and Life Sciences, Xiamen University, Xiamen, Fujian, ChinaEngineering Research Centre of Molecular Diagnostics of the Ministry of Education, State Key Laboratory of Cellular Stress Biology, State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, School of Life Sciences, Faculty of Medicine and Life Sciences, Xiamen University, Xiamen, Fujian, ChinaEngineering Research Centre of Molecular Diagnostics of the Ministry of Education, State Key Laboratory of Cellular Stress Biology, State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, School of Life Sciences, Faculty of Medicine and Life Sciences, Xiamen University, Xiamen, Fujian, ChinaEngineering Research Centre of Molecular Diagnostics of the Ministry of Education, State Key Laboratory of Cellular Stress Biology, State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, School of Life Sciences, Faculty of Medicine and Life Sciences, Xiamen University, Xiamen, Fujian, ChinaABSTRACT Rapid identification and continuous surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants are critical for guiding the response to the COVID-19 pandemic. Whole-genome sequencing (WGS) is a preferred tool for this aim, but many laboratories suffer from a lack of resources to support population-level sequencing. Here, we describe two PCR strategies targeting spike protein mutations to identify the Alpha, Delta, and Omicron variants. Signature mutations were selected using a dedicated bioinformatic program. The selected mutations in Alpha and Delta variants were detected using multicolor melting curve analysis (MMCA). Thirty-two mutations of the Omicron variant were targeted using the MeltArray approach in one reaction, which was able to detect the Omicron subvariants BA.1, BA.2, BA.3, and BA.4/5. The limits of detection varied from five to 50 copies of RNA templates/reactions. No cross-reactivity was observed with nine other respiratory viruses, including other coronaviruses. We validated the MMCA and MeltArray assays using 309 SARS-CoV-2 positive samples collected at different time points. These assays exhibited 98.3% to 100% sensitivity and 100% specificity compared with WGS. Multiplexed real-time PCR strategies represent an alternative tool capable of identifying current SARS-CoV-2 VOCs, adaptable for emerging variants and accessible for laboratories using existing equipment and personnel. IMPORTANCE Rapid detection and mutation surveillance of SARS-CoV-2 VOCs is crucial for COVID-19 control, management, and prevention. We developed two rapid molecular assays based on the real-time PCR platform to identify important variants of concern, including the Omicron variant with a large number of mutations. Signature mutations were selected by an R program. Then, MMCA assays were established for Alpha and Delta variants, and a MeltArray assay targeting 32 mutations was developed for Omicron variant. These multiplexed PCR assays could be performed in a 96-well real-time PCR instrument within 2.5 h, offering a high-throughput choice for dynamic monitoring of SARS-CoV-2 VOCs in a standard microbiology laboratory.https://journals.asm.org/doi/10.1128/spectrum.03222-22SARS-CoV-2 VOCsrapid genotypingmultiplex PCROmicron variant |
| spellingShingle | Ting Yan Ye Xu Rongrong Zheng Xiaohong Zeng Zehui Chen Su Lin Zihan Xia Yiqun Liao Yongyou Zhang Qingge Li Accessible and Adaptable Multiplexed Real-Time PCR Approaches to Identify SARS-CoV-2 Variants of Concern Microbiology Spectrum SARS-CoV-2 VOCs rapid genotyping multiplex PCR Omicron variant |
| title | Accessible and Adaptable Multiplexed Real-Time PCR Approaches to Identify SARS-CoV-2 Variants of Concern |
| title_full | Accessible and Adaptable Multiplexed Real-Time PCR Approaches to Identify SARS-CoV-2 Variants of Concern |
| title_fullStr | Accessible and Adaptable Multiplexed Real-Time PCR Approaches to Identify SARS-CoV-2 Variants of Concern |
| title_full_unstemmed | Accessible and Adaptable Multiplexed Real-Time PCR Approaches to Identify SARS-CoV-2 Variants of Concern |
| title_short | Accessible and Adaptable Multiplexed Real-Time PCR Approaches to Identify SARS-CoV-2 Variants of Concern |
| title_sort | accessible and adaptable multiplexed real time pcr approaches to identify sars cov 2 variants of concern |
| topic | SARS-CoV-2 VOCs rapid genotyping multiplex PCR Omicron variant |
| url | https://journals.asm.org/doi/10.1128/spectrum.03222-22 |
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