A Ligation/Recombinase Polymerase Amplification Assay for Rapid Detection of SARS-CoV−2
The pandemic of COVID-19 caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has led to more than 117 million reported cases and 2.6 million deaths. Accurate diagnosis technologies are vital for controlling this pandemic. Reverse transcription (RT)-based nucleic acid detection ass...
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| Format: | Article |
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Frontiers Media S.A.
2021-05-01
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| Series: | Frontiers in Cellular and Infection Microbiology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fcimb.2021.680728/full |
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| author | Pei Wang Chao Ma Xue Zhang Lizhan Chen Longyu Yi Xin Liu Qunwei Lu Yang Cao Song Gao |
| author_facet | Pei Wang Chao Ma Xue Zhang Lizhan Chen Longyu Yi Xin Liu Qunwei Lu Yang Cao Song Gao |
| author_sort | Pei Wang |
| collection | DOAJ |
| description | The pandemic of COVID-19 caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has led to more than 117 million reported cases and 2.6 million deaths. Accurate diagnosis technologies are vital for controlling this pandemic. Reverse transcription (RT)-based nucleic acid detection assays have been developed, but the strict sample processing requirement of RT has posed obstacles on wider applications. This study established a ligation and recombinase polymerase amplification (L/RPA) combined assay for rapid detection of SARS-CoV−2 on genes N and ORF1ab targeting the specific biomarkers recommended by the China CDC. Ligase-based strategies usually have a low-efficiency problem on RNA templates. This study has addressed this problem by using a high concentration of the T4 DNA ligase and exploiting the high sensitivity of RPA. Through selection of the ligation probes and optimization of the RPA primers, the assay achieved a satisfactory sensitivity of 101 viral RNA copies per reaction, which was comparable to RT-quantitative polymerase chain reaction (RT-qPCR) and other nucleic acid detection assays for SARS-CoV−2. The assay could be finished in less than 30 min with a simple procedure, in which the requirement for sophisticated thermocycling equipment had been avoided. In addition, it avoided the RT procedure and could potentially ease the requirement for sample processing. Once validated with clinical samples, the L/RPA assay would increase the practical testing availability of SARS-CoV-2. Moreover, the principle of L/RPA has an application potential to the identification of concerned mutations of the virus. |
| first_indexed | 2024-12-17T00:28:02Z |
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| institution | Directory Open Access Journal |
| issn | 2235-2988 |
| language | English |
| last_indexed | 2024-12-17T00:28:02Z |
| publishDate | 2021-05-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Cellular and Infection Microbiology |
| spelling | doaj.art-43b67d3754954cb990d045a9cf28781b2022-12-21T22:10:25ZengFrontiers Media S.A.Frontiers in Cellular and Infection Microbiology2235-29882021-05-011110.3389/fcimb.2021.680728680728A Ligation/Recombinase Polymerase Amplification Assay for Rapid Detection of SARS-CoV−2Pei Wang0Chao Ma1Xue Zhang2Lizhan Chen3Longyu Yi4Xin Liu5Qunwei Lu6Yang Cao7Song Gao8Key Laboratory of Molecular Biophysics of Ministry of Education, Department of Biomedical Engineering, College of Life Science and Technology, Center for Human Genome Research, Huazhong University of Science and Technology, Wuhan, ChinaJiangsu Key Laboratory of Marine Pharmaceutical Compound Screening, Jiangsu Key Laboratory of Marine Biological Resources and Environment, Co-Innovation Center of Jiangsu Marine Bio-industry Technology, School of Pharmacy, Jiangsu Ocean University, Lianyungang, ChinaJiangsu Key Laboratory of Marine Pharmaceutical Compound Screening, Jiangsu Key Laboratory of Marine Biological Resources and Environment, Co-Innovation Center of Jiangsu Marine Bio-industry Technology, School of Pharmacy, Jiangsu Ocean University, Lianyungang, ChinaJiangsu Key Laboratory of Marine Pharmaceutical Compound Screening, Jiangsu Key Laboratory of Marine Biological Resources and Environment, Co-Innovation Center of Jiangsu Marine Bio-industry Technology, School of Pharmacy, Jiangsu Ocean University, Lianyungang, ChinaKey Laboratory of Molecular Biophysics of Ministry of Education, Department of Biomedical Engineering, College of Life Science and Technology, Center for Human Genome Research, Huazhong University of Science and Technology, Wuhan, ChinaKey Laboratory of Molecular Biophysics of Ministry of Education, Department of Biomedical Engineering, College of Life Science and Technology, Center for Human Genome Research, Huazhong University of Science and Technology, Wuhan, ChinaKey Laboratory of Molecular Biophysics of Ministry of Education, Department of Biomedical Engineering, College of Life Science and Technology, Center for Human Genome Research, Huazhong University of Science and Technology, Wuhan, ChinaJiangsu Key Laboratory of Marine Pharmaceutical Compound Screening, Jiangsu Key Laboratory of Marine Biological Resources and Environment, Co-Innovation Center of Jiangsu Marine Bio-industry Technology, School of Pharmacy, Jiangsu Ocean University, Lianyungang, ChinaJiangsu Key Laboratory of Marine Pharmaceutical Compound Screening, Jiangsu Key Laboratory of Marine Biological Resources and Environment, Co-Innovation Center of Jiangsu Marine Bio-industry Technology, School of Pharmacy, Jiangsu Ocean University, Lianyungang, ChinaThe pandemic of COVID-19 caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has led to more than 117 million reported cases and 2.6 million deaths. Accurate diagnosis technologies are vital for controlling this pandemic. Reverse transcription (RT)-based nucleic acid detection assays have been developed, but the strict sample processing requirement of RT has posed obstacles on wider applications. This study established a ligation and recombinase polymerase amplification (L/RPA) combined assay for rapid detection of SARS-CoV−2 on genes N and ORF1ab targeting the specific biomarkers recommended by the China CDC. Ligase-based strategies usually have a low-efficiency problem on RNA templates. This study has addressed this problem by using a high concentration of the T4 DNA ligase and exploiting the high sensitivity of RPA. Through selection of the ligation probes and optimization of the RPA primers, the assay achieved a satisfactory sensitivity of 101 viral RNA copies per reaction, which was comparable to RT-quantitative polymerase chain reaction (RT-qPCR) and other nucleic acid detection assays for SARS-CoV−2. The assay could be finished in less than 30 min with a simple procedure, in which the requirement for sophisticated thermocycling equipment had been avoided. In addition, it avoided the RT procedure and could potentially ease the requirement for sample processing. Once validated with clinical samples, the L/RPA assay would increase the practical testing availability of SARS-CoV-2. Moreover, the principle of L/RPA has an application potential to the identification of concerned mutations of the virus.https://www.frontiersin.org/articles/10.3389/fcimb.2021.680728/fullSARS-CoV-2T4 DNA ligaseligationrecombinase polymerase amplificationnucleic acid detection |
| spellingShingle | Pei Wang Chao Ma Xue Zhang Lizhan Chen Longyu Yi Xin Liu Qunwei Lu Yang Cao Song Gao A Ligation/Recombinase Polymerase Amplification Assay for Rapid Detection of SARS-CoV−2 Frontiers in Cellular and Infection Microbiology SARS-CoV-2 T4 DNA ligase ligation recombinase polymerase amplification nucleic acid detection |
| title | A Ligation/Recombinase Polymerase Amplification Assay for Rapid Detection of SARS-CoV−2 |
| title_full | A Ligation/Recombinase Polymerase Amplification Assay for Rapid Detection of SARS-CoV−2 |
| title_fullStr | A Ligation/Recombinase Polymerase Amplification Assay for Rapid Detection of SARS-CoV−2 |
| title_full_unstemmed | A Ligation/Recombinase Polymerase Amplification Assay for Rapid Detection of SARS-CoV−2 |
| title_short | A Ligation/Recombinase Polymerase Amplification Assay for Rapid Detection of SARS-CoV−2 |
| title_sort | ligation recombinase polymerase amplification assay for rapid detection of sars cov 2 |
| topic | SARS-CoV-2 T4 DNA ligase ligation recombinase polymerase amplification nucleic acid detection |
| url | https://www.frontiersin.org/articles/10.3389/fcimb.2021.680728/full |
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