RAP: A Novel Approach to the Rapid and Highly Sensitive Detection of Respiratory Viruses
Recombinase aided amplification (RAA) is an emerging isothermal amplification method used for detecting various pathogens. However, RAA requires a complex and long probe to ensure high sensitivity during fluorescence assay. TaqMan probe used for quantitative PCR (qPCR) is simple and universal. Herei...
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2021-11-01
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| Series: | Frontiers in Bioengineering and Biotechnology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fbioe.2021.766411/full |
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| author | Guohao Fan Ruiqing Zhang Xiaozhou He Fengyu Tian Mingzhu Nie Xinxin Shen Xuejun Ma Xuejun Ma |
| author_facet | Guohao Fan Ruiqing Zhang Xiaozhou He Fengyu Tian Mingzhu Nie Xinxin Shen Xuejun Ma Xuejun Ma |
| author_sort | Guohao Fan |
| collection | DOAJ |
| description | Recombinase aided amplification (RAA) is an emerging isothermal amplification method used for detecting various pathogens. However, RAA requires a complex and long probe to ensure high sensitivity during fluorescence assay. TaqMan probe used for quantitative PCR (qPCR) is simple and universal. Herein, we developed a new approach for detecting nucleic acids of pathogens, known as RAP (Recombinase aided PCR). The method combines RAA and qPCR to ensure a rapid and highly sensitive detection using a conventional qPCR device. RAP is a two-stage amplification process performed in a single tube within 1 hour. The method involves an RAA reaction for 10 min at 39°C (first stage) followed by 15 cycles of qPCR (second stage). Using human adenovirus 3 (HADV3) and human adenovirus 7 (HADV7) plasmids, the sensitivities of RAP assays for detecting HADV3 and HADV7 were 6 and 17 copies per reaction, respectively. The limit of RAP detection was at least 16-fold lower than the corresponding qPCR, and no-cross reaction with other respiratory viruses was observed. The results of RAP analysis revealed 100% consistency with qPCR assay. This study shows that RAP assay is a rapid, specific, and highly sensitive detection method with a potential for clinical and laboratory application. |
| first_indexed | 2024-12-21T01:47:36Z |
| format | Article |
| id | doaj.art-cdc56d24b79d42efa2188dfa5acb76fe |
| institution | Directory Open Access Journal |
| issn | 2296-4185 |
| language | English |
| last_indexed | 2024-12-21T01:47:36Z |
| publishDate | 2021-11-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Bioengineering and Biotechnology |
| spelling | doaj.art-cdc56d24b79d42efa2188dfa5acb76fe2022-12-21T19:19:59ZengFrontiers Media S.A.Frontiers in Bioengineering and Biotechnology2296-41852021-11-01910.3389/fbioe.2021.766411766411RAP: A Novel Approach to the Rapid and Highly Sensitive Detection of Respiratory VirusesGuohao Fan0Ruiqing Zhang1Xiaozhou He2Fengyu Tian3Mingzhu Nie4Xinxin Shen5Xuejun Ma6Xuejun Ma7National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, ChinaNational Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, ChinaNational Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, ChinaNational Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, ChinaNational Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, ChinaNational Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, ChinaNational Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, ChinaCenter for Biosafety Mega-Science, Chinese Academy of Sciences, Wuhan, ChinaRecombinase aided amplification (RAA) is an emerging isothermal amplification method used for detecting various pathogens. However, RAA requires a complex and long probe to ensure high sensitivity during fluorescence assay. TaqMan probe used for quantitative PCR (qPCR) is simple and universal. Herein, we developed a new approach for detecting nucleic acids of pathogens, known as RAP (Recombinase aided PCR). The method combines RAA and qPCR to ensure a rapid and highly sensitive detection using a conventional qPCR device. RAP is a two-stage amplification process performed in a single tube within 1 hour. The method involves an RAA reaction for 10 min at 39°C (first stage) followed by 15 cycles of qPCR (second stage). Using human adenovirus 3 (HADV3) and human adenovirus 7 (HADV7) plasmids, the sensitivities of RAP assays for detecting HADV3 and HADV7 were 6 and 17 copies per reaction, respectively. The limit of RAP detection was at least 16-fold lower than the corresponding qPCR, and no-cross reaction with other respiratory viruses was observed. The results of RAP analysis revealed 100% consistency with qPCR assay. This study shows that RAP assay is a rapid, specific, and highly sensitive detection method with a potential for clinical and laboratory application.https://www.frontiersin.org/articles/10.3389/fbioe.2021.766411/fullrespiratory virusesqPCRRAAhighly sensitiveclinical detection |
| spellingShingle | Guohao Fan Ruiqing Zhang Xiaozhou He Fengyu Tian Mingzhu Nie Xinxin Shen Xuejun Ma Xuejun Ma RAP: A Novel Approach to the Rapid and Highly Sensitive Detection of Respiratory Viruses Frontiers in Bioengineering and Biotechnology respiratory viruses qPCR RAA highly sensitive clinical detection |
| title | RAP: A Novel Approach to the Rapid and Highly Sensitive Detection of Respiratory Viruses |
| title_full | RAP: A Novel Approach to the Rapid and Highly Sensitive Detection of Respiratory Viruses |
| title_fullStr | RAP: A Novel Approach to the Rapid and Highly Sensitive Detection of Respiratory Viruses |
| title_full_unstemmed | RAP: A Novel Approach to the Rapid and Highly Sensitive Detection of Respiratory Viruses |
| title_short | RAP: A Novel Approach to the Rapid and Highly Sensitive Detection of Respiratory Viruses |
| title_sort | rap a novel approach to the rapid and highly sensitive detection of respiratory viruses |
| topic | respiratory viruses qPCR RAA highly sensitive clinical detection |
| url | https://www.frontiersin.org/articles/10.3389/fbioe.2021.766411/full |
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