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Reverse Transcription Recombinase-Aided Amplification Assay With Lateral Flow Dipstick Assay for Rapid Detection of 2019 Novel Coronavirus.
Front Cell Infect Microbiol. 2021; 11:613304.FC

Abstract

Background

The emerging Coronavirus Disease-2019 (COVID-19) has challenged the public health globally. With the increasing requirement of detection for SARS-CoV-2 outside of the laboratory setting, a rapid and precise Point of Care Test (POCT) is urgently needed.

Methods

Targeting the nucleocapsid (N) gene of SARS-CoV-2, specific primers, and probes for reverse transcription recombinase-aided amplification coupled with lateral flow dipstick (RT-RAA/LFD) platform were designed. For specificity evaluation, it was tested with human coronaviruses, human influenza A virus, influenza B viruses, respiratory syncytial virus, and hepatitis B virus, respectively. For sensitivity assay, it was estimated by templates of recombinant plasmid and pseudovirus of SARS-CoV-2 RNA. For clinical assessment, 100 clinical samples (13 positive and 87 negatives for SARS-CoV-2) were tested via quantitative reverse transcription PCR (RT-qPCR) and RT-RAA/LFD, respectively.

Results

The limit of detection was 1 copies/μl in RT-RAA/LFD assay, which could be conducted within 30 min at 39°C, without any cross-reaction with other human coronaviruses and clinical respiratory pathogens. Compared with RT-qPCR, the established POCT assay offered 100% specificity and 100% sensitivity in the detection of clinical samples.

Conclusion

This work provides a convenient POCT tool for rapid screening, diagnosis, and monitoring of suspected patients in SARS-CoV-2 endemic areas.

Authors+Show Affiliations

School of Food Engineering and Biotechnology, Hanshan Normal University, Chaozhou, China.Department of Medical Laboratory, Huizhou Central People's Hospital, Huizhou, China.Department of Human Parasitology, School of Basic Medical Sciences, Hubei University of Medicine, Shiyan, China.Department of Medical Laboratory, Huizhou Central People's Hospital, Huizhou, China.Department of Medical Laboratory, Center for Disease Control and Prevention, Chaozhou, China.Department of Medical Laboratory, Chaozhou People's Hospital, Shantou University Medical College, Chaozhou, China.School of Food Engineering and Biotechnology, Hanshan Normal University, Chaozhou, China.Department of Medical Laboratory, Huizhou Central People's Hospital, Huizhou, China. Department of Medical Laboratory, Chaozhou People's Hospital, Shantou University Medical College, Chaozhou, China.Department of Medical Laboratory, Chaozhou People's Hospital, Shantou University Medical College, Chaozhou, China.School of Food Engineering and Biotechnology, Hanshan Normal University, Chaozhou, China.School of Food Engineering and Biotechnology, Hanshan Normal University, Chaozhou, China.Department of Research and Development, Chaozhou Hybribio Limited Corporation, Chaozhou, China.Department of Laboratory Medicine, First Affiliated Hospital of Gannan Medical University, Ganzhou, China.Department of Human Parasitology, School of Basic Medical Sciences, Hubei University of Medicine, Shiyan, China.Department of Human Parasitology, School of Basic Medical Sciences, Hubei University of Medicine, Shiyan, China.Department of Human Parasitology, School of Basic Medical Sciences, Hubei University of Medicine, Shiyan, China.School of Food Engineering and Biotechnology, Hanshan Normal University, Chaozhou, China.

Pub Type(s)

Evaluation Study
Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

33598439

Citation

Zheng, Yu-Zhong, et al. "Reverse Transcription Recombinase-Aided Amplification Assay With Lateral Flow Dipstick Assay for Rapid Detection of 2019 Novel Coronavirus." Frontiers in Cellular and Infection Microbiology, vol. 11, 2021, p. 613304.
Zheng YZ, Chen JT, Li J, et al. Reverse Transcription Recombinase-Aided Amplification Assay With Lateral Flow Dipstick Assay for Rapid Detection of 2019 Novel Coronavirus. Front Cell Infect Microbiol. 2021;11:613304.
Zheng, Y. Z., Chen, J. T., Li, J., Wu, X. J., Wen, J. Z., Liu, X. Z., Lin, L. Y., Liang, X. Y., Huang, H. Y., Zha, G. C., Yang, P. K., Li, L. J., Zhong, T. Y., Liu, L., Cheng, W. J., Song, X. N., & Lin, M. (2021). Reverse Transcription Recombinase-Aided Amplification Assay With Lateral Flow Dipstick Assay for Rapid Detection of 2019 Novel Coronavirus. Frontiers in Cellular and Infection Microbiology, 11, 613304. https://doi.org/10.3389/fcimb.2021.613304
Zheng YZ, et al. Reverse Transcription Recombinase-Aided Amplification Assay With Lateral Flow Dipstick Assay for Rapid Detection of 2019 Novel Coronavirus. Front Cell Infect Microbiol. 2021;11:613304. PubMed PMID: 33598439.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Reverse Transcription Recombinase-Aided Amplification Assay With Lateral Flow Dipstick Assay for Rapid Detection of 2019 Novel Coronavirus. AU - Zheng,Yu-Zhong, AU - Chen,Jiang-Tao, AU - Li,Jian, AU - Wu,Xian-Jing, AU - Wen,Jin-Zhou, AU - Liu,Xiang-Zhi, AU - Lin,Li-Yun, AU - Liang,Xue-Yan, AU - Huang,Hui-Ying, AU - Zha,Guang-Cai, AU - Yang,Pei-Kui, AU - Li,Lie-Jun, AU - Zhong,Tian-Yu, AU - Liu,Long, AU - Cheng,Wei-Jia, AU - Song,Xiao-Nan, AU - Lin,Min, Y1 - 2021/02/01/ PY - 2020/10/12/received PY - 2021/01/08/accepted PY - 2021/2/18/entrez PY - 2021/2/19/pubmed PY - 2021/2/26/medline KW - Coronavirus Disease-2019 KW - Severe Acute Respiratory Syndrome Coronavirus 2 KW - lateral flow dipstick KW - point of care test KW - reverse-transcription recombinase-aided amplification SP - 613304 EP - 613304 JF - Frontiers in cellular and infection microbiology JO - Front Cell Infect Microbiol VL - 11 N2 - Background: The emerging Coronavirus Disease-2019 (COVID-19) has challenged the public health globally. With the increasing requirement of detection for SARS-CoV-2 outside of the laboratory setting, a rapid and precise Point of Care Test (POCT) is urgently needed. Methods: Targeting the nucleocapsid (N) gene of SARS-CoV-2, specific primers, and probes for reverse transcription recombinase-aided amplification coupled with lateral flow dipstick (RT-RAA/LFD) platform were designed. For specificity evaluation, it was tested with human coronaviruses, human influenza A virus, influenza B viruses, respiratory syncytial virus, and hepatitis B virus, respectively. For sensitivity assay, it was estimated by templates of recombinant plasmid and pseudovirus of SARS-CoV-2 RNA. For clinical assessment, 100 clinical samples (13 positive and 87 negatives for SARS-CoV-2) were tested via quantitative reverse transcription PCR (RT-qPCR) and RT-RAA/LFD, respectively. Results: The limit of detection was 1 copies/μl in RT-RAA/LFD assay, which could be conducted within 30 min at 39°C, without any cross-reaction with other human coronaviruses and clinical respiratory pathogens. Compared with RT-qPCR, the established POCT assay offered 100% specificity and 100% sensitivity in the detection of clinical samples. Conclusion: This work provides a convenient POCT tool for rapid screening, diagnosis, and monitoring of suspected patients in SARS-CoV-2 endemic areas. SN - 2235-2988 UR - https://www.unboundmedicine.com/medline/citation/33598439/Reverse_Transcription_Recombinase_Aided_Amplification_Assay_With_Lateral_Flow_Dipstick_Assay_for_Rapid_Detection_of_2019_Novel_Coronavirus_ L2 - https://www.ncbi.nlm.nih.gov/pmc/articles/pmid/33598439/ DB - PRIME DP - Unbound Medicine ER -