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Development and Clinical Application of a Rapid and Sensitive Loop-Mediated Isothermal Amplification Test for SARS-CoV-2 Infection.
mSphere. 2020 08 26; 5(4)M

Abstract

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak urgently necessitates sensitive and convenient COVID-19 diagnostics for the containment and timely treatment of patients. We aimed to develop and validate a novel reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay to detect SARS-CoV-2. Patients with suspected COVID-19 and close contacts were recruited from two hospitals between 26 January and 8 April 2020. Respiratory samples were collected and tested using RT-LAMP, and the results were compared with those obtained by reverse transcription-quantitative PCR (RT-qPCR). Samples yielding inconsistent results between these two methods were subjected to next-generation sequencing for confirmation. RT-LAMP was also applied to an asymptomatic COVID-19 carrier and patients with other respiratory viral infections. Samples were collected from a cohort of 129 cases (329 nasopharyngeal swabs) and an independent cohort of 76 patients (152 nasopharyngeal swabs and sputum samples). The RT-LAMP assay was validated to be accurate (overall sensitivity and specificity of 88.89% and 99.00%, respectively) and diagnostically useful (positive and negative likelihood ratios of 88.89 and 0.11, respectively). RT-LAMP showed increased sensitivity (88.89% versus 81.48%) and high consistency (kappa, 0.92) compared to those of RT-qPCR for SARS-CoV-2 screening while requiring only constant-temperature heating and visual inspection. The time required for RT-LAMP was less than 1 h from sample preparation to the result. In addition, RT-LAMP was feasible for use with asymptomatic patients and did not cross-react with other respiratory pathogens. The developed RT-LAMP assay offers rapid, sensitive, and straightforward detection of SARS-CoV-2 infection and may aid the expansion of COVID-19 testing in the public domain and hospitals.IMPORTANCE We developed a visual and rapid reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay targeting the S gene for SARS-CoV-2 infection. The strength of our study was that we validated the RT-LAMP assay using 481 clinical respiratory samples from two prospective cohorts of suspected COVID-19 patients and on the serial samples from an asymptomatic carrier. The developed RT-LAMP approach showed an increased sensitivity (88.89%) and high consistency (kappa, 0.92) compared with those of reverse transcription-quantitative PCR (RT-qPCR) for SARS-CoV-2 screening while requiring only constant-temperature heating and visual inspection, facilitating SARS-CoV-2 screening in well-equipped labs as well as in the field. The time required for RT-LAMP was less than 1 h from sample preparation to the result (more than 2 h for RT-qPCR). This study showed that the RT-LAMP assay was a simple, rapid, and sensitive approach for SARS-CoV-2 infection and can facilitate COVID-19 diagnosis, especially in resource-poor settings.

Authors+Show Affiliations

Division of Laboratory Medicine, Guangdong Provincial People's Hospital, Guangdong Academy of Medical Sciences, Guangzhou, People's Republic of China.Division of Laboratory Medicine, Guangdong Provincial People's Hospital, Guangdong Academy of Medical Sciences, Guangzhou, People's Republic of China.MOE International Joint Lab for Synthetic Biology and Medicine, School of Biology and Biological Engineering, South China University of Technology, Guangzhou, People's Republic of China.Division of Laboratory Medicine, Guangdong Provincial People's Hospital, Guangdong Academy of Medical Sciences, Guangzhou, People's Republic of China.Division of Laboratory Medicine, Guangdong Provincial People's Hospital, Guangdong Academy of Medical Sciences, Guangzhou, People's Republic of China.Division of Laboratory Medicine, Guangdong Provincial People's Hospital, Guangdong Academy of Medical Sciences, Guangzhou, People's Republic of China.Department of Laboratory Medicine and Central Laboratories, Guangdong Second Provincial General Hospital, Guangzhou, People's Republic of China.Department of Laboratory Medicine and Central Laboratories, Guangdong Second Provincial General Hospital, Guangzhou, People's Republic of China.Division of Laboratory Medicine, Guangdong Provincial People's Hospital, Guangdong Academy of Medical Sciences, Guangzhou, People's Republic of China.Genskey Biotechnology Co. Ltd., Beijing, People's Republic of China.Division of Laboratory Medicine, Guangdong Provincial People's Hospital, Guangdong Academy of Medical Sciences, Guangzhou, People's Republic of China.MOE International Joint Lab for Synthetic Biology and Medicine, School of Biology and Biological Engineering, South China University of Technology, Guangzhou, People's Republic of China.MOE International Joint Lab for Synthetic Biology and Medicine, School of Biology and Biological Engineering, South China University of Technology, Guangzhou, People's Republic of China.MOE International Joint Lab for Synthetic Biology and Medicine, School of Biology and Biological Engineering, South China University of Technology, Guangzhou, People's Republic of China lzhangce@scut.edu.cn houtieying@gdph.org.cn.Division of Laboratory Medicine, Guangdong Provincial People's Hospital, Guangdong Academy of Medical Sciences, Guangzhou, People's Republic of China lzhangce@scut.edu.cn houtieying@gdph.org.cn.

Pub Type(s)

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

Language

eng

PubMed ID

32848011

Citation

Hu, Xuejiao, et al. "Development and Clinical Application of a Rapid and Sensitive Loop-Mediated Isothermal Amplification Test for SARS-CoV-2 Infection." MSphere, vol. 5, no. 4, 2020.
Hu X, Deng Q, Li J, et al. Development and Clinical Application of a Rapid and Sensitive Loop-Mediated Isothermal Amplification Test for SARS-CoV-2 Infection. mSphere. 2020;5(4).
Hu, X., Deng, Q., Li, J., Chen, J., Wang, Z., Zhang, X., Fang, Z., Li, H., Zhao, Y., Yu, P., Li, W., Wang, X., Li, S., Zhang, L., & Hou, T. (2020). Development and Clinical Application of a Rapid and Sensitive Loop-Mediated Isothermal Amplification Test for SARS-CoV-2 Infection. MSphere, 5(4). https://doi.org/10.1128/mSphere.00808-20
Hu X, et al. Development and Clinical Application of a Rapid and Sensitive Loop-Mediated Isothermal Amplification Test for SARS-CoV-2 Infection. mSphere. 2020 08 26;5(4) PubMed PMID: 32848011.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Development and Clinical Application of a Rapid and Sensitive Loop-Mediated Isothermal Amplification Test for SARS-CoV-2 Infection. AU - Hu,Xuejiao, AU - Deng,Qianyun, AU - Li,Junmin, AU - Chen,Jierong, AU - Wang,Zixia, AU - Zhang,Xiqin, AU - Fang,Zhixin, AU - Li,Haijian, AU - Zhao,Yunhu, AU - Yu,Pan, AU - Li,Wenmin, AU - Wang,Xiaoming, AU - Li,Shan, AU - Zhang,Lei, AU - Hou,Tieying, Y1 - 2020/08/26/ PY - 2020/8/28/entrez PY - 2020/8/28/pubmed PY - 2020/9/8/medline KW - COVID-19 KW - RT-LAMP KW - SARS-CoV-2 KW - asymptomatic carriers KW - clinical diagnosis JF - mSphere JO - mSphere VL - 5 IS - 4 N2 - The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak urgently necessitates sensitive and convenient COVID-19 diagnostics for the containment and timely treatment of patients. We aimed to develop and validate a novel reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay to detect SARS-CoV-2. Patients with suspected COVID-19 and close contacts were recruited from two hospitals between 26 January and 8 April 2020. Respiratory samples were collected and tested using RT-LAMP, and the results were compared with those obtained by reverse transcription-quantitative PCR (RT-qPCR). Samples yielding inconsistent results between these two methods were subjected to next-generation sequencing for confirmation. RT-LAMP was also applied to an asymptomatic COVID-19 carrier and patients with other respiratory viral infections. Samples were collected from a cohort of 129 cases (329 nasopharyngeal swabs) and an independent cohort of 76 patients (152 nasopharyngeal swabs and sputum samples). The RT-LAMP assay was validated to be accurate (overall sensitivity and specificity of 88.89% and 99.00%, respectively) and diagnostically useful (positive and negative likelihood ratios of 88.89 and 0.11, respectively). RT-LAMP showed increased sensitivity (88.89% versus 81.48%) and high consistency (kappa, 0.92) compared to those of RT-qPCR for SARS-CoV-2 screening while requiring only constant-temperature heating and visual inspection. The time required for RT-LAMP was less than 1 h from sample preparation to the result. In addition, RT-LAMP was feasible for use with asymptomatic patients and did not cross-react with other respiratory pathogens. The developed RT-LAMP assay offers rapid, sensitive, and straightforward detection of SARS-CoV-2 infection and may aid the expansion of COVID-19 testing in the public domain and hospitals.IMPORTANCE We developed a visual and rapid reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay targeting the S gene for SARS-CoV-2 infection. The strength of our study was that we validated the RT-LAMP assay using 481 clinical respiratory samples from two prospective cohorts of suspected COVID-19 patients and on the serial samples from an asymptomatic carrier. The developed RT-LAMP approach showed an increased sensitivity (88.89%) and high consistency (kappa, 0.92) compared with those of reverse transcription-quantitative PCR (RT-qPCR) for SARS-CoV-2 screening while requiring only constant-temperature heating and visual inspection, facilitating SARS-CoV-2 screening in well-equipped labs as well as in the field. The time required for RT-LAMP was less than 1 h from sample preparation to the result (more than 2 h for RT-qPCR). This study showed that the RT-LAMP assay was a simple, rapid, and sensitive approach for SARS-CoV-2 infection and can facilitate COVID-19 diagnosis, especially in resource-poor settings. SN - 2379-5042 UR - https://www.unboundmedicine.com/medline/citation/32848011/Development_and_Clinical_Application_of_a_Rapid_and_Sensitive_Loop_Mediated_Isothermal_Amplification_Test_for_SARS_CoV_2_Infection_ L2 - https://doi.org/10.1128/mSphere.00808-20 DB - PRIME DP - Unbound Medicine ER -