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Rapid and Visual Detection of SARS-CoV-2 Using Multiplex Reverse Transcription Loop-Mediated Isothermal Amplification Linked With Gold Nanoparticle-Based Lateral Flow Biosensor.
Front Cell Infect Microbiol. 2021; 11:581239.FC

Abstract

Background

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel coronavirus that has caused the outbreak of coronavirus disease 2019 (COVID-19) all over the world. In the absence of appropriate antiviral drugs or vaccines, developing a simple, rapid, and reliable assay for SARS-CoV-2 is necessary for the prevention and control of the COVID-19 transmission.

Methods

A novel molecular diagnosis technique, named multiplex reverse transcription loop-mediated isothermal amplification, that has been linked to a nanoparticle-based lateral flow biosensor (mRT-LAMP-LFB) was applied to detect SARS-CoV-2 based on the SARS-CoV-2 RdRp and N genes, and the mRT-LAMP products were analyzed using nanoparticle-based lateral flow biosensor. The mRT-LAMP-LFB amplification conditions, including the target RNA concentration, amplification temperature, and time were optimized. The sensitivity and specificity of the mRT-LAMP-LFB method were tested in the current study, and the mRT-LAMP-LFB assay was applied to detect the SARS-CoV-2 virus from clinical samples and artificial sputum samples.

Results

The SARS-CoV-2 specific primers based on the RdRp and N genes were valid for the establishment of mRT-LAMP-LFB assay to detect the SARS-CoV-2 virus. The multiple-RT-LAMP amplification condition was optimized at 63°C for 30 min. The full process, including reaction preparation, viral RNA extraction, RT-LAMP, and product identification, could be achieved in 80 min. The limit of detection (LoD) of the mRT-LAMP-LFB technology was 20 copies per reaction. The specificity of mRT-LAMP-LFB detection was 100%, and no cross-reactions to other respiratory pathogens were observed.

Conclusion

The mRT-LAMP-LFB technique developed in the current study is a simple, rapid, and reliable method with great specificity and sensitivity when it comes to identifying SARS-CoV-2 virus for prevention and control of the COVID-19 disease, especially in resource-constrained regions of the world.

Authors+Show Affiliations

The Second Clinical College, Guizhou University of Traditional Chinese Medicine, Guiyang, China. Central Laboratory of the Second Affiliated Hospital, Guizhou University of Traditional Chinese Medicine, Guiyang, China. Laboratory of Bacterial Infectious Disease of Experimental Centre, Guizhou Provincial Centre for Disease Control and Prevention, Guiyang, China.Clinical Laboratory, Hangzhou Women's Hospital, Hangzhou, China.Laboratory of Bacterial Infectious Disease of Experimental Centre, Guizhou Provincial Centre for Disease Control and Prevention, Guiyang, China.Department of Microbiology, Zhejiang Provincial Center for Disease Control and Prevention, Hangzhou, China.The Second Clinical College, Guizhou University of Traditional Chinese Medicine, Guiyang, China. Central Laboratory of the Second Affiliated Hospital, Guizhou University of Traditional Chinese Medicine, Guiyang, China.TCM Research Institute, Zhejiang Chinese Medical University, Hangzhou, China.Department of Clinical Laboratory, Zhejiang Hospital, Hangzhou, China.

Pub Type(s)

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

Language

eng

PubMed ID

34336708

Citation

Chen, Xu, et al. "Rapid and Visual Detection of SARS-CoV-2 Using Multiplex Reverse Transcription Loop-Mediated Isothermal Amplification Linked With Gold Nanoparticle-Based Lateral Flow Biosensor." Frontiers in Cellular and Infection Microbiology, vol. 11, 2021, p. 581239.
Chen X, Zhou Q, Li S, et al. Rapid and Visual Detection of SARS-CoV-2 Using Multiplex Reverse Transcription Loop-Mediated Isothermal Amplification Linked With Gold Nanoparticle-Based Lateral Flow Biosensor. Front Cell Infect Microbiol. 2021;11:581239.
Chen, X., Zhou, Q., Li, S., Yan, H., Chang, B., Wang, Y., & Dong, S. (2021). Rapid and Visual Detection of SARS-CoV-2 Using Multiplex Reverse Transcription Loop-Mediated Isothermal Amplification Linked With Gold Nanoparticle-Based Lateral Flow Biosensor. Frontiers in Cellular and Infection Microbiology, 11, 581239. https://doi.org/10.3389/fcimb.2021.581239
Chen X, et al. Rapid and Visual Detection of SARS-CoV-2 Using Multiplex Reverse Transcription Loop-Mediated Isothermal Amplification Linked With Gold Nanoparticle-Based Lateral Flow Biosensor. Front Cell Infect Microbiol. 2021;11:581239. PubMed PMID: 34336708.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Rapid and Visual Detection of SARS-CoV-2 Using Multiplex Reverse Transcription Loop-Mediated Isothermal Amplification Linked With Gold Nanoparticle-Based Lateral Flow Biosensor. AU - Chen,Xu, AU - Zhou,Qingxue, AU - Li,Shijun, AU - Yan,Hao, AU - Chang,Bingcheng, AU - Wang,Yuexia, AU - Dong,Shilei, Y1 - 2021/07/14/ PY - 2020/07/08/received PY - 2021/06/29/accepted PY - 2021/8/2/entrez PY - 2021/8/3/pubmed PY - 2021/8/5/medline KW - COVID-19 KW - SARS-CoV-2 KW - lateral flow biosensor KW - limit of detection KW - reverse transcription-loop-mediated isothermal amplification SP - 581239 EP - 581239 JF - Frontiers in cellular and infection microbiology JO - Front Cell Infect Microbiol VL - 11 N2 - Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel coronavirus that has caused the outbreak of coronavirus disease 2019 (COVID-19) all over the world. In the absence of appropriate antiviral drugs or vaccines, developing a simple, rapid, and reliable assay for SARS-CoV-2 is necessary for the prevention and control of the COVID-19 transmission. Methods: A novel molecular diagnosis technique, named multiplex reverse transcription loop-mediated isothermal amplification, that has been linked to a nanoparticle-based lateral flow biosensor (mRT-LAMP-LFB) was applied to detect SARS-CoV-2 based on the SARS-CoV-2 RdRp and N genes, and the mRT-LAMP products were analyzed using nanoparticle-based lateral flow biosensor. The mRT-LAMP-LFB amplification conditions, including the target RNA concentration, amplification temperature, and time were optimized. The sensitivity and specificity of the mRT-LAMP-LFB method were tested in the current study, and the mRT-LAMP-LFB assay was applied to detect the SARS-CoV-2 virus from clinical samples and artificial sputum samples. Results: The SARS-CoV-2 specific primers based on the RdRp and N genes were valid for the establishment of mRT-LAMP-LFB assay to detect the SARS-CoV-2 virus. The multiple-RT-LAMP amplification condition was optimized at 63°C for 30 min. The full process, including reaction preparation, viral RNA extraction, RT-LAMP, and product identification, could be achieved in 80 min. The limit of detection (LoD) of the mRT-LAMP-LFB technology was 20 copies per reaction. The specificity of mRT-LAMP-LFB detection was 100%, and no cross-reactions to other respiratory pathogens were observed. Conclusion: The mRT-LAMP-LFB technique developed in the current study is a simple, rapid, and reliable method with great specificity and sensitivity when it comes to identifying SARS-CoV-2 virus for prevention and control of the COVID-19 disease, especially in resource-constrained regions of the world. SN - 2235-2988 UR - https://www.unboundmedicine.com/medline/citation/34336708/Rapid_and_Visual_Detection_of_SARS_CoV_2_Using_Multiplex_Reverse_Transcription_Loop_Mediated_Isothermal_Amplification_Linked_With_Gold_Nanoparticle_Based_Lateral_Flow_Biosensor_ L2 - https://www.ncbi.nlm.nih.gov/pmc/articles/pmid/34336708/ DB - PRIME DP - Unbound Medicine ER -