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      Development and clinical application of loop-mediated isothermal amplification combined with lateral flow assay for rapid diagnosis of SARS-CoV-2

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          Abstract

          Background

          The diagnostic assay leveraging multiple reverse transcription loop-mediated isothermal amplification (RT-LAMP) could meet the requirements for rapid nucleic acid detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

          Methods

          The devised assay merged the lateral flow assay with the RT-LAMP technology and designed specific primers for the simultaneous detection of the target and human-derived internal reference genes within a single reaction. An inquiry into the assay's limit of detection (LOD), sensitivity, and specificity was carried out. The effectiveness of this assay was validated using 498 clinical specimens.

          Results

          This LOD of the assay was determined to be 500 copies/mL, and there was no observed cross-reaction with other respiratory pathogens. The detection results derived from clinical specimens showed substantial concordance with those from real-time reverse transcription-polymerase chain reaction (RT-qPCR) (Cohen's kappa, 0.876; 95% CI: 0.833-0.919; p<0.005). The diagnostic sensitivity and specificity were 87.1% and 100%, respectively.

          Conclusion

          The RT-LAMP assay, paired with a straightforward and disposable lateral immunochromatographic strip, achieves visual detection of dual targets for SARS-CoV-2 immediatly. Moreover, the entire procedure abstains from nucleic acids extraction. The samples are lysed at room temperature and subsequently proceed directly to the RT-LAMP reaction, which can be executed within 30 minutes at a constant temperature of 60-65°C. Then, the RT-LAMP amplification products are visualized using colloidal gold test strips.

          Trial registration

          This study was registered at the Chinese Clinical Trial Registry (Registration number: ChiCTR2200060495, Date of registration 2022-06-03).

          Supplementary Information

          The online version contains supplementary material available at 10.1186/s12879-023-08924-3.

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          Most cited references18

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          Loop-mediated isothermal amplification of DNA.

          T. Notomi (2000)
          We have developed a novel method, termed loop-mediated isothermal amplification (LAMP), that amplifies DNA with high specificity, efficiency and rapidity under isothermal conditions. This method employs a DNA polymerase and a set of four specially designed primers that recognize a total of six distinct sequences on the target DNA. An inner primer containing sequences of the sense and antisense strands of the target DNA initiates LAMP. The following strand displacement DNA synthesis primed by an outer primer releases a single-stranded DNA. This serves as template for DNA synthesis primed by the second inner and outer primers that hybridize to the other end of the target, which produces a stem-loop DNA structure. In subsequent LAMP cycling one inner primer hybridizes to the loop on the product and initiates displacement DNA synthesis, yielding the original stem-loop DNA and a new stem-loop DNA with a stem twice as long. The cycling reaction continues with accumulation of 10(9) copies of target in less than an hour. The final products are stem-loop DNAs with several inverted repeats of the target and cauliflower-like structures with multiple loops formed by annealing between alternately inverted repeats of the target in the same strand. Because LAMP recognizes the target by six distinct sequences initially and by four distinct sequences afterwards, it is expected to amplify the target sequence with high selectivity.
          Article 0 comments Cited 1337 times – based on 0 reviews     Review now
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            Loop-mediated isothermal amplification (LAMP) of gene sequences and simple visual detection of products.

            Norihiro Tomita, Yasuyoshi Mori, Hidetoshi Kanda … (2008)
            As the human genome is decoded and its involvement in diseases is being revealed through postgenome research, increased adoption of genetic testing is expected. Critical to such testing methods is the ease of implementation and comprehensible presentation of amplification results. Loop-mediated isothermal amplification (LAMP) is a simple, rapid, specific and cost-effective nucleic acid amplification method when compared to PCR, nucleic acid sequence-based amplification, self-sustained sequence replication and strand displacement amplification. This protocol details an improved simple visual detection system for the results of the LAMP reaction. In LAMP, a large amount of DNA is synthesized, yielding a large pyrophosphate ion by-product. Pyrophosphate ion combines with divalent metallic ion to form an insoluble salt. Adding manganous ion and calcein, a fluorescent metal indicator, to the reaction solution allows a visualization of substantial alteration of the fluorescence during the one-step amplification reaction, which takes 30-60 min. As the signal recognition is highly sensitive, this system enables visual discrimination of results without costly specialized equipment. This detection method should be helpful in basic research on medicine and pharmacy, environmental hygiene, point-of-care testing and more.
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              COVID-19: A Review on the Novel Coronavirus Disease Evolution, Transmission, Detection, Control and Prevention

              Anshika Sharma, Isra Ahmad Farouk, Sunil Kumar Lal (2021)
              Three major outbreaks of the coronavirus, a zoonotic virus known to cause respiratory disease, have been reported since 2002, including SARS-CoV, MERS-CoV and the most recent 2019-nCoV, or more recently known as SARS-CoV-2. Bats are known to be the primary animal reservoir for coronaviruses. However, in the past few decades, the virus has been able to mutate and adapt to infect humans, resulting in an animal-to-human species barrier jump. The emergence of a novel coronavirus poses a serious global public health threat and possibly carries the potential of causing a major pandemic outbreak in the naïve human population. The recent outbreak of COVID-19, the disease caused by SARS-CoV-2, in Wuhan, Hubei Province, China has infected over 36.5 million individuals and claimed over one million lives worldwide, as of 8 October 2020. The novel virus is rapidly spreading across China and has been transmitted to 213 other countries/territories across the globe. Researchers have reported that the virus is constantly evolving and spreading through asymptomatic carriers, further suggesting a high global health threat. To this end, current up-to-date information on the coronavirus evolution and SARS-CoV-2 modes of transmission, detection techniques and current control and prevention strategies are summarized in this review.
              Article 0 comments Cited 219 times – based on 0 reviews     Review now
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                Author and article information

                Contributors
                Yuan Fang: yuan.fang@genoxor.com
                Weifeng Huang: breeze-huang@hotmail.com
                Journal
                Journal ID (nlm-ta): BMC Infect Dis
                Journal ID (iso-abbrev): BMC Infect Dis
                Title: BMC Infectious Diseases
                Publisher: BioMed Central (London )
                ISSN (Electronic): 1471-2334
                Publication date (Electronic): 15 January 2024
                Publication date PMC-release: 15 January 2024
                Publication date Collection: 2024
                Volume: 24
                Electronic Location Identifier: 81
                Affiliations
                [1 ]Department of Clinical Laboratory, Shanghai Sixth People’s Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, ( https://ror.org/0220qvk04) Shanghai, 200233 China
                [2 ]Shanghai Sixth People’s Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, ( https://ror.org/0220qvk04) Shanghai, 200233 China
                [3 ]Shanghai Jiao Tong University affiliated the Eighth People’s Hospital, ( https://ror.org/0220qvk04) Shanghai, 200235 China
                [4 ]Shanghai Fengxian District Central Hospital, Shanghai, 201406 China
                [5 ]GRID grid.508079.3, Shanghai Fengxian District Guhua Hospital, ; Shanghai, 201499 China
                [6 ]Department of Intensive Care Medicine, Shanghai Sixth People’s Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, ( https://ror.org/0220qvk04) Shanghai, 200233 China
                [7 ]Genoxor Medical Science and Technology Inc., No 555 Wangfang Road, Minhang District, Shanghai, 201112 China
                Article
                Publisher ID: 8924
                DOI: 10.1186/s12879-023-08924-3
                PMC ID: 10788970
                PubMed ID: 38225546
                SO-VID: ec5ad3aa-c040-4a04-ad3c-176effda5818
                Copyright © © The Author(s) 2024
                License:

                Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

                History
                Date received : 29 May 2023
                Date accepted : 17 December 2023
                Funding
                Funded by: Shanghai Sixth People's Hospital
                Award ID: ynxg202205
                Award Recipient :
                Categories
                Subject: Research
                Custom metadata
                issue-copyright-statement © BioMed Central Ltd., part of Springer Nature 2024

                ScienceOpen disciplines: Infectious disease & Microbiology
                Keywords: sars-cov-2,covid-19,rt-lamp,isothermal amplification,diagnostics,molecular testing
                Data availability:
                ScienceOpen disciplines: Infectious disease & Microbiology
                Keywords: sars-cov-2, covid-19, rt-lamp, isothermal amplification, diagnostics, molecular testing

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