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A highly effective reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay for the rapid detection of SARS-CoV-2 infection.

ABSTRACT: The COVID-19 pandemic has illustrated the importance of simple, rapid and accurate diagnostic testing. This study describes the validation of a new rapid SARS-CoV-2 RT-LAMP assay for use on extracted RNA or directly from swab offering an alternative diagnostic pathway that does not rely on traditional reagents that are often in short supply during a pandemic. Analytical specificity (ASp) of this new RT-LAMP assay was 100% and analytical sensitivity (ASe) was between 1 × 10¹ and 1 × 10² copies per reaction when using a synthetic DNA target. The overall diagnostic sensitivity (DSe) and specificity (DSp) of RNA RT-LAMP was 97% and 99% respectively, relative to the standard of care rRT-PCR. When a C_T cut-off of 33 was employed, above which increasingly evidence suggests there is a low risk of patients shedding infectious virus, the diagnostic sensitivity was 100%. The DSe and DSp of Direct RT-LAMP (that does not require RNA extraction) was 67% and 97%, respectively. When setting C_T cut-offs of ≤33 and ≤25, the DSe increased to 75% and 100%, respectively, time from swab-to-result, C_T < 25, was < 15 min. We propose that RNA RT-LAMP could replace rRT-PCR where there is a need for increased sample throughput and Direct RT-LAMP as a near-patient screening tool to rapidly identify highly contagious individuals within emergency departments and care homes during times of increased disease prevalence ensuring negative results still get laboratory confirmation.

SUBMITTER: Fowler VL

PROVIDER: S-EPMC7703389 | biostudies-literature | 2021 Jan

REPOSITORIES: biostudies-literature

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A highly effective reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay for the rapid detection of SARS-CoV-2 infection.

Fowler Veronica L VL Armson Bryony B Gonzales Jose L JL Wise Emma L EL Howson Emma L A ELA Vincent-Mistiaen Zoe Z Fouch Sarah S Maltby Connor J CJ Grippon Seden S Munro Simon S Jones Lisa L Holmes Tom T Tillyer Claire C Elwell Joanne J Sowood Amy A de Peyer Oliver O Dixon Sophie S Hatcher Thomas T Patrick Helen H Laxman Shailen S Walsh Charlotte C Andreou Michael M Morant Nick N Clark Duncan D Moore Nathan N Houghton Rebecca R Cortes Nicholas J NJ Kidd Stephen P SP

The Journal of infection 20201130 1

The COVID-19 pandemic has illustrated the importance of simple, rapid and accurate diagnostic testing. This study describes the validation of a new rapid SARS-CoV-2 RT-LAMP assay for use on extracted RNA or directly from swab offering an alternative diagnostic pathway that does not rely on traditional reagents that are often in short supply during a pandemic. Analytical specificity (ASp) of this new RT-LAMP assay was 100% and analytical sensitivity (ASe) was between 1 × 10<sup>1</sup> and 1 × 10 ...[more]

PMID: 33271166

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Project description:In December 2019, the Chinese Center for Disease Control (CDC of China) reported an outbreak of pneumonia in the city of Wuhan (Hubei province, China) that haunted the world, resulting in a global pandemic. This outbreak was caused by a betacoronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Several of these cases have been observed in healthcare professionals working in hospitals and providing care on the pandemic's frontline. In the present study, nasopharyngeal swab samples of healthcare workers were used to assess the performance of the reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay and subsequently compared with the real-time reverse-transcription quantitative PCR (RT-qPCR) method. Thus, in this study, we validated a method for detecting SARS-CoV-2 based on RT-LAMP that can be used to diagnose these workers. The methodology used was based on analyzing the sensitivity, specificity, evaluation of the detection limit, and cross-reaction with other respiratory viruses. The agreement was estimated using a dispersion diagram designed using the Bland-Altman method. A total of 100 clinical specimens of nasopharyngeal swabs were collected from symptomatic and asymptomatic healthcare workers in Pelotas, Brazil, during the SARS-CoV-2 outbreak. RT-LAMP assay, it was possible to detect SARS-CoV-2 in 96.7% of the healthcare professionals tested using the E gene and N gene primers approximately and 100% for the gene of human β-actin. The observed agreement was considered excellent for the primer set of the E and N genes (k = 0.957 and k = 0.896), respectively. The sensitivity of the RT-LAMP assay was positive for the primer set of the E gene, detected to approximately 2 copies per reaction. For the primer set of the N gene, the assay was possible to verify an LoD of approximately 253 copies per reaction. After executing the RT-LAMP assay, no positive reactions were observed for any of the virus respiratory tested. Therefore, we conclude that RT-LAMP is effective for rapid molecular diagnosis during the COVID-19 outbreak period in healthcare professionals.

Dataset Information

A highly effective reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay for the rapid detection of SARS-CoV-2 infection.

Publications

A highly effective reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay for the rapid detection of SARS-CoV-2 infection.

Similar Datasets

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