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Dry loop mediated isothermal amplification assay for detection of SARS-CoV-2 from clinical specimens
Preprint
en En
| PREPRINT-MEDRXIV
| ID: ppmedrxiv-20204297
ABSTRACT
Coronavirus disease 2019 (COVID-19) has had a major disease burden on many countries around the world. The spread of COVID-19 is anticipated to have a major impact on developing countries including African nations. To establish a point-of-care test for COVID-19, we developed a dry loop-mediated isothermal amplification (LAMP) method to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA. We carried out reverse transcription (RT)-LAMP using the Loopamp SARS-CoV-2 Detection kit (Eiken Chemical, Tokyo, Japan). The entire mixture except for the primers is dried and immobilized inside the tube lid. To determine the specificity of the kit, 22 viral genomes associated with respiratory infections, including the SARS coronavirus, were tested. No LAMP product was detected in reactions performed with RNA from these pathogens. The sensitivity of this assay, determined by either a real-time turbidity assay or colorimetric change of the reaction mixture, as evaluated by the naked eye or under illumination with ultraviolet light, was 10 copies/reaction. After the initial validation analysis, we analyzed 24 nasopharyngeal swab specimens collected from patients suspected to have COVID-19. Nineteen (79.2%) of the 24 samples were positive for SARS-CoV-2 RNA, as determined by real-time RT-PCR analysis. Using the Loopamp SARS-CoV-2 Detection kit, we detected SARS-CoV-2 RNA in 15 (62.5%) of the 24 samples. Thus, the sensitivity, specificity, positive predictive value, and negative predictive value of the Loopamp 2019-CoV-2 detection reagent kit were 94.0%, 96.0%, 95.9%, and 94.1%, respectively. The dry LAMP method for detection of SARS-CoV-2 RNA was fast and easy to use, solves the cold chain problem, and therefore represents a promising tool for diagnosis of COVID-19 in developing countries. Author summaryCoronavirus disease 2019 (COVID-19) is a major public health problem around the world. A reliable point-of-care (POC) test for severe acute respiratory syndrome coronavirus 2 (SARS CoV-2) is urgently needed, especially in developing countries. The loop-mediated isothermal amplification (LAMP) method amplifies template nucleotides under isothermal conditions with high efficiency and specificity, both of which are major advantages for a POC test. In addition, because dry LAMP reagents can be stored at 4{degrees}C, it is suitable for use in developing countries. We evaluated the specificity and sensitivity of the Loopamp SARS-CoV-2 Detection kit (Eiken Chemical, Tokyo, Japan), a dry LAMP method for amplifying viral RNA. The initial validation study revealed that the method was highly specific and sensitive (lower detection limit 10 copies/reaction). We then analyzed 24 nasopharyngeal swab specimens from patients suspected to have COVID-19. Using the Loopamp SARS-CoV-2 Detection kit, SARS-CoV-2 RNA was detected in 15 (62.5%) of the 24 samples. Compared with the standard real-time reverse transcription PCR, the sensitivity, specificity, positive predictive value, and negative predictive value of the Loopamp SARS-CoV-2 Detection kit were 78.9%, 100%, 100%, and 55.6%, respectively.
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Texto completo:
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Colección:
09-preprints
Base de datos:
PREPRINT-MEDRXIV
Tipo de estudio:
Diagnostic_studies
/
Experimental_studies
/
Prognostic_studies
Idioma:
En
Año:
2020
Tipo del documento:
Preprint