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Development and evaluation of a rapid and simple diagnostic assay for COVID-19 based on loop-mediated isothermal amplification.

ABSTRACT: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly pathogenic novel coronavirus that has caused a worldwide outbreak. Here we describe a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay that uses a portable device for efficient detection of SARS-CoV-2. This RT-LAMP assay specifically detected SARS-CoV-2 without cross-reacting with the most closely related human coronavirus, SARS-CoV. Clinical evaluation of nasal swab samples from suspected SARS-CoV-2 pneumonia (COVID-19) patients showed that the assay could detect over 23.7 copies within 15 min with a 100% probability. Since the RT-LAMP assay can be performed with a portable battery-supported device, it is a rapid, simple, and sensitive diagnostic assay for COVID-19 that can be available at point-of-care. We also developed the RT-LAMP assay without the RNA extraction step-Direct RT-LAMP, which could detect more than 1.43 x 103 copies within 15 min with a 100% probability in clinical evaluation test. Although the Direct RT-LAMP assay was less sensitive than the standard RT-LAMP, the Direct RT-LAMP assay can be available as the rapid first screening of COVID-19 in poorly equipped areas, such as rural areas in developing countries.

SUBMITTER: Yoshikawa R

PROVIDER: S-EPMC7668588 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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Development and evaluation of a rapid and simple diagnostic assay for COVID-19 based on loop-mediated isothermal amplification.

Yoshikawa Rokusuke R Abe Haruka H Igasaki Yui Y Negishi Saeki S Goto Hiroaki H Yasuda Jiro J

PLoS neglected tropical diseases 20201104 11

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly pathogenic novel coronavirus that has caused a worldwide outbreak. Here we describe a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay that uses a portable device for efficient detection of SARS-CoV-2. This RT-LAMP assay specifically detected SARS-CoV-2 without cross-reacting with the most closely related human coronavirus, SARS-CoV. Clinical evaluation of nasal swab samples from suspected SARS-C ...[more]

PMID: 33147214

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Project description:Cystic echinococcosis (CE) or hydatidosis, caused by the larval stage of Echinococcus granulosus (EG)-complex, is a neglected parasitic disease of public health importance. The disease is endemic in many African and Mediterranean countries including the Sudan. The objective of the present study was to develop and evaluate a real-time loop-mediated isothermal amplification (LAMP) assay for simple and rapid detection of CE in humans and domestic live stock in Sudan.A set of six LAMP primers, designed from the mitochondrial NADH-1 gene of EG cattle strain of genotype 5 (G5), was used as a target for LAMP assay. The assay was performed at a constant temperature (63 °C), with a real-time follow-up using a LightCycler and fluorochrome dye. Following amplification cycles in a simple water bath, LAMP products were observed for color change by naked eye and were visualized under UV light source using agarose gel electrophoresis.The real-time LAMP assay identified a variety of hydatid cysts strains recovered in the Sudan, including Echinococcus canadenses (G6) and Echinococcus ortleppi (G5). Real-time LAMP positive results were detected by the presence of an amplification curve, whereas negative results were indicated by absence of fluorescence detection. Positive LAMP results appeared as a bluish-colored reaction as observed by naked eye, whereas negative LAMP results were observed as purple-colored reaction. The sensitivity studies indicated that the LAMP assay detected as little as a 10 fg of parasite DNA. There was 100 % agreement between results of the LAMP assay and our previously described nested PCR when testing 10-fold serial dilution of DNA extracted from EG-complex hydatid cyst. However, there was no cross-reactivity with other parasites including cysticercus bovis, Fasciola gigantica, and Schistosoma bovis and nucleic acid free samples.The developed LAMP assay would be expected to prove highly significant in epidemiological surveys of CE in developing countries or areas of resource-poor settings for both ease of use and cost.

Dataset Information

Development and evaluation of a rapid and simple diagnostic assay for COVID-19 based on loop-mediated isothermal amplification.

Publications

Development and evaluation of a rapid and simple diagnostic assay for COVID-19 based on loop-mediated isothermal amplification.

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