Le, U. P. | Search bioRxiv

bioRxiv2020

Direct colorimetric LAMP assay for in-field detection of African swine fever virus: a validation study during an outbreak in Vietnam

African Swine Fever (ASF) is a highly infectious viral disease with high mortality. The most recent ASF outbreak in Vietnam occurred in 2019, posing a threat to spread to the neighboring Asian countries. Without a commercial vaccine or efficient chemotherapeutics successfully developed, rapid diagnosis and necessary biosecurity procedures are required to control the disease. While the diagnosis method of ASF recommended by the World Organization of Animal Health is real-time PCR, it is not suitable for in-field detection of the disease. In this study, a colorimetric Loop-Mediated Isothermal Amplification (LAMP) assay was developed and evaluated for ASF virus detection using crude serum samples collected from domestic pigs in Vietnam during the 2019 outbreak. The LAMP results can be readily visualized to naked eyes within 30 minutes without the requirement of DNA extraction and sophisticated equipment. The sensitivity, specificity, and limit of detection of colorimetric LAMP assay were comparable to a commercial diagnostic real-time PCR kit. Results strongly indicate that the developed colorimetric LAMP assay is highly recommended for the in-field diagnosis of ASF.

molecular biology↗

bioRxiv2020

A comparative study of isothermal nucleic acid amplification methods for SARS-CoV-2 detection at point of care

COVID-19, caused by the novel coronavirus SARS-CoV-2, has spread worldwide and put most of the world under lockdown. Despite that there have been emergently approved vaccines for SARS-CoV-2, COVID-19 cases, hospitalizations, and deaths have remained rising. Thus, rapid diagnosis and necessary public health measures are still key parts to contain the pandemic. In this study, the colorimetric isothermal nucleic acid amplification tests (iNAATs) for SARS-CoV-2 detection based on loop-mediated isothermal amplification (LAMP), cross-priming amplification (CPA), and polymerase spiral reaction (PSR) were designed and evaluated. The three methods showed the same limit of detection (LOD) value of 1 copy of the targeted gene per reaction. However, for the direct detection of SARS-CoV-2 genomic-RNA, LAMP outperformed both CPA and PSR, exhibiting the LOD value of roughly 43.14 genome copies/reaction. The results can be read with the naked eye within 45 minutes, without cross-reactivity to closely related coronaviruses. Moreover, the direct detection of SARS-CoV-2 RNA in simulated patient specimens by iNAATs was also successful. Finally, the ready-to-use lyophilized reagents for LAMP reactions were shown to maintain the sensitivity and LOD value of the liquid assays. The results indicate that the colorimetric lyophilized LAMP kit developed herein is highly suitable for detecting SARS-CoV-2 nucleic acids at point-of-care.

molecular biology↗