The new coronavirus infection (COVID-19) is a major public health concern, with a high burden and risk for infection among patients and healthcare workers. Saliva droplets containing SARS-COV-2 are a major vector for COVID-19 infection, making saliva a promising alternative for COVID-19 testing using nasopharyngeal swab samples. To diagnose COVID-19 patients in the field, a point-of-care test (POCT) using saliva was conceptualized. We have developed a simple method for extracting RNA from saliva samples using semi-alkaline proteinase, a sputum homogenizer typically used for preparing samples for tuberculosis testing, and a subsequent simple heating step with no need for centrifugation or RNA extraction. Further, we newly developed a triplex reverse transcription loop-mediated isothermal amplification approach (RT-LAMP) which utilizes colorimetric readout using a heat block, with results evaluated with the unaided eye. In 44 clinical patients suspected of having COVID-19 infection, the test took 45 minutes, and resulted in a diagnostic sensitivity of 82.6% (19/23) and diagnostic specificity of 100% (21/21), compared to the reference standard. The limit of detection was 250 copies/reaction (25,000 copies/ml). Our newly developed POCT approach achieved simple RNA extraction and constant RT-LAMP detection. This POCT has the potential to be used for simple inspection stations in a field setting, helping reduce the risk of infection by simplifying and accelerating testing for COVID-19.

Significance and Impact of the Study: We developed and evaluated a point-of-care test (POCT) with a combination of new rapid DNA extraction method and real-time PCR and LAMP detection suitable for field settings that does not require advanced laboratory equipment and expensive DNA extraction kits. The developed POCT achieves crude DNA extraction within 10 min at extremely low cost, and high diagnostic performance under combined use of real-time PCR and LAMP analyses. This economical, easy, rapid, highly sensitive and specific POCT promises to provide an important new tool for routine veterinary medicine as well as zoonotic diseases in a field setting.

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To diagnose COVID-19 patients in the field, a sensitive point-of-care test using saliva was developed. Using a heat block without centrifuge, the test took 45 minutes. Naked eye judgement with color change dye outperformed the reference standard, with a diagnostic sensitivity of 82.6% (19/23) and diagnostic specificity of 100% (21/21).

Highly sensitive detection of pathogens is effective for screening meat during quarantine inspection and export. The "micro-amount of virion enrichment technique" (MiVET) was recently developed, which is a new method combining virus concentration with immunomagnetic beads and simple RNA extraction with sodium dodecyl benzenesulfonate (SDBS) for the specific and sensitive detection of avian influenza viruses (AIVs). AIV subtypes H3N2 and H4N2 were used to spike the surface of chicken breast meat samples. The modified MiVET protocol was tested by comparing it against three different homogenate preparation conditions, as well as in samples with added α-amylase and collagenase to digest inhibitors. The performance of the modified MiVET was evaluated by real-time RT-PCR assay targeting the matrix gene. Compared with conventional RNA extraction, the modified MiVET reproducibly concentrated AIVs in chicken meat samples with 100-1000-fold improvement by 60 s-hand homogenization. The 30 s-and 60 s-4 2 1 2 3 4stomacher homogenizations resulted 100-fold and 10-100-fold improvement, respectively. The modified MiVET required < 60 min from homogenate preparation to final RNA elution. Further, use of the modified MiVET also decreased the rate of false-negative results. The modified MiVET is effective for the rapid and highly sensitive detection of AIVs in chicken meat samples, and can be applied to quarantine and export inspection at airports and seaports.