Machine Learning-Assisted Real-Time Polymerase Chain Reaction and High-Resolution Melt Analysis for SARS-CoV-2 Variant Identification.

Since the declaration of COVID-19 as a pandemic in early 2020, multiple variants of the severe acute respiratory syndrome-related coronavirus (SARS-CoV-2) have been detected. The emergence of multiple variants has raised concerns due to their impact on public health. Therefore, it is crucial to distinguish between different viral variants. Here, we developed a machine learning web-based application for SARS-CoV-2 variant identification via duplex real-time polymerase chain reaction (PCR) coupled with high-resolution melt (qPCR-HRM) analysis. As a proof-of-concept, we investigated the platform's ability to identify the Alpha, Delta, and wild-type strains using two sets of primers. The duplex qPCR-HRM could identify the two variants reliably in as low as 100 copies/µL. Finally, the platform was validated with 167 nasopharyngeal swab samples, which gave a sensitivity of 95.2%. This work demonstrates the potential for use as automated, cost-effective, and large-scale viral variant surveillance.

A case series report on successful management of patients with COVID-19-associated lymphopenia and potential application of PG2.

Background: Lymphopenia and the resultant high neutrophil-to-lymphocyte ratio (NLR) are hallmark signs of severe COVID-19, and effective treatment remains unavailable. We retrospectively reviewed the outcomes of COVID-19 in a cohort of 26 patients admitted to Chung Shan Medical University Hospital (Taichung City, Taiwan). Twenty-five of the 26 patients recovered, including 9 patients with mild/moderate illness and 16 patients with severe/critical illness recovered. One patient died after refusing treatment. Case presentation: We report the cases of four patients with high NLRs and marked lymphopenia, despite receiving standard care. A novel injectable botanical drug, PG2, containing Astragalus polysaccharides, was administered to them as an immune modulator. The decrease in the NLR in these four patients was faster than that of other patients in the cohort (0.80 vs. 0.34 per day). Conclusion: All patients recovered from severe COVID-19 showed decreased NLR and normalized lymphocyte counts before discharge. Administration of PG2 may be of benefit to patients with moderate to severe COVID-19 and lymphopenia.

Rapid electrochemical detection of coronavirus SARS-CoV-2.

Coronavirus disease 2019 (COVID-19) is a highly contagious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Diagnosis of COVID-19 depends on quantitative reverse transcription PCR (qRT-PCR), which is time-consuming and requires expensive instrumentation. Here, we report an ultrasensitive electrochemical biosensor based on isothermal rolling circle amplification (RCA) for rapid detection of SARS-CoV-2. The assay involves the hybridization of the RCA amplicons with probes that were functionalized with redox active labels that are detectable by an electrochemical biosensor. The one-step sandwich hybridization assay could detect as low as 1 copy/µL of N and S genes, in less than 2 h. Sensor evaluation with 106 clinical samples, including 41 SARS-CoV-2 positive and 9 samples positive for other respiratory viruses, gave a 100% concordance result with qRT-PCR, with complete correlation between the biosensor current signals and quantitation cycle (Cq) values. In summary, this biosensor could be used as an on-site, real-time diagnostic test for COVID-19.

Rapid Electrochemical Detection of Coronavirus SARS-CoV-2. (preprint)

COVID-19 is a highly contagious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Diagnosis of COVID-19 depends on quantitative reverse transcription PCR (qRT-PCR), which is time-consuming and requires expensive instrumentation. Here, we report an ultrasensitive electrochemical biosensor based on isothermal rolling circle amplification (RCA) for rapid detection of SARS-CoV-2. The assay involves the hybridization of the RCA amplicons with probes that were functionalized with redox active labels that are detectable by an electrochemical biosensor. The one-step sandwich hybridization assay could detect as low as 1 copy/mL of N and S genes, in less than 2 hours. Sensor evaluation with 105 clinical samples, including 40 SARS-CoV-2 positive and 9 samples positive for other respiratory viruses, gave a 100% concordance result with qRT-PCR, with complete  correlation between the biosensor current signals and quantitation cycle (Cq) values. In summary, this biosensor could be used as an on-site, real-time diagnostic test for COVID-19.