Development of a Multiplex One-step RT-qPCR Diagnostic Test for SARS- CoV-2 Using a Portable Biomeme Thermocycler

초록

Coronavirus disease-19 (COVID-19), caused by Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), poses a critical public health threat due to its high trans- missibility, infectivity, and prolonged incubation in humans. Timely identification of SARS-CoV-2 is crucial to control COVID-19 in the lack of effective antiviral medications in low resource regions. However, an accurate assay and portable diag- nostic method for SARS-CoV-2 remains to be developed. To address this issue, we developed a multiplex one-step reverse transcription quantitative polymerase chain reaction (RT-qPCR) for diagnosis of SARS-CoV-2 using a portable Biomeme Franklin™ Real-Time PCR thermocycler. The performance of the Biomeme assay was evaluated and validated for the testing of SARS-CoV-2 based on multiplex detection of the nucleocapsid, envelope, and spike genes. The Biomeme assay demonstrated high sensitivity by detecting the RNA of wild type 19 A and multiple SARS-CoV-2 variants, including B.1.617.2, BA.1, BA.2, BA.2.75, BA.5, and BN.1 from 0.01 ng/µl of total RNA and showed no cross-reactivity with other human coronaviruses, such as HCoV-OC43, HCoV-NL63, and HCoV-229E. The diagnostic method exhibited an analytical sensitivity of 95% and a specificity of 100%. The Biomeme Franklin platform generates rapid results within 3-4 h and allows smartphone-based output quantification, making it highly suitable as a Point of Care (POC) diagnostic tool. Therefore, this study reports on a diagnostic testing method for the prompt and reliable molecular diagnosis of SARS-CoV-2.

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