Development of Multiplex One-step Point-of-Care Diagnostic Test for SARS- CoV-2 Using Portable Biomeme Thermocycler

Abstract

Coronavirus disease-19 (COVID-19), caused by Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), poses a critical public health threat due to the high transmissibility, infectivity, and prolonged incubation in humans. The urgent demand for swift and efficient detection assays during the pandemic led to the establishment of Point-of-Care Testing (POCT) methods using RT-qPCR for SARS-CoV-2 detection. However, an accurate and portable diagnostic method for SARS-CoV-2 remains to be developed. To address this challenge, we developed the multiplex one-step RT-qPCR for POC 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 POCT 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 multiple SARS-CoV-2 variants, including 19A, 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 a clinical sensitivity of 95% and a specificity of 100%. Therefore, this study reports a POCT method for the prompt and reliable molecular diagnosis of SARS-CoV-2 in resource-limited settings.