This study presents a rapid and versatile low-cost sample-to-answer system for SARS-CoV-2 diagnostics. The system integrates the extraction and purification of nucleic acids, followed by amplification via either reverse transcription-quantitative polymerase chain reaction (RT-qPCR) or reverse transcription loop-mediated isothermal amplification (RT-LAMP). By meeting diverse diagnostic and reagent needs, the platform yields testing results that closely align with those of commercial RT-LAMP and RT‒qPCR systems. Notable advantages of our system include its speed and cost-effectiveness. The assay is completed within 28 min, including sample loading (5 min), ribonucleic acid (RNA) extraction (3 min), and RT-LAMP (20 min). The cost of each assay is ≈ $9.5, and this pricing is competitive against that of Food and Drug Administration (FDA)-approved commercial alternatives. Although some RNA loss during on-chip extraction is observed, the platform maintains a potential limit of detection lower than 297 copies. Portability makes the system particularly useful in environments where centralized laboratories are either unavailable or inconveniently located. Another key feature is the platform's versatility, allowing users to choose between RT‒qPCR or RT‒LAMP tests based on specific requirements.

• Keywords
• Chemistry, Electrical and electronic engineering, Microfluidics,
• Publication type
• Journal Article MeSH

Since its invention in 1986, the polymerase chain reaction (PCR), has become a well-established method for the detection and amplification of deoxyribonucleic acid (DNA) with a specific sequence. Incorporating fluorescent probes, known as TaqMan probes, or DNA intercalating dyes, such as SYBR Green, into the PCR mixture allows real-time monitoring of the reaction progress and extraction of quantitative information. Previously reported real-time PCR product detection using intercalating dyes required melting curve analysis (MCA) to be performed following thermal cycling. Here, we propose a technique to perform dynamic MCA during each thermal cycle, based on a continuous fluorescence monitoring method, providing qualitative and quantitative sample information. We applied the proposed method in multiplexing detection of hepatitis B virus DNA and complementary DNA of human immunodeficiency virus as well as glyceraldehyde 3-phosphate dehydrogenase in different concentration ratios. We extracted the DNA melting curve and its derivative from each PCR cycle during the transition from the elongation to the denaturation temperature with a set heating rate of 0.8 K·s-1and then used the data to construct individual PCR amplification curves for each gene to determine the initial concentration of DNA in the sample. Our proposed method allows researchers to look inside the PCR in each thermal cycle, determining the PCR product specificity in real time instead of waiting until the end of the PCR. Additionally, the slow transition rate from elongation to denaturation provides a dynamic multiplexing assay, allowing the detection of at least three genes in real time.

• Publication type
• Journal Article MeSH