The need for tools that facilitate rapid detection and continuous monitoring of SARS-CoV-2 variants of concern (VOCs) is greater than ever, as these variants are more transmissible and therefore increase the pressure of COVID-19 on healthcare systems. To address this demand, we aimed at developing and evaluating a robust and fast diagnostic approach for the identification of SARS-CoV-2 VOC-associated spike gene mutations. Our diagnostic assays detect the E484K and N501Y single-nucleotide polymorphisms (SNPs) as well as a spike gene deletion (HV69/70) and can be run on standard laboratory equipment or on the portable rapid diagnostic technology platform peak PCR. The assays achieved excellent diagnostic performance when tested with RNA extracted from culture-derived SARS-CoV-2 VOC lineages and clinical samples collected in Equatorial Guinea, Central-West Africa. Simplicity of usage and the relatively low cost are advantages that make our approach well suitable for decentralized and rapid testing, especially in resource-limited settings.

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We report improvements on the heat transfer and on the control strategy of a thermal cycler implemented on a novel device for performing a fast polymerase chain reaction (PCR). The reduction of the thermal mass of the sample holder and the direct contact with the sample allow for a significant reduction of the transition times, while the hybrid feedforward/feedback controller can rely on inexpensive components (actuators, sensors, processor) and still achieve minimal over/undershooting and good temperature stability. The design of the device has been improved by performing transient heat conduction analysis on the highly heat-conductive sample holder and on the solid metal body of the device which rapidly dissipates the excess heat produced during the thermal cycling. In the current setup, the sample holder hosts nine 1 μL samples covered with mineral oil, which can be simultaneously read in real time by the detection module designed in-house and installed on the device. An increase in speed of the PCR amplification was achieved with a reduction of the transition time of 63.8% when compared against a commercial real-time PCR machine. Our work shows that a complete, stand-alone, and ready-to-use quantitative PCR instrument can be fabricated from inexpensive and easily available components and it can achieve fast thermal cycling thanks to a hybrid control strategy.