Portable and accurate diagnostics for COVID-19: Combined use of the miniPCR thermocycler and a well-plate reader for SARS-CoV-2 virus detection

Abstract

<jats:p>The coronavirus disease 2019 (COVID-19) pandemic has crudely demonstrated the need for massive and rapid diagnostics. By the first week of July, more than 10,000,000 positive cases of COVID-19 have been reported worldwide, although this number could be greatly underestimated. In the case of an epidemic emergency, the first line of response should be based on commercially available and validated resources. Here, we demonstrate the use of the miniPCR, a commercial compact and portable PCR device recently available on the market, in combination with a commercial well-plate reader as a diagnostic system for detecting genetic material of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causal agent of COVID-19. We used the miniPCR to detect and amplify SARS-CoV-2 DNA sequences using the sets of initiators recommended by the World Health Organization (WHO) for targeting three different regions that encode for the N protein. Prior to amplification, samples were combined with a DNA intercalating reagent (i.e., EvaGreen Dye). Sample fluorescence after amplification was then read using a commercial 96-well plate reader. This straightforward method allows the detection and amplification of SARS-CoV-2 nucleic acids in the range of ~625 to 2X105 DNA copies. The accuracy and simplicity of this diagnostics strategy may provide a cost-efficient and reliable alternative for COVID-19 pandemic testing, particularly in underdeveloped regions where RT-QPCR instrument availability may be limited. The portability, ease of use, and reproducibility of the miniPCR makes it a reliable alternative for deployment in point-of-care SARS-CoV-2 detection efforts during pandemics.</jats:p>

Scaling diagnostics in times of COVID-19: Colorimetric Loop-mediated Isothermal Amplification (LAMP) assisted by a 3D-printed incubator for cost-effective and scalable detection of SARS-CoV-2

“…The sequences of our LAMP primers are presented in Table 1. The sequences of the PCR primers (N1) have been reported elsewhere[24,53].…”

“…To finalize our analysis, we simulated some real-time amplification experiments to compare the performance of LAMP and PCR in similar conditions (Figure 6A). To that end, we conducted amplification reactions using initial amounts of 4 × 10 4 copies of synthetic SARS-CoV2 in a commercial miniPCR cycler[24,51] (using primer set N1) and in our LAMP 3D-printed incubator (using primer set β). We added the intercalating agent, EvaGreen ® Dye, to the reaction mix at the initial time and extracted samples after 0, 7, 14, 21, 28, 35, 42, and 51 minutes.…”

“…These samples form PCR and LAMP experiments were dispensed in 96-weel plates. The fluorescence from these samples was then measured in a commercial plate reader[24] (Figure 5C). We observed an exponential increase in fluorescence as more LAMP or PCR cycles were performed, which highlights the quantitative nature of the intercalating reaction.…”

Biomedical Science to Tackle the COVID-19 Pandemic: Current Status and Future Perspectives

“…To date, several publications have detailed molecular methods and medical tools to accelerate the detection process of the coronavirus responsible of COVID-19 pandemic [ 3 , 7 , 174 ]. For instance, Chu et al developed a single-step real-time reverse-transcription polymerase chain reaction (RT-qPCR) assay to detect two different regions of the viral genome [ 175 ] while González-González et al [ 176 ] found no significant differences when they compared the efficiency of a mini-PCR equipment to its lab-bench counterpart. On the other hand, the same group designed a 3D-printed incubator for a faster amplification of the COVID-19 genome using loop-mediated isothermal amplification (LAMP) reaction that is detected using qRT-PCR [ 177 ].…”

COVID-19 en Latinoamérica: una revisión sistemática de la literatura y análisis bibliométrico

“…Solo se encontró un estudio que abordó los aspectos terapéuticos de la de la enfermedad y, además, aportaba, el mayor nivel de evidencia (ensayo clínico aleatorizado, doble-ciego) de todos los reportados en esta revisión (36). Cuatro investigaciones de ciencias básicas fueron publicadas en la región (37)(38)(39)(40).…”