key: cord-0687254-avo5jfaj authors: Gonzalez-Gonzalez, Everardo; Trujillo-de Santiago, Grissel; Lara-Mayorga, Itzel Montserrat; Martinez-Chapa, Sergio Omar; Alvarez, Mario Moises title: Portable and accurate diagnostics for COVID-19: Combined use of the miniPCR thermocycler and a well-plate reader for SARS-CoV-2 virus detection date: 2020-04-07 journal: nan DOI: 10.1101/2020.04.03.20052860 sha: a0530745a1b48b0a45d93155f9878f561ce5778e doc_id: 687254 cord_uid: avo5jfaj The COVID-19 pandemic has crudely demonstrated the value of massive and rapid diagnostics. By the first week of April, more than 900,000 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 combined use of the miniPCR, a commercial compact and portable PCR device recently available on the market, and a commercial well-plate reader as a diagnostic system for detecting SARS-CoV-2 nucleic acids. We used the miniPCR to detect and amplify SARS-CoV-2 DNA sequences using the sets of initiators recommended by the World Health Organization 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 2 x 105 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. Many methodologies have been proposed to deliver cost-effective and accurate diagnosis 55 (i.e., methods based on immunoassays or specific gene hybridization [15, 16] ); however, 56 nucleic acid amplification, and particularly real-time quantitative PCR (RT-qPCR), 57 continues to be the gold standard for the detection of viral diseases in early stages [17, 18] . CC-BY-NC-ND 4.0 International license It is made available under a author/funder, who has granted medRxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) The copyright holder for this preprint . CC-BY-NC-ND 4.0 International license It is made available under a author/funder, who has granted medRxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) The copyright holder for this preprint . (see Fig 1D) consisting of a denat ration sta e at for 5 min, followed y 25 cycles of 136 94 °C for 20s, 60 °C for 30s, and 72 °C for 20s, and then a final stage at 72 °C for 5 min, 137 for a total duration of 60 minutes in the miniPCR® thermocycler. In some of our experiments, we also used the blueGel unit, a portable electrophoresis unit 147 sold by MiniPCR from Amplyus (MA, USA). This is a compact electrophoresis unit (23 × 148 10 × 7 cm) that weighs 350 g. In these experiments, we analyzed 10 μL of PCR product 149 using 2% agarose electrophoresis tris-borate-EDTA buffer (TBE). Gels were dyed with 150 Gel-Green (CA, USA) using a 1:10,000 dilution, and a current of 48 V was supplied by the 151 blueGel built-in power supply (AC 100-240 V, 50-60 Hz). 152 As a third method of detection and to read the amplification product, we evaluated the 153 amplification products by detecting the fluorescence emitted by a DNA intercalating agent, 154 the EvaGreen® Dye, in the Synergy HT microplate reader (BioTek Instruments, VT, USA). 155 Briefly, 20 μL of the PCR reaction mix was placed in distinct wells of a 96-well plate, after 156 completion of the PCR program. Each well was made to a final volume of 150 μL by 157 adding 130 μL of n clease free water and the samples were well mixed by pipetting. These 158 . CC-BY-NC-ND 4.0 International license It is made available under a author/funder, who has granted medRxiv a license to display the preprint in perpetuity. We conducted a series of experiments to assess the sensitivity of the PCR reactions 173 conducted in the miniPCR thermocycler using the three sets of primers recommended by 174 CDC to diagnose infection by SARS-CoV2. Table 1 shows the sets of primers used to 175 target genetic sequences that code for the expression of the SARS-CoV2 N protein. Table 2 176 shows the sequence of the DNA products (amplicons) generated by successful targeting of 177 these regions with the N1, N2, and N3 primer pairs. 178 material, in the range of 2.0 × 10 5 to 625 DNA copies, were used as reaction templates. If 181 . CC-BY-NC-ND 4.0 International license It is made available under a author/funder, who has granted medRxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) The copyright holder for this preprint . https://doi.org/10.1101/2020.04.03.20052860 doi: medRxiv preprint swabs from patients has been estimated to fall within the range of 10 5 to 10 6 viral copies 183 per mL [19] . The amplification proceeds with sufficient quality to allow proper 184 visualization of the amplification products in electrophoresis gels, even at low nucleic acid 185 concentrations. Figure . CC-BY-NC-ND 4.0 International license It is made available under a author/funder, who has granted medRxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) The copyright holder for this preprint . https://doi.org/10.1101/2020.04.03.20052860 doi: medRxiv preprint quantitatively evaluated using a commercial 96-well plate reader. To do this, we used an 206 intercalating agent during amplification in the miniPCR apparatus. Figure 2a instance, we observed variance coefficients of 2.31%, 2.15%, and 3.34% when using 225 primer sets N1, N2, and N3, respectively. If we considered only fluorescence readings from 226 positive samples, we observed variance coefficients of 2.23%, 2.34%, and 1.31% when 227 using primer sets N1, N2, and N3, respectively. 228 . CC-BY-NC-ND 4.0 International license It is made available under a author/funder, who has granted medRxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) The copyright holder for this preprint . https://doi.org/10.1101/2020.04.03.20052860 doi: medRxiv preprint (i.e., exposed to the same number of cycles). For example, for amplifications using primer 262 set N3, we observe a linear relationship between the natural logarithm of the number of 263 viral copies and the natural logarithm of fluorescence signal for the range of 625 to 40,000 264 . CC-BY-NC-ND 4.0 International license It is made available under a author/funder, who has granted medRxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) The copyright holder for this preprint . https://doi.org/10.1101/2020.04.03.20052860 doi: medRxiv preprint where F o is the fluorescence reading exhibited by a blank (i.e., a negative sample prepared 268 and processed in the same way than the positive samples) and α = 8.897 (as determined by 269 fitting of the data presented in Figure 2C ). For instance, we believe we can adjust the Using the primers and methods described here, we were able to consistently detect the 281 presence of SARS-CoV2 synthetic DNA using a miniPCR and a simple plate reader. In the 282 current context of the COVID-19 pandemics, the importance of communicating this result 283 . CC-BY-NC-ND 4.0 International license It is made available under a author/funder, who has granted medRxiv a license to display the preprint in perpetuity. To further illustrate the deterministic and quantitative dependence between the 302 concentration of amplification product and the fluorescence signal, as measured in a plate 303 reader, we simulated some real-time amplification experiments. To that end, we conducted 304 amplification reactions using initial amounts of 4 × 10 4 copies of synthetic SARS-CoV2 in 305 the miniPCR cycler. We added the intercalating agent, EvaGreen® Dye, to the reaction mix 306 at the initial time and extracted samples after 1, 5, 10, 15, 20, 25, and 30 PCR cycles. The 307 . CC-BY-NC-ND 4.0 International license It is made available under a author/funder, who has granted medRxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) The copyright holder for this preprint . increase in fluorescence as more PCR cycles were performed, which highlights the 309 quantitative nature of the intercalating reaction. 310 Our results suggest that using a commercial plate reader to determine the extent of advance 311 of PCR amplifications is a practical, reliable, reproducible, and robust alternative to the use 312 of gel electrophoresis. Moreover, fluorescence reading of PCR products may lead to precise 313 quantification of viral loads. we corroborate that the miniPCR apparatus is capable of amplifying small amounts of 327 SARS-CoV2 synthetic nucleic acids. We were able to detect and amplify 64 copies of 328 genes encoding for the N protein of SARS-CoV2. 329 . CC-BY-NC-ND 4.0 International license It is made available under a author/funder, who has granted medRxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) The copyright holder for this preprint . https://doi.org/10.1101/2020.04.03.20052860 doi: medRxiv preprint the iterative temperature cycling using a laptop. Despite its compact size, the miniPCR® 331 allows the performance of a full amplification protocol in a similar time as achieved with a 332 conventional thermocycler. The mini-PCR® thermocycler exhibits the essential attributes 333 of a POC system: (a) the use of small volumes, (b) low capital cost, (c) portability, (d) and 334 a fast, accurate, and selective response. 335 Moreover, the combined use of the miniPCR thermocycler and a 96-well plate reader 336 enables the possibility of obtaining immediate readings of the amplification products, 337 thereby providing faster (and potentially quantitative) diagnostic results in shorter times 338 than when gel electrophoresis techniques are used. Therefore, the combined use of these 339 already two commercially available devices-a miniPCR thermocycler and a 96-well plate 340 reader-has great potential for use during epidemic emergencies. Excel data file S1: Excel file that contains raw data 351 352 . CC-BY-NC-ND 4.0 International license It is made available under a author/funder, who has granted medRxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) The copyright holder for this preprint . CC-BY-NC-ND 4.0 International license It is made available under a author/funder, who has granted medRxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) The copyright holder for this preprint . https://doi.org/10.1101/2020.04.03.20052860 doi: medRxiv preprint Point-of-Care Diagnostics for Global Health Emerging Trends in Micro-and Nanoscale Technologies in Medicine: From Basic 358 Discoveries to Translation Emergence 361 of a Novel Swine-Origin Influenza A (H1N1) Virus in Humans Towards detection and 365 diagnosis of Ebola virus disease at point-of-care Towards detection and 377 diagnosis of Ebola virus disease at point-of-care Comparative Evaluation of the Diagnostic Performance of the Prototype Cepheid 381 Innovative technologies for point-of-care testing of viral 384 hepatitis in low-resource and decentralized settings Countries test tactics in "war" against COVID-19 Articles 390 Investigation of three clusters of COVID-19 in Singapore: implications for 391 surveillance and response measures Response to COVID-19 in Taiwan: Big Data 394 New Technology, and Proactive Testing COVID-398 19 epidemic in Switzerland: on the importance of testing Antibodies in Human Serum: A Simple Virus-Free ELISA Method. 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