key: cord-0430491-89q0smn0
authors: Ferreira, B. I. d. S.; Gomes, N. L. d. S.; Coelho, W. L. d. C. N. P.; Costa, V. D.; Carneiro, V. C. d. S.; Kader, R. L.; Amaro, M. P.; Villar, L. M.; Miyajima, F.; Alves-Leon, S. V.; de Paula, V. S.; Leon, L. A. A.; Moreira, O. C.
title: Validation of a Novel Molecular Assay to the Diagnostic of COVID-19 Based on Real Time PCR with High Resolution Melting
date: 2021-07-18
journal: nan
DOI: 10.1101/2021.07.14.21260471
sha: 1c44f0d8c4acebd1e96231bb63abe23a69e3e6a9
doc_id: 430491
cord_uid: 89q0smn0

With the emergence of the Covid-19 pandemic, the world faced an unprecedented need for RT-qPCR-based molecular diagnostic tests, leading to a lack of kits and inputs, especially in developing countries. Hence, the costs for commercial kits and inputs were overrated, stimulating the development of alternative methods to detect SARS-CoV-2 in clinical specimens. The availability of the complete SARS-CoV-2 genome at the beginning of the pandemic facilitated the development of specific primers and standardized laboratory protocols for Covid-19 molecular diagnostic. High-sensitive and cost-effective molecular biology technique based on the Melting Temperature differences between purine and pyrimidine bases can be used to the detection and genotyping of pathogens in clinical specimens. Here, a RT-qPCR assays with High Resolution Melting (HRM-RTqPCR) was developed for different regions of the SARS-CoV-2 genome (RdRp, E and N) and an internal control (human RNAse P gene). The assays were validated using synthetic sequences from the viral genome and clinical specimens (nasopharyngeal swabs, serum and saliva) of sixty-five patients with severe or moderate COVID-19 from different states in Brazil, in comparison to a commercial TaqMan RT-qPCR assay, as gold standard. The sensitivity of the HRM-RTqPCR assays targeting N, RdRp and E were 94.12, 98.04 and 92.16%, with 100% specificity to the 3 targets, and diagnostic accuracy of 95.38, 98.46 and 93.85%, respectively. Thus, the HRM-RTqPCR emerges as an alternative and low-cost methodology to increase the molecular diagnostic of patients suspicious for Covid-19, especially in restricted-budget laboratories.

TaqMan assays or DNA sequencing to the molecular diagnostic or pathogens genotyping 100 from clinical samples. 101 In this study, real-time RT-PCR assays with High Resolution Melting were 102 developed for different regions of the SARS-CoV-2 genome (RdRp, E and N) and an 103 internal control (human RNAse P gene). The assays were standardized and validated 104 using synthetic sequences from the viral genome and clinical specimens (nasopharyngeal 105 swabs, serum and saliva) of patients with clinical suspicion of COVID-19 from different 106 states of Brazil, in comparison to TaqMan RT-qPCR assays, as gold standard. Therefore, 107 we aimed to obtain an sensitive and specific assay to COVID-19 diagnosis as an 108 alternative to TaqMan assay to overcome high costs and the limited supplies that might 109 delay accurate diagnosis. women and with cancer were excluded from this study. 128 To the nasopharyngeal samples collection, swab was inserted through the nostril 129 with a rotation movement until the nasopharynx was reached, and the sample was 130 obtained by rotating the swab gently for 2-3 seconds. Then, the swab was placed into a 5 131 ml tube containing 3 ml viral transport medium (VTM). After transportation to the 132 laboratory, tubes containing the swabs were vortexed vigorously, VTM was transferred 133 to 1.5mL tubes and stored at -80°C until RNA extraction. 134 To the saliva samples collection, patients were asked to self-collect the saliva in a 135 sterile dry tube, closing the lid after placing the saliva in it. The staff cleaned the outside 136 of the tube with 1/10 diluted bleach-impregnated cloth, after taking the container while 137 wearing gloves. After taking both samples, they were delivered to the laboratory inside 138 the triple transport system. 139 To the serum samples collection, around five milliliters of blood were harvested 140 in a BD Vacutainer® Plus Plastic Serum tube and remained resting for 30 min at room 141 temperature, to generate the clot from blood cells and the serum phase. After that, tubes 142 were centrifuged at 1,000 x g for 10 minutes at 4 °C and serum was collected and stored 143 at -80°C until use. To select the primers for HRM-RTqPCR assays ( 

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The copyright holder for this preprint this version posted July 18, 2021. and E regions of SARS-CoV-2 from the Severe acute respiratory syndrome coronavirus 213 2 isolate Wuhan-Hu-1 (GenBank NC_045512.2). To the linearity assay, a 1:10 serial 214 dilution of the synthetic DNA was performed from 10 6 to 1 copies/µL in TE buffer. To 215 the precision assay, three concentrations of the synthetic SARS-CoV-2 templates were 216 used (12, 10 and 8 copies/µL). Forty technical replicates were assayed in each 217 concentration to the same operator in the same day, and the results were compared. To 218 the Reproducibility assay, the same three concentrations of the synthetic sequences, at 12, 219 10 and 8 copies/µL, were also used. The amplification was performed in 40 technical 220 replicates for each template concentration, divided in two consecutive days, by the same 221 operator.

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Primers selection 236 To select the appropriate primers to develop the HRM-RTqPCR assays, the primers 237 designed for COVID-19 molecular diagnostic described by WHO 8 were evaluated based 238 on the Tm similarity between primer pairs, low hairpin, self-dimer and hetero-dimer 239 formation tendency, small amplicon size (below 120 bp), and PCR efficiency. Therefore, (Table 1) . Those primers were selected based on the Tm similarity between 245 primer pairs, low hairpin, self-dimer and hetero-dimer formation tendency and small 246 amplicon size (below 120 bp).

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The copyright holder for this preprint this version posted July 18, 2021. ; https://doi.org/10.1101/2021.07.14.21260471 doi: medRxiv preprint 248 Analytical Validation of HRM-RTqPCR assays 249 After standardization, HRM-qPCR assays were initially validated using synthetic DNA 250 sequences of SARS-CoV-2 (GenBank NC_045512.2). A single and sharp peak at the 251 derivative HRM curve was generated to each target, with Tm 80.8, 83.5, 77.6 and 86°C 252 to N, RdRp, E and RNAse P, respectively (Table 1 CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted July 18, 2021. ; https://doi.org/10.1101/2021.07.14.21260471 doi: medRxiv preprint were compared (Table 2) target, the medians of Ct values for days one and two, at 12 copies/µL, were respectively 295 28.58 and 28.93. At 10 copies/µL, the medians were 28.25 and 28.62 and, at 8 copies/µL, 296 the medians were 28.42 and 28.61, respectively. It was possible to observe a little 297 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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dispersion of Ct values in the replicates, and a small number of outliers in all 298 concentrations tested. In addition, no significant difference was observed in Ct values 299 between day 1 and day 2 for all the targets, which means a good reproducibility of the 300 HRM-qPCR assays. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted July 18, 2021. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted July 18, 2021. ; https://doi.org/10.1101/2021.07.14.21260471 doi: medRxiv preprint outliers were observed. In all comparisons, those parameters represent a very good 348 agreement between the assays, which resulted in a performance of the HRM-RTqPCR 349 similar to the gold standard TaqMan RTqPCR assay. 

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The copyright holder for this preprint this version posted July 18, 2021. ; https://doi.org/10.1101/2021.07.14.21260471 doi: medRxiv preprint Based on the TaqMan in-house assays, we first evaluated all the primers 373 sequences in silico, selecting three primers pairs targeting the N, RdRp and E regions of 374 SARS-CoV-2 genome. As the main objective of this study was to develop HRM-375 RTqPCR assays to the molecular detection of the virus, regardless the mutations 376 accumulated by the variants of concern, it was important to select the most stable genes 377 to the diagnostic assays, to avoid the presence of insertions, deletions or SNPs that can 378 lead to primer mismatches or differences in the shape of the HRM curves, used to certify 379 the specificity of the RT-qPCR amplifications. compared between day one and day two for each target, but no statistical difference was 412 observed (Fig. 2) , assuring the good reproducibility of the method.

The clinical validation of the HRM-RTqPCR assays was performed with 65 414 samples, mostly nasopharyngeal swabs, but also saliva and serum. The human RNAse P CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted July 18, 2021. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted July 18, 2021. ; https://doi.org/10.1101/2021.07.14.21260471 doi: medRxiv preprint Table 1 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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Natural History, Pathobiology, and Clinical Manifestations of SARS-CoV-2

The species Severe acute respiratory syndrome-related coronavirus: 492 classifying 2019-nCoV and naming it SARS-CoV-2

World 495 Health Organization 2021