This study was approved by the Research Ethics Committees of the Industrial University of Santander, the Chicamocha Clinic, and the Chicamocha Clinical Laboratory (L3C) (Bucaramanga, Santander, Colombia). The study participants were patients either hospitalized-diagnosed with SARS-CoV-2 or persons that presented to the emergency room or volunteers. All participants provided written informed consent and voluntarily participated in our study. Further, all participants were kept anonymous and were informed that the present study was conducted strictly for research purposes and that its outcomes were not intended to serve as treatment or diagnosis.

Nasopharyngeal swab samples were acquired by L3C personnel. Two samples were obtained per study participant, one for clinical diagnosis, as authorized by the Ministry of Health and Social Protection of Colombia, and the other for our study. The samples were placed in Universal Transport Medium (UTM) and stored at −80°C until required for downstream analyses ( Rogers et al., 2020). The samples were collected over the course of one week and then appropriately packaged according to the sanitary legislation of Colombia ( Ministry of Health and Social Protection, 2020) prior to their transportation and processing. All samples were shipped to the Central Research Laboratory of the Industrial University of Santander Health Faculty (LCI-FS-UIS) and maintained at 4°C while the informed consents were processed and digitalized.

Afterwards, the samples were aliquoted in a Class II, Type A2 Biosafety Cabinet (Thermo Fisher Scientific). A total of three aliquots were obtained from each sample, including a 200-μL aliquot for total RNA extraction, which was used immediately, and two 650-μL aliquots used as backup samples, which were stored at −80°C.

Publicly available SARS-CoV-2 genome sequences were obtained from the GenBank and GISAID databases (RRID:SCR_002760; RRID:SCR_018251) ( Sayers et al., 2021; Shu & McCauley, 2017). To gather data from the GenBank database, a Python script (RRID:SCR_008394) was executed in the GUANE-1 High Performance and Scientific Computing Center of the Industrial University of Santander (SC3UIS) through the Entrez Programming Utilities interface using the Biopython package (RRID:SCR_013249; RRID:SCR_007173) ( Cock et al., 2009; Geer et al., 2010), whereas all GISAID data were manually downloaded. Genomes with uncharacterized regions/nucleotides were discarded using another Python script (RRID:SCR_008394), which was used to conduct monthly FASTA sequence alignments using the MAFFT software (RRID:SCR_011811) ( Katoh & Standley, 2013) coupled with previously described DNA loss model parameters ( Martínez-Pérez et al., 2002, 2007). The consensus sequences were generated using BioEdit version 7.2 (RRID:SCR_007361) with a 100% threshold frequency ( Hall, 1999).

Using the SARS-CoV-2 (NC_045512) reference genome and consensus sequences from January to April 2020 ( Zhu et al., 2020), an approximately 150-base pair (bp) region of the ORF8 gene was selected based on the secondary structure of its RNA transcript, which in turn was predicted using the algorithms proposed by Zuker ( Zuker & Jacobson, 1998) via the Mflod software (RRID:SCR_001360) ( Zuker, 2003). The obtained sequence was used as a template to create primers, a TaqMan FAM-BBQ probe, and substrate oligos for RT-qPCR. The specificity of the aforementioned molecules was confirmed via GenBank BLAST analyses (RRID:SCR_004870) ( Ye et al., 2006). Genes E and N from the Berlin and CDC protocols were used as controls ( Biotek, 2020; Corman et al., 2020).

All molecules were synthesized by Bioneer (Korea). The primers were purified via separation on a reverse-phase cartridge, whereas the probe and substrate oligos were purified via high-performance liquid chromatography (HPLC) and polyacrylamide gel electrophoresis (PAGE), respectively. The ORF8 RT-PCR conditions were implemented as described by the Berlin protocol ( Corman et al., 2020) using the aforementioned molecules coupled with the 2019-nCoV TaqMan RT-PCR Kit (Norgen Biotek Corp) developed by the CDC ( Biotek, 2020).

A:T and G:C ratios were calculated based on the monthly SARS-CoV-2 consensus sequences to obtain an average for each nucleotide. These averages were then used to determine the minimum concentrations of TEA (ABCAM-USA), DMSO (Scharlab-Spain), and dNTPs (100 mM each, Promega-USA) in molecular-grade ultrapure water (Promega-USA), in addition to the MgSO ₄ concentration recommended by the Berlin protocol.

Total RNA extraction was conducted using the MagMAX™ Viral/Pathogen II (MVP II) Nucleic Acid Isolation Kit (2000 RXNs) (Applied Biosystems-USA) using a KingFisher Duo Prime (5400110) DNA/RNA extraction system according to the manufacturer’s instructions (Thermo Fisher Scientific-USA) ( Fang et al., 2007).

RT-qPCR was conducted using the ORF8-specific primers and probe designed herein, in addition to the E (Berlin protocol) and N genes (N1-N2; CDC protocol). The RNase P gene was used as an external control, as proposed by both of the aforementioned protocols. The reactions were conducted using the SuperScript III One-Step RT-PCR System with Platinum Taq DNA Polymerase (Ref. 11732088; Invitrogen-USA) and the 2019-nCoV TaqMan RT-PCR Kit (Ref. TM67100; Norgen-Biotek-Canada). Each reaction for each diagnostic system was conducted in either 15- or 25-μL reaction volumes consisting of 2 μL of patient-derived purified RNA, 2× One-Step RT-PCR Master Mix, 2× nuclease-free buffer, and the respective primers/probe at the concentrations recommended by the CDC. Moreover, a denaturing stock solution was added to obtain a final concentration of 0.7% TEA, 0.2% DMSO, and 0.8 mMol MgSO ₄. The dNTP reagent had a final concentration of 12 mMol dATP-dTTP, 10 mMol dCTP-dGTP, and 0.8 mMol MgSO ₄. The reaction conditions were the following: 55°C for 15 minutes, 95°C for 3 minutes followed by 45 cycles of 95°C for 15 s and 58°C for 30 s for the SuperScript™ III One-Step RT-PCR kit; and 95°C for 3 s followed by 55°C for 20 s for the 2019-nCoV TaqMan RT-PCR kit. Fluorescence signals were quantified using a QuantStudio 1 Real-Time PCR System (No. A40427) in a 96-well 0.2 μL block (Thermo Fisher Scientific-USA).

The above-described procedure was conducted using two different Multiplex One-Step RT-qPCR protocols. In the first instance, the primers and probes for the E, ORF8, and N (N1) genes were mixed, whereas the other reaction was performed by mixing the N1 and N2 sets of the N gene. The RNase P gene was independently assessed in both cases. All reactions were performed as described above.

A total of 19,317 genomes were retrieved from the GenBank database from January to October 2020 based on our search criteria, whereas the GISAID database rendered 107,259 full genomes from January to December 2020. In both cases, the number of available sequences increased substantially each month. However, the frequency of base substitutions in the monthly consensus sequences for the E, ORF8, and N genes was higher in the GISAID database compared to the GenBank database ( Figure 1).

The region of the E gene employed herein exhibited a 113 bp length, whereas the amplicons of the N1-N2 system of the N gene were 72 and 67 bp in length, respectively, all of which exhibited loop-bubble structures. Interestingly, 154 bp of the first half of the ORF8 gene presented a loop-bubble structure, which was similar to that of the other two genes. Nevertheless, the third loop of the N1 system and the second loop of the ORF8 gene were formed via four and seven canonical pairings, respectively. These structures can be considered scorpion-type primers or probes, which are often used in RT-qPCR; however, the loop-bubble structures used for these purposes are typically formed via 2–6 canonical pairings ( Figure 2). Table 1 summarizes the primer sequences used in this study.

A total of 49 samples were collected from October 25, 2020, to January 21, 2021, of which 22 tested positive and 27 were negative. The October samples were used to confirm that the E, ORF8, and N genes could be used as effective indicators to detect SARS-CoV-2 via RT-qPCR ( Table 2, Figure 3). However, given that our secondary structure and nucleotide ratio analyses indicated that the A:T ratio was always between 63% and 70% regardless of the consensus sequence, denaturing and dNTP solutions were also incorporated in these reactions (see the Methods for more details). These conditions generally improved the Ct values of the reactions compared to those of the commercial procedures; however, some exceptions were observed ( Table 2, Figure 3).

Given that all samples were freshly acquired and never stored, we concluded that the gradual loss of amplicon systems and adjuvant solutions began in December. This was confirmed first for the E gene system, then for the N2 system, and finally for ORF8. This trend was determined using ten samples collected from December 14 to 21, 2020. The results of our experiments could be generally classified into three outcomes depending on the viral detection system: (1) the system worked following the manufacturer’s instructions with the addition of the denaturing solution (2); the system rendered positive results with the denaturing solution but negative without it; and (3) the opposite of the second outcome ( Table 2).

Our analysis of the global SARS-CoV-2 mutation patterns based on the GenBank and GISAID databases indicated that most mutations began in March 2020 and increased thereafter by a factor of millions or even thousands of millions. This trend was particularly noticeable for the quencher and forward primer sequences of the ORF8 and N genes. Moreover, the potential primer and probe combinations for each gene increased substantially starting in November according to the GISAID database and were significantly higher in December. For example, the ORF8 system and the N1 set exhibited 3.4×10 ¹¹ and 1.7×10 ¹² combinations, respectively, whereas the N2 set and E had only 1.4×10 ⁵ and 32 combinations ( Figure 1).

The multiplex RT-qPCR controls of systems E, ORF8, and N (N1) with the positive samples 02 and 03 rendered the expected results, which were thereafter confirmed using samples 05 and 06 obtained in October 2020. Nevertheless, using the same reaction conditions, the denaturing solution decreased the Ct values for the positive samples obtained in October but enhanced those of the samples obtained in November. In contrast, the dNTP solution had the opposite effect ( Table 3, Figure 4).

The influence of the denaturing and dNTP solutions was further confirmed using nine samples obtained from December 4 to 7, 2020, and from January 14 to 15, 2021, using the N1–N2 primers and probe as described by the CDC. A total of six samples exhibited the originally documented pattern and the three remaining samples tested negative, both in terms of curve trends and Ct values ( Table 3, Figure 5). Therefore, the initial multiplex reaction effectively detected the SARS-CoV-2 virus in the nine samples obtained from January 19 to 29, 2021 ( Table 3).

This genomic sequence is available in GenBank:

GenBank: Severe acute respiratory syndrome coronavirus 2 isolate Wuhan-Hu-1, complete genome. Accession number NC_045512; https://www.ncbi.nlm.nih.gov/nuccore/NC_045512.

GitHub: Steps to download and align genomic sequences from GenBank in the GUANE-1 supercomputer from High Performance and Scientific Computing Center of the Industrial University of Santander (SC3UIS). This software is also used for manually downloaded sequences such as GISAID. For both databases or others, the script eliminates the undetermined sequences indicated with “n” within the genome or study sequence.

https://github.com/druedaplata/bio

Zenodo: SARS-CoV-2 genomic data obtained for this study are available in: Denaturing and dNTPs reagents improve SARS-CoV-2 detection via single and multiplex RT-qPCR. https://doi.org/10.5281/zenodo.6337537 ( Cadena-Caballero et al., 2022).

This project contains the following extended data: -

SARS-CoV-2 genomic sequences from the GenBank and GISAID databases and their alignments.

Data are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).