Development and Optimization of a Multiplex Real-Time RT-PCR to Detect SARS-CoV-2 in Human Samples

PCR and its variants (RT-PCR and qRT-PCR) are valuable and innovative molecular techniques for studying nucleic acids. qPCR has proven to be highly sensitive, efficient, and reproducible, generating reliable results that are easy to analyze. During the COVID-19 pandemic, qPCR became the gold standard technique for detecting the SARS-CoV-2 virus that allowed to confirm the infection event, and those asymptomatic ones, and thus save millions of lives. In-house multiplex qPCR tests were developed worldwide to detect different viral targets and ensure results, follow the infections, and favor the containment of a pandemic. Here, we present the detailed fundamentals of the qPCR technique based on fluorogenic probes and processes to develop and optimize a successful multiplex RT-qPCR test for detecting SARS-CoV-2 that could be used to diagnose COVID-19 accurately.


Introduction
In late 2019, a new Betacoronavirus was identifed in humans, causing a severe pneumonia disease known as COVID-19 [1].Since its emergence, the virus has infected almost 700 million people and caused over 6.5 million deaths worldwide.Te excess mortality associated with the pandemic was estimated to be 15 million between 2020 and 2021.Te rapid spread of the virus and its high mortality rate have created an urgent need to control viral transmission.However, the transmission of the virus from asymptomatic individuals has made it challenging to trace SARS-CoV-2 based solely on clinical symptoms.Tis highlights the need to implement rapid and specifc techniques to efciently detect SARS-CoV-2.Molecular tests such as reverse transcription-polymerase chain reaction (RT-PCR) and its quantitative variant RT-qPCR are excellent diagnostic options, given their ability to detect target nucleic acids with high sensitivity [2] (Figure S1).
Multiplex PCR is a technique that involves amplifying and detecting two or more gene sequences in the same reaction, using more than one primer pair in the amplifcation tube [3].Some of the benefts of this technique include higher throughput (potentially more samples analyzed per plate), lower sample usage, and lower reagent usage, depending on the number of targets in the experiment, reducing the time and cost of analysis [4].Te development of PCR detection equipment with simultaneous multitarget detection and advances in probe chemistry have made comparative analyses standard in many areas of research and testing [5].
Te qPCR based in probe hydrolysis is one of the most important tools for diagnosing and studying the SARS-CoV-2 due to its high sensitivity, specifcity, and reliability as it depends upon the primer binding to its specifc target sequences and fuorescent-based quantitative PCR assays to allow sensitive detection (Figure S2).Additionally, the one-step RT-qPCR became the preferred method over the two-step method owing to it being fast and efcient and involving limited sample handling, minimal experimental errors, and reduced bench time, allowing high-throughput testing necessary for the pandemic control [6].Another advantage is the capability of hydrolysis probes in qPCR to detect multiple genes in a single reaction, which was important in a context of shortage of enzymes, probes, and plastic ware and the rising cost of enzymes and dual-labeled probes, due to the enormous demand for assays.Additionally, the hydrolysis probe qPCR allows to quantify and compare transcripts between control and experimental samples, to evaluate changes in gene expression (Figure S3).Low-income countries were the most afected by that limitation.Tis paper presents the fundamentals and principles of qPCR (in the Supplementary File) to better understand the technique, and additionally we describe the development of a multiplex RT-qPCR system for detecting SARS-CoV-2 virus during the onset and evolution of the COVID-19 pandemic in Colombia that allows the simultaneous detection of two viral genes and a human internal control for a rapid and specifc diagnosis of SARS-CoV-2, obtaining a procedure sensitive, rapid, and accessible to track the pandemic virus.

Clinical Samples and RNA Extraction.
In this study, 155 RNA samples obtained from nasopharyngeal swab samples of respiratory symptomatic patients with clinical suspicion of SARS-CoV-2 infection were selected and collected by a private diagnostic laboratory (Approval by Ethics Committee UEB-560-2020).Tese samples were previously evaluated using the commercial GeneFinder ® COVID-19   Plus RealAmp RT-PCR kit, and 79 negative and 76 positive samples were confrmed.RNA extraction from clinical samples was performed by an in-house method using SpeedBead Magnetic Carboxylate Modifed Particles and lysis bufer with guanidine salts developed and standardized at the Virology Laboratory of Universidad El Bosque.Te elution volume used for this method was 50 µL.

Primers and Probes and In Silico Evaluation. Primers and probes for E and N viral genes published by Corman et al. in
the Charité University protocol were used [7].Te RNase P gene was used as a human internal control to assess the presence of amplifcation-susceptible RNA in the samples [7].Te probe for the E gene was labeled with FAM fuorophore, the N probe with Texas Red, and RNase P with HEX, and their respective quenchers.In silico evaluation of primers and probes included in the multiplex system was performed using 20 Colombian genome sequences reported in the GISAID database, with each sequence representing one of the lineages circulating in the country from 2020 to December 2021, including the Mu variant described in Colombia and other variants of interest in the region such as P1 and P2 in Brazil and C37 in Peru.Te results showed that all primers and probes presented a 100% identity with those SARS-CoV-2 lineages (Figure 1).During frst steps of standardization, amplicons were cloned and sequenced to confrm their identity.

RT-qPCR Assays for the Detection of SARS-CoV-2 and Single vs. Multiplex Performance Comparison.
Reverse transcription and amplifcation of the viral genome were performed using the Luna ® Universal One-Step RT-qPCR enzyme kit.Te E and N viral genes were amplifed individually (single reaction) and in combination with the RNase P gene (triplex reaction).Several concentrations of primers and probes were tested and fnally selected at 0.2 μM for both reactions (single and multiplex).Samples were considered positive when fuorescence exceeded the detection threshold at Cq less than 37 and a gradual increase during the amplifcation cycle, generating a typical sigmoidal amplifcation curve.Samples with Cq values greater than 37 were considered negative.Te study also compared the performance of the E and N single reactions with that of the triplex reaction (E, N, and RNAse P) using a commercial SARS-CoV-2 RNA control, diluted to obtain concentrations of 100, 50, and 20 viral copies/μL, and for the latter, serial two-fold dilutions were made to obtain concentrations of 10 and 5 viral copies/μL, using nuclease-free water as the diluent.Te single reactions were performed in duplicate and six replicates of the triplex reaction were performed to obtain the analytical sensitivity (LoD).Finally, to evaluate the performance of triplex RT-qPCR in clinical samples, 155 RNA samples from patients with suspected COVID-19 were selected and analyzed in a CFX-96 thermal cycler.
Calculations of sensitivity and specifcity were performed by a classical approach, counting those samples positive that were obtained from confrmed SARS-CoV-2 patients (true positives) and negative samples that were from discarded cases (true negatives).We use the commercial and validated system GeneFinder Plus RealAmp Kit to compare the results in concordance with the developed RT-qPCR system described in the article.Te reported performance of the commercial kit was sensitivity of 100% (95% CI: 88.6-100%) and specifcity of 100% (95% CI: 88.6-100%).

Evaluation of the Analytical Sensitivity of the Single E and
N and E-N-RNAseP P Multiplex Reactions.Te study evaluated the analytical sensitivity of the single E and N qPCR and E-N-RNAse P multiplex reaction.Te results showed no signifcant diferences in the Cq and relative fuorescence units (p � 0.1) between the single reactions of 2 International Journal of Microbiology the E and N genes and the multiplex reaction (viral E-N in combination with RNAse P) (Figure 2).Te mean and standard deviations of Cq were similar between groups at all dilutions, and the system reliably detected up to 2 viral copies/μL, corresponding to 10 genomic viral copies per reaction.Table 1 presents the data obtained after evaluation of limit of detection.

Evaluation of the Clinical Sensitivity and Specifcity of the E-N-RNAse P Multiplex System.
Te study evaluated the clinical sensitivity and specifcity of the developed E-N-RNAse P multiplex system by testing 155 clinical samples and comparing the results with those obtained by the commercial GeneFinder ® COVID-19 Plus RealAmp RT-PCR kit.Te commercial kit detected three SARS-CoV-2 genes (E, N, and RNA-dependent RNA polymerase, RdRp) and confrmed 76 positive cases, and 73 out of these were also confrmed with the standardized multiplex system.Final analysis resulted in 96% specifcity and 100% sensitivity regarding the commercial kit (Table 2).To verify that the amplifcation signal obtained in the RT-qPCR reaction corresponded to the expected products for each amplifcation target, the products obtained in the single reaction (E or N), duplex (E-N), and multiplex (E-N-RNAse P) were evaluated using 3% agarose gel electrophoresis.Te bands corresponding to the fragments: E with a size of 115 bp, N of 70 bp, and the fragment corresponding to RNAse P of 64 bp, were observed (Figure 3).However, because of the closeness between the N and RNAse P amplicons, the three products of the E-N-RNase P reaction were not simultaneously observed.

Discussion
During the COVID-19 pandemic, RT-qPCR became an essential tool for managing and providing epidemiological information in all countries [8].In Latin American countries, such as Colombia, this technique is well established, but mainly in research centers and laboratories belonging to universities.Tese became a support network for diagnosing SARS-CoV-2 infections in the Colombian health system.However, one of the challenges was access to commercial kits for in vitro diagnosis, as there was little availability in this region.Likewise, the price of these commercial kits exceeds USD $1600 (100 reactions), such International Journal of Microbiology a single reaction for detecting two viral genes and a human control gene, which helped improve the diagnostic processes [11].Te study presents a rapid and simple method for developing and adapting a multiplex system for real-time PCR diagnostics with retrotranscription, using independently reported and evaluated primer-probe sets as a starting point.Using this specifc protocol, our laboratory and others processed almost 100 000 samples during the frst year of the pandemic.
Te multiplex reaction did not individually afect the sensitivity and specifcity reported by the protocols.Te annealing temperature of the recommended primers and probes (58 °C) was conserved, which contributed to the regulation of nonspecifc amplifcation during the amplifcation protocol.Te results showed that the multiplex system can discriminate between true positives and negatives, identical to a commercial kit (GeneFinder ® COVID-19   Plus RealAmp RT-PCR), which shows a LoD of 0.5 cp/μL.Adapting protocols to each laboratory and geographic region is essential to preserve the technical-scientifc recommendations and optimize them for each context [12][13][14].
Latin America, despite contributing less than 10% of the world's population, has experienced one-third of COVID-19 cases and 25% of deaths worldwide.Tis is due in part to the economic and health system challenges faced by  4 International Journal of Microbiology underdeveloped countries, as well as the weakness and asymmetry of molecular diagnosis laboratories and a global shortage of reagents and supplies, which explain the low rates of confrmatory tests done for each reported case in Latin America [15].However, during the frst year of the pandemic, many Latin American university research laboratories transferred their molecular skills to SARS-CoV-2 diagnosis, helping public health authorities to follow and control the virus transmission.For example, Peruvian academic and researchers standardized and validated a multiplex qPCR [16], while Ecuadorian and Uruguayan universities developed new qPCR systems that reached optimal clinical performance [17].In Colombia and Brazil, university laboratories improved alternative low-cost multiplex PCR or Sybr green protocols to confrm positive samples [11,18,19].Te protocols mentioned above used the reported primers and probes tested until then and demonstrated optimal performance in terms of sensitivity and specifcity.Tese eforts shed light on the conditions required to develop a molecular diagnosis assay and allowed for the gain of scientifc and technical capabilities for public health surveillance.Te developed protocols were important to cope with the sanitary crisis derived from the pandemic and to unblock the historic backlog in Latin American biotechnology.Tis academic and technical work presented the hydrolysis probe qPCR fundamentals to spread the principles to scientists and diagnostic laboratories and resume the necessary steps to implement the technique for clinical diagnosis [20].

Conclusions
Tis paper presents the fundamentals and principles of quantitative real-time PCR (qPCR) as a learning tool in both academic and diagnostic environments (Supplement).Additionally, the study established a diagnostic method for SARS-CoV-2 using multiplex amplifcation and found that primers and probes were still adequate for the SARS-CoV-2 strains circulating in Colombia.Te mixed preparation of reagents for detecting two viral genes and one human gene detected the virus from respiratory patients with high accuracy, specifcity, and sensitivity, which showed low cost and high clinical performance compared with a commercial molecular system.Te standardized protocol was useful in following the SARS-CoV-2 circulation in Colombia during the challenging pandemic when the shortage of reagents and lab ware was characteristic.

asFigure 1 :
Figure 1: Multiple sequence alignment.Primers and probes for SARS-CoV-2 used in the multiplex reaction are highlighted at the frst sequence.Each dot indicates a match or identity between the analyzed sequences.Main strains and variants circulating in Colombia were compared.

Table 1 :
Cq (and standard deviation) obtained for diferent copy number of templates to evaluate the limit of detection (LoD).

Table 2 :
Clinical sensitivity and specifcity of the developed multiplex system.