In the context of strengthening local and international diagnostic facilities and technical capacity for pathogen detection during pandemics, epidemics or outbreaks, our study aims to contribute to the development of a simple and rapid method that does not require the use of sophisticated equipment and can be implemented in minimally equipped healthcare centers. Indeed, the emergence of infectious diseases, such as COVID-19, has stressed the importance and necessity of rapid and effective diagnosis for detecting pathogens and controlling their spread. COVID-19 represented an international public health emergency due to its pathogenesis and rapid spread. Simple, rapid, sensitive, specific, and equipment-free diagnostic techniques were essential to meet the needs of epidemic control strategies and thus limit the spread of the disease. While different approaches have been used for disease diagnosis based on serology and computing tomography imaging, the WHO recommended real-time reverse transcription polymerase chain reaction (RT-qPCR) as the reference method [1]. The classical RT-qPCR is a sensitive and accurate technique but has several disadvantages, including the high cost of kits, the prolonged time to result delivery, the need for trained personnel, and the requirement for a suitable and costly piece of equipment that is usually missing in low-resource setting areas. [35]. The development of a simple, rapid, accurate, and field-applicable methods for detecting SARS-CoV-2 and future emerging pathogens remains a pressing need. Various molecular diagnostic alternatives, such as RT-LAMP [9], RT-RPA [15], and tests based on the CRISPR-Cas systems [19, 21, 22], have been developed for detecting the SARS-CoV-2 virus. These tests allow for rapid, specific, and effective detection under isothermal conditions, therefore, eliminating the need for sophisticated equipment. However, despite their advantages, these techniques have drawbacks, namely complex primer design and optimization of reaction conditions in case of LAMP [4], the risk of false positives with RPA [36], and multiple workflow steps for the CRISPR Cas systems [37]. As PCR is still one of the most valuable methods used in different fields, including food security, forensics, research and biomedicine, in this study, we chose to develop PCR assays using newly designed primers or re-adapt the WHO and published real time PCRs into simpler PCR-based tests. The test here reported is based on a one-step fast multiplex RT-PCR using a ready-to-use master mix and two pairs of already described primers that is coupled to lateral flow PCRD immunoassay detection. It is faster than the classical real time PCR. It is also easy to perform and read, and it obviates need for skilled operators and costly equipment mobilization that makes RT-qPCR only available in central laboratories and the ensuing transfer of samples to these central laboratories, which results in delays in result delivery. Indeed, for example, in Tunisia during the pandemics, real time PCR facilities and expert technicians were missing in different regions of the country. Therefore, in the beginning of the COVID-19 pandemics, all the samples from most regions of the country were transferred to the Institut Pasteur de Tunis. Personnel, including MDs, researchers and lab technicians from different departments and labs of the institute, were called to join the COVID-19 testing team and were organized into three daily shifts, which led to postponing all other research and/or diagnostic activities. Therefore, one of the strategic actions for preparedness for future pandemics, epidemics or outbreaks must be the investment in laboratory infrastructure and diagnostic capabilities [38]. Here, we bring a proof of concept that the one-step fast multiplex RT-PCR coupled to PCRD detection could be a good alternative when there is a shortage in reagents or at points of need where real time PCR facilities are missing. In addition, this technology could be easily applied for the diagnosis of other pathogens [24]. Furthermore, lateral flow immuno-chromatographic assay devices are commercially available. They represent a simple method for the generic detection of nucleic acids without the need for specialized and costly equipment. Their use relies on the detection of dual-labeled amplicons. Primers’ labels are chosen according to the device used; like in this study, we used different labels associations, Fam/biotin and Dig/biotin for each primer pair, which target two different genomic viral regions, to be able to detect the amplicons on their respective test line on the PCRD cassette. Compared to agarose gel electrophoresis, the PCRD assay is rapid (5 min), easy to use and read and does not require extensive molecular biology expertise. Our study showed that the results observed in the agarose gel upon electrophoresis are concordant with those obtained with the PCRD analysis of the E target amplification. However, for the N target, where the amplicon is smaller in length (67 bp), the PCRD assay was able to resolve interpretation ambiguity of the read outs on agarose gels, as the amplicons were not distinguishable from excess primers and/or primer dimers upon electrophoresis.
The most commonly used genomic regions of SARS-CoV-2 for RT-qPCR diagnosis are highly conserved and/or highly expressed genes [39]. Notably, they include the ORF1ab regions, the genes encoding for nucleocapsid (N), envelope (E), spike protein (S), membrane protein (M), RNA-dependent RNA polymerase (RdRp), hemagglutinin-esterase (HE), and helicase genes. In this study, we considered most of the mentioned genes to develop the assay aimed for. We selected and designed our primers in the N, S, E, RdRp and ORF1ab viral genomic sequences. Our selection criteria for the primers were based, namely, on the stability and reproducibility of the fast-PCR as analyzed by the agarose gel electrophoresis, and then on the absence of background noise on the PCRD. The two targets that meet these criteria were the N (US-CDC-N2) and E genes [32]. The multiplexing of the amplification of these two targets within a same reaction was also successful, but we noticed that there was an imbalance in terms of band intensity on both the agarose gel and PCRD. The E amplicon band was more intense compared to the N one, despite our attempts to improve the reaction conditions. This could be due to a sort of competition between primers for the same mix of reagents [25, 40]. The GC content of the target (40,4% for E and 52,2% for N) could also lead to preferential denaturation, resulting in preferential amplification [40], or secondary structures within genomes could induce differential accessibility of targets [40, 41]. Nevertheless, PCRD results were easily read by the naked eye for both the E and N targets. The two targets showed the same sensitivity and specificity when tested in the multiplexed assay but gave discordant results for two patients: patient 47 was positive for the E target but negative for the N target, and patient 311 was negative for the E target but positive for the N target. For these patients, mutations in the priming sites could have occurred, leading to negative results. Or, SARS-CoV-2 gene dynamic lead to differential genes expression depending on the patient status and disease stage [42, 43]. This is the interest in using multiple targets to define the infected patient’s status and minimize false negative results [1, 42]. However, our results showed that six patients who were positive according to RT-qPCR were negative with our assay. These patients had Ct values greater than 33, except for patient 285, who had Ct values of 37/38 for the two targets tested in RT-qPCR but showed positive results for the two targets tested in our one-step fast multiplex RT-PCR/PCRD. In this case, we could come up with two hypotheses. First, our assay is not sensitive enough and is able to detect only patients with a high viral load and a Ct below 33. The second hypothesis, given the fact that the RNAs in this study were extracted in 2021 (and our assays were performed in 2024), long-term stability of the RNAs constitutes a factor influencing the performance of the DNA assays, especially in case of samples with a low viral load. Indeed, it was demonstrated that RNA with a low viral load is more prone to a reduction in its RNA content than RNA with a high viral load [44]. Therefore, our one-step fast multiplex RT-PCR assay should be evaluated with freshly extracted RNAs and, at the same time when RT-qPCR is performed, to accurately determine their performances.
The main limitation of the study is that the internal control (human β-globin) was not included in our multiplex assay. We have attempted to minimize this drawback by setting in parallel this internal control on the same day and run. The one-step fast simplex PCR targeting the human β-globin was performed in a separate reaction as one cannot visualize more than 2 amplicons on the PCRD lateral flow device. Indeed, to our knowledge, immunoassay devices with 3 test lines are not yet commercially available. For future test developments, it would be more interesting to have all the targets (viral targets and internal control) in the same reaction.