Early detection of pathogens is a crucial step to disease prevention  and containment, especially during epidemic outbreaks . PCR is one of the reliable and relatively accessible molecular methods that directly recognize pathogen-derived
material from patients samples . However, the optimization of PCR protocols is strongly dependent on primers specificity and efficiency . This reason, combined with the increasing number of SARS-CoV-2 sequences available and its crescent polymorphism, led us to design a set of new primers that can address very conserved regions of the virus genomes.
Therefore, in order to aid PCR optimization, the UFRN_primers were designed to present Tm values that were as close as possible. It is expected that these configurations will allow the use of at least two systems using the same thermal cycling parameters. In this way, it would be possible to perform the PCR test identifying different regions of the viral genome at the same time, according to the protocols already described for the PD_primers. In this context, possibly the systems UFRN_3 and UFRN_4, will have different thermal cycling parameters comparing to the other systems since, in this case, the probe Tm is similar to the primers (Table 1). Probably these systems will depend on more annealing time in order to ensure that the probe has interacted in DNA template before the amplification starts.
The higher specificity of UFRN_primers confirmed by in silico analysis is mainly due to the availability of 2.341 genome sequences, which made it possible to identify the conserved regions with greater accuracy from the alignment. Quite possibly, at the time of publication of this work, a considerably larger number of additional sequences will be available, which may reveal new polymorphic sites in the target regions of UFRN_primers and PD_primers. In this way, our research group will continue this bioinformatics work, and whenever relevant, new updates on the primer sequences or new primers set will be reported.
Another critical point is that primers presented here were tested against the updated databases of RNA sequences from bacteria, fungi, and protozoa, and did not generate non-specific amplicons in any case. Although executed through in silico analyses, this negative prediction increases the potential for applying these primers to different samples such as blood, feces, or even in environmental samples. Currently, the most suitable sample for detecting SARS-CoV-2 is the human nasal swab; however, there are already studies that have shown digestive symptoms (e.g. diarrhea and vomiting) [9,10] and other less frequent symptoms (e.g. conjunctivitis) in patients who tested positive for SARS-CoV-2 [11-13]. This diversity of symptoms makes clinical diagnosis difficult, and testing new types of samples may be needed in a short time. The application of UFRN_primers to detect SARS-CoV-2 in blood or fecal samples is likely
efficient since these primers should not interact non-specifically with RNAs of the main protozoa and bacteria that cause health problems in humans.
The use of universal primers makes it possible to identify several variants of the virus using the same PCR protocol. UFRN_primers are strong candidates to help simplify the procedures and supply chain for detecting SARS-CoV-2, allowing, for example, the mass production of primers and kits that could be applied in different parts of the world with equivalent efficiency. However, the primers presented here still depend on in vitro validation. The availability of these sequences at this time will be crucial so that these new protocols can be validated promptly to assist in the control of the SARS-CoV-2 pandemic.