Rapid System to Detect the Main Variants of SARS-CoV-2 in Biological Specimens

Since its appearance in late 2019, SARS-CoV-2 has been reported to acquire substitutions more slowly than other RNA viruses, but its tendency to manifest recurrent deletions/mutations in the spike glycoprotein exceeds this slow replacement rate. To date, variants have been identied in many countries, some of which are transmitted eciently and also present several lineages. The rapid identication of such variants is paramount to quickly implement containment measures. We developed a novel assay using traditional real-time PCR to detect the main reported variants of the spike gene of SARS CoV-2. Primers and probes were designed to detect the following deletions and mutations as well as to cover all lineages known to date (B.1.617, B.1.617.1, B.1.617.2, B.1.617.3 and B.1.618): delta 69:70 and delta 144:145 deletions, which denote the UK variant (VOC 202012/01, now called Alpha); delta 242:244 deletion, which identies the South African variant (now named Beta); delta 3675:3677 deletion in the ORF1a gene, which denotes the Brazilian variant (now called Gamma); and P681R mutation as well as delta 145:146 and delta 157:158 deletions, which identify the Indian variant (also known as Delta). Our assay will help clinical microbiologists and clinicians to rapidly recognize the presence of variants in biological samples (particularly nasopharyngeal swabs), and it may also be useful for epidemiological purposes in the early selection for successive tracing of patients harbouring virus variants that may be more diffusive and/or not responsive to vaccines.


Introduction
Since the appearance of SARS-CoV-2 in late 2019, people worldwide have presented with severe pneumonia at hospitals (1). Over time, the number of patients has rapidly increased. Community transmission of the virus, as well as antiviral treatments, can promote mutations in the virus, resulting in more virulent and/or more diffusive viruses with potentially higher mortality rates (2,3). Although most of the emerging mutations do not have a signi cant impact on the spread of the virus or on its virulence, many others may provide selective advantages, including increased transmissibility, the ability to escape from the host immune response and resistance to antiviral drugs and vaccine effectiveness (2,4,5). To date are known at least eleven variants of SARS-CoV-2 named with the letters of the Greek alphabet to simplify their identi cation (from Alfa to Kappa) (5). Some variants pose an increased risk to global public health, and they are identi ed as Variants of Concern (VOCs: Alpha, Beta, Gamma and Delta,) in order to prioritize global monitoring and research (6).
The world is still facing these four main variants of SARS-CoV-2 which in detail are: VOC 202012/01 (501Y.V2; UK or Alpha variant); B1.351 (South African variant, or Beta); variant P.1 (Brazilian and Japanese variant or Gamma); and the Indian variant (speci cally the B.1.617 lineage), known as Delta (5,(7)(8)(9)(10)(11)(12)(13)(14)(15). These variants likely have no impact on the mortality rate but have led to increased transmissibility, especially for the Brazilian and South African variants in which the K417N and E484K point mutations affect the e cacy of vaccines; in fact, a worsening epidemiological situation has been observed in many countries worldwide (7)(8)(9). The Delta variant may have been present for some time, but the rst B.1.617 genome was recorded on October 5, 2020 in the global database (GISAID) (14). The Delta variant comprises the B.1.617.1, B.1.617.2 and B.1.617.3 SARS-CoV-2 lineages, which have been increasingly detected in many countries (15). B.1.617 has several mutations (approximately thirteen) that are present in other variants of interest/concern, and it is controversial whether it has antigenic escape (15). To identify and trace these variants, researchers are using the whole-genome sequencing approach, which helps to de ne the emerging clades and identify single point mutations, but it is time consuming, expensive and available only in large laboratories or in national reference laboratories (16)(17)(18)(19). Therefore, for the early identi cation of such variants, it is desirable to develop a molecular assay that is easy to use and cost e cient.
Here, we present a novel assay based on real-time PCR to detect SARS-CoV-2 variants located in the spike gene. Our assay detects the main deletions/mutations associated with the variants reported above,

Samples
The present study did not include human participants but included leftover samples. For the assay, we used nucleic acids (NCs) extracted from 400 nasopharyngeal swabs (NFWs) routinely processed using a Nimbus instrument (Seegene Inc; Songpa-gu, Seoul 05548, Republic of Korea) and also con rmed using a qRT-PCR of our design as reported by Favaro et al (20). 5 µl of the eluate was used for the assay. NFWs were routinely delivered to the microbiology laboratory of our hospital from March to May 2021. Positive NFWs were established to be positive based on the results obtained using a commercial system (Allplex TM 2019-nCov Assay-Seegene) and the method described in our previous work (20). NCs were randomly selected among positive NFs and then processed using our assay.

Primers and Probes
The PCR assays used ve sets of primers and probes of our own design. Four sets were used for the identi cation of the speci c deletions, and one set of primers and probes, targeting human β−actin, was used as an internal control (IC). Table 1 shows the primer and probe sequences as well as labelling uorophores for each probe. The primers and probes were synthesized by Metabion International AG (Planegg, Germany) and Bio-Fab (Rome, Italy). and 30 seconds at 60°C. An Amplilab real-time machine (Adaltis SRL, Guidonia Montecelio, Italy) was used for the real-time PCR, and the results are shown in Table 2.

Sequence analysis
Amplicons from our assay were sequenced by the Sanger method using the Bio-Fab Research sequencing service (Rome, Italy). The following primers were used: S seq F 5'CCA CTA GTC TCT AGT CAG TGT GT 3' and S seq R 5'GAG AGG GTC AAG TGC ACA GT 3' (this work).
To con rm the nature of the variants identi ed by our assay, all samples were analysed by sequence analysis.
Seventy-nine samples showed sequences compatible with Δ69:70 and Δ144:145 deletions, while ten samples showed mixed electropherograms with overlapping peaks. Figure 1 shows the UK variant Δ69:70 sequence, while Figure 2 shows a mixed electropherogram, resulting from the presence of two viral genomes (wild type and variant) in the same sample. The presence of two different lineages has previously been reported in the literature (3,21).

Discussion
VOC 202012/01 was the rst variant identi ed in the United Kingdom in December 2020, but it has been traced back to September 2020 (22,23). VOC 202012/01 is the predominant variant circulating in the UK, and it has become a great concern due to its increased transmissibility (2-23). The UK has implemented stricter nonpharmaceutical interventions (NPIs) to reduce risk of transmission (23, 24). Additionally, community transmission of VOC 202012/01 has been observed in Denmark, and in its response, the country has strengthened and prolonged the measures of containment. In December 2020, the 501Y.V2 variant was rst identi ed in South Africa, and again, it is now one of the most prevalent. This V2 variant is characterized by increased transmissibility, and starting in January 2021, it has been identi ed in ten EU/EEA countries (France is at the top), but also in Israel and the UK (24)(25)(26)(27)(28). Starting in December 2020, the B.1.617.1, B.1.617.2 and B.1.617.3 SARS-CoV-2 lineages were reported in India, and they have been increasingly detected in many countries (5,6,15). Therefore, early identi cation is extremely urgent to contain the spread of such variants (5,6,29). The reference method for identifying variants of SARS-CoV-2 is whole-genome sequencing, but it is expensive, time consuming and limited to use in large laboratories and reference laboratories. Our system has been demonstrated to be a rapid and costeffective method to detect the main variants of the virus. Our assay is a simple real-time PCR and does not require expensive instrumentation. Furthermore, it is easy to use and can be introduced in any laboratory even in those that may not have advanced sequencing systems available. The advantage of our assay is that every hospital may quickly obtain the result of the variant circulation to promptly implement the infection control measures required to prevent further transmission in their setting (25)(26)(27)(28)(29).
Our assay has helped to quickly con rm/exclude the presence of the main SARS-CoV-2 variants, because our test is based on the direct detection of the presence of deletions/mutations, it is not affected by the potential co-presence of the wild-type SARS-CoV-2 virus in the specimens (which indicates a coinfection). Importantly, some of the commercially available tests have based their detection of the variants on the lack of ampli cation of the S gene. For both instances of a co-infection and re-infection (conditions that may generate the co-presence of two types of virus in the same sample/patient), the commercial tests may show a curve for the spike gene, but the latter belongs to ampli cation of the SARS-CoV-2 wild type virus, thereby masking the co-presence of a viral variant.
Finally, the results of our assay have provided information for the circulation of such variants in our location. Surprisingly, we found that the UK variant is widespread in our country, but it explains the massive and prolonged second longwave despite the lockdown measures implemented by our authorities (26). Moreover at the time of study also Indian variant was rising, and up to date it is dominant in our country (30). The global and rapid diffusion of SARS-CoV-2, combined with its ability to mutate, provides a terrible example of the prediction of the Nobel Laureate, Joshua Lederberg, who de ned the ght against microbes as "Our wits versus their genes". Figure 1 Electropherogram peak analyses showing the sequence of "mixed type sample" blue arrows indicate the presence of overlapped peaks in the eletropherogram due to the co-presence of wild type as well as variant of SARS-CoV-2 Figure 2