Several methods have been developed for early detection of SARS-CoV-2 variants (VOCs, VOI or VUM). These methods for diagnostic screening mainly consist of nucleic acid amplification technique-based assays able to generate preliminary results in a few hours.

Many of these methods can also accurately identify the variants, while others will require subsequent verification/confirmation by sequencing [82]. We here show and discuss some of the most widely used in diagnostic routine commercially available and home-made methods for variants screening and identification.

One of the commercially available methods for identifying variants of SARS-CoV-2 is “COVID-19 Variant Catcher” developed by Clonit S.r.l. This is a qualitative CE-IVD test, based on Real Time RT-PCR, that allows the identification of the S gene mutations 69–70del, E484K and N501Y. The COVID-19 Variant Catcher kit must be used on extracted RNA from SARS-CoV-2 positive samples that have shown amplification within the 35th Cycle Threshold (Ct) in a previous Real Time RT-PCR-based method. The COVID-19 Variant Catcher kit was developed and validated to be used with the following instruments: Rotor Gene Q MDx from QIAGEN, CFX96 from Biorad and 7500 from LifeTechnologies. (Instructions For Use COVID-19 Variant Catcher, Clonit distribuited by Biomedica). An updated version called “COVID-19 Ultra Variant Catcher” discriminates the L452R, E484K, E484Q and N501Y mutations allowing the identification of a larger number of lineages (see Table 3).

Another rapid method for SARS-CoV-2 variants identifications is the “Simplexa™ SARS-CoV-2 Variants Direct assay” (RUO) by Diasorin Molecular, that can identify the SARS-CoV-2 spike mutations N501Y, E484K, E484Q, and L452R. The advantage of this assay consists on its use on nasopharyngeal and nasal swab specimens without previous RNA extraction. Fluorescent probes specific for each mutation are used to perform a post-amplification melting analysis to identify the presence of mutant or wild-type nucleotides at specific locations. This test requires the LIAISON MDX instrument and his appropriate software for the analysis and interpretation of the results. The Limit of Detection (LoD) was determined to be the lowest detectable concentration of quantitated extracted viral genomic RNA (copies/mL) at which ≥ 95% of all replicates test positive and it turned out to be 500 copies/mL. (Instructions For Use of Simplexa™ COVID-19 Direct, by DiaSorin Molecular). We previously examined 118 positive nasopharyngeal swabs (NPS) first characterized by Sanger sequencing, using Simplexa^® SARS-CoV-2 Variants Direct assay, with the aim of evaluating the performance of the assay. Results for 111/118 NPS were in complete agreement with the Sanger sequencing, while the remaining 7 samples were not amplified, due to the low viral load. For the 7 NPS not amplified by the assay, a Real Time RT-PCR on thawed samples showed positive results although with high Ct values (median Ct were > 30.for S and ORF1ab gene) [108].

During the last year and a half, with the aim to identify the circulating variants, the Seegene Inc developed a series of multiplex Real Time RT-PCR based typing tests called “Novaplex SARS-CoV-2 Variants Assay” detecting different panels of spike protein mutations such as: 69–70del, E484K, N501Y (Variants I); L452R, W152C, K417T and K417N (Variants II), L452R, P681R, AND K417N (Variants IV), L452Q, F490S,P681R, and L452R (Variants V), L452Q, F490S, R346K, and D950N (Variants VI), and 69–70del, E484A, N501Y and RdRp (Variants VII). The system provides an automated extraction and Real Time RT-PCR setup followed by an automated interpretation reporting data for each specific mutation probe as a Ct value. These assays must be used with Seegene instruments: STARIet (for the extraction and PCR setup), CFX96Dx (for the Real Time RT PCR) and a specific Seegene Viewer (for the automated interpretation). A recent paper compared the results obtained from the Novaplex Variants I, II, and IV assays with S gene Sanger sequencing resulting in a 100% overall agreement in variants identification when using extracted RNA, while a RNA-extraction free protocol was less sensitive in detecting some mutations especially with Ct values > 30 [109].

The ABL (Advanced Biological Laboratories) “UltraGene Assay SARS-CoV-2 Multi Variants Deletions V1” (CE-IVD), a Real Time RT-PCR test intended to be used for the qualitative detection of SARS-CoV-2 deletions 69/70 (Δ69), Y144 (Δ144) and 242–244 (Δ242) on the Spike (S) gene and the deletion 3675–3677 (Δ3675) on the ORF1ab gene in upper respiratory specimens from patients already diagnosed positive to SARS-CoV-2. This system needs extracted RNA by a compatible extraction method (such as Roche MagNA Pure) and, for the amplification stage, is usable with any qPCR instrument compatible with the FAM, HEX, ROX, Cy5 channels. The LoD is 1150 TCID50/mL for the QuantStudio 5 Real Time RT-PCR instrument and 115 TCID50/mL with the UltraGene qPCR 48 instrument.

The “SARS-CoV-2 Extended ELITe MGB” (ELITech Group) molecular kit is a RUO multi-target designed assay intended for use as a reflex test for the detection and discrimination of the mutations L452R, E484K, E484Q and N501Y of the S gene of SARS-CoV-2. This assay foresees a Real Time RT-PCR performed using the ELITe InGenius instrument (ELITechGroup) and the identification of mutations by analysis of the melting curve.

One of the first commercially available methods intended for the qualitative detection of nucleic acid from SARS-CoV-2 in upper respiratory specimens was “TaqPath™ COVID-19 CE-IVD RT-PCR Kit” (by ThermoFisher Scientific, Waltham, Massachusetts, Stati Uniti). TaqPath™ COVID-19 is a multiplexed assay that contains three primer/probe sets specific to ORF1ab, N gene and S gene of SARS-CoV-2. This system needs extracted RNA (the minimum recommended elution volume is 50 µL). The TaqPath COVID-19 was developed and validated to be used with the following instruments: QuantStudio 5, QuantStudio 7 Flex, 7500 Fast Dx, 7500 Fast, and 7500 from LifeTechnologies. The LoD study established that the lowest SARS-CoV-2 viral concentration (Genomic Copy Equivalents or GCE) that can be detected by the TaqPath™ was 10 Genomic copy equivalent/reaction (GCE/reaction) for both nasopharyngeal swab and bronchoalveolar lavage specimens. The Ct cut-off value for clinical target was ≤37 (TaqPath™ COVID-19 CE-IVD RT-PCR Kit Instructions For Use). Interestingly, although this test was developed with the diagnostic intent of quickly diagnose COVID-19 caused by SARS-CoV-2 infection, if a sample with the 69–70del S-gene mutation is tested using this kit, it will result in an S-gene dropout in presence of ORF1ab and N gene amplifications (also indicated as a S gene Target Failure or SGTF). Considering the importance of this mutation in identifying some SARS-CoV-2 variants, namely the Alpha and Omicron BA.1, this assay, has been used as a fast screening method. On 26 November 2021, the ECDC released a document (“Implications of the emergence and spread of the SARS-CoV-2 B.1.1.529 variant of concern (Omicron) for the EU/EEA”) [110] in which the SGTF from the Thermo Fisher TaqPath assay was indicated as a good proxy for Omicron identification in the scenario present at the time with the Delta variant dominating and the Omicron (BA.1) rising.

De Pace at al. evaluated the diagnostic performance of five qualitative Real Time RT-PCR based tests (SARS-CoV-2 Variants II Assay—Allplex-Seegene Inc.; UltraGene Assay SARS-CoV-2 452R & 484K & 484Q Mutations V1.x—Advanced Biological Laboratories; COVID-19 Ultra Variant Catcher—Clonit S.r.l; SARS-CoV-2 Extended ELITe MGB—ELITechGroup; Simplexa SARS-CoV-2 Variants Direct - Diasorin Molecular) as compared with NGS finding that the overall accuracy of these assays ranged from 96.9% to 100% and specificity and sensitivity were 100% and 96–100%, respectively. The authors recommend the use of these assays as second-level tests in the routine workflow of SARS-CoV-2 laboratory diagnostics, as they are accurate, user friendly, low cost, may identify specific mutations in about 2–3 h and, therefore, optimize the surveillance of SARS-CoV-2 variants [111].

Vice versa Alejo-Cancho et al. describe three cases in which a Mu strain containing the mutation K417N was initially misclassified as the Beta variant using a multiplex Real Time RT-PCR (Allplex SARS-CoV-2 Variants Assay -Seegene), in this case the authors recommend to use NGS or other methods for the detection of P681H to distinguish between these two variants [112] (Table 3).

Viral genome sequencing procedures are expensive and time-consuming, so with a view to reducing labor intensive and to quickly screen for the different SARS-CoV-2 circulating variants, many laboratories have developed an in house rapid methods for SARS-CoV-2 variants identification.

Several studies reported delectable results by using in-house molecular tests for identifying a specific single SARS-CoV-2 variant screening, in the contest of knowledge of variants circulating in a given period. Hamill et al. developed a Real Time RT-PCR for deletions of Δ156–157 in the spike gene that is characteristic of the Delta variant with the purpose, primarily to monitor and identify the Delta variant strains, but data analysis indicated that to increase the identification of both Delta and Omicron variants this newly designed assay need to be used in combination with CDC N1 target [119].

Barua et al. developed a reverse transcription fluorescence resonance energy transfer-polymerase chain reaction (RT-FRET-PCR) designed to identify the T478K mutation (present in 99.73% of the Delta variant) that can be used both to diagnose COVID-19 patients and simultaneously identify if they are infected with the Delta variant; but the Delta variant is just one of several SARS-CoV-2 variants, so a continuous monitoring of strains will still be necessary [120].

Many other groups have also developed homemade techniques for the identification of single position mutations in order to quickly identify a single variant circulating on the territory [121] but the major limitation of all this kind of assays is the inability to detect all the other major variants because of each variant is characterized by a multitude of mutations.

Erster et al. focused their attention on developing a test that was able to discriminate between two variants such as Alpha (B.1.1.7) and Beta (B.1.351) variants. This kind of approach can be useful in a territorial context in which two variants mainly circulate but does not allow the identification of new mutation or to the advent of new variants [122].

Because of tracking SARS-CoV-2 variants through WGS can be time consuming and resource-heavy, some laboratories describe an in-house validation of an allele-specific polymerase chain reaction (ASP) variant assay to detect SARS-CoV-2 VOC’s. For example, Brito-Mutunayagam et al. described an ASP based three mutation targets: E484 K, L452R and P681R (based on the circulating variant epidemiology at the time) [123]. Despite the considerable advantage in terms of time, this type of methodology can help to identify a variant only in certain historical periods, when you already know what circulates in a specific area and when you want to discriminate between specific variants.

Other groups describe multiplex Real Time RT-PCR homemade methods capable to detect 9 mutations with specific primers and probes, these PCR typing strategy allowed the detection of the major variants and also provided an open-source PCR assay which could rapidly be deployed in laboratories around the world [124].

Given that genotyping approaches are rapid methods for monitoring SARS-CoV-2 variants but require continuous adaptation, fragment analysis may represent an approach for improved SARS-CoV-2 variant detection. Clark et al. described a multiplex fragment analysis approach using PCR targeting variants by size and fluorescent color. Eight SARS-CoV-2 mutational hot spots in VOCs were targeted. This kind of method could classify a variant with similar accuracy as sequencing without frequent target modification [125].

Moreover, given the possibility that Emerging variants pose the risk for target dropout and false-negative results secondary to primer/probe binding site (PBS) mismatches. Hernandez et al. described a method that combine RT-PCR and matrix-assisted laser desorption/ionization time-of-flight mass-spectrometry to probe for five targets across N and ORF1ab genes, which provides a robust platform to accommodate PBS mismatches in divergent viruses [126].

Another important goal could be to develop a single platform with both diagnostic and surveillance capabilities for comprehensive SARS-CoV-2 Spike gene mutations. This is what was made by Welch et al. that selected up to 26 mutations to distinguish between or detect mutations shared among the Alpha, Beta, Gamma, Delta, Epsilon, and Omicron variant lineages in a cost-effective virus and variant detection platform, which combines CRISPR-based diagnostics and microfluidics with a streamlined workflow for clinical use [127] (Table 4).