Rapid identification of SARS-CoV-2 variants: Validation of the simplexa SARS-CoV-2 variant direct assay.

The circulation of certain SARS-CoV-2 variants may have a great impact on the epidemiological status of a geographical area; therefore, it is important that their presence is monitored. Currently, the gold standard method used to identify newly emerged variants is sequencing of either genes or whole genomes. However, since this method is relatively expensive and has a long turnaround time, other rapid strategies should also be employed. The current study aimed to evaluate the performance of the Simplexa® SARS-CoV-2 Variants Direct assay, which is a RT-PCR that determines the variant present in a nasopharyngeal swab sample in approximately two hours. Totally, 527 positive samples for SARS-CoV-2 were analyzed from January until December 2022 and next-generation sequencing (NGS) was used as the reference method. The assay showed high sensitivity, ranging from 94.12 % to 100 %, depending on the variant. The assay also showed high specificity, reaching 100 % for Delta and BA.1 variants, and 99.74 % and 98.67 % for BA.2 and BA.4/BA.5 variants, respectively. Moreover, the assay was able to identify the correct variant category in the presence of any subvariant in the sample. We conclude that the assay can be used to facilitate faster monitoring of circulating SARS-CoV-2 variants, however sequencing cannot be completely replaced, since new variants always emerge, and constant updates are needed, so that the user is able to interpret the melting curve patterns.

Rapid Detection of Predominant SARS-CoV-2 Variants Using Multiplex High-Resolution Melting Analysis.

Coronavirus disease 2019, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), poses a considerable threat to global public health. This study developed and evaluated a rapid, low-cost, expandable, and sequencing-free high-resolution melting (HRM) assay for the direct detection of SARS-CoV-2 variants. A panel of 64 common bacterial and viral pathogens that can cause respiratory tract infections was employed to evaluate our method's specificity. Serial dilutions of viral isolates determined the sensitivity of the method. Finally, the assay's clinical performance was assessed using 324 clinical samples with potential SARS-CoV-2 infection. Multiplex HRM analysis accurately identified SARS-CoV-2 (as confirmed with parallel reverse transcription-quantitative PCR [qRT-PCR] tests), differentiating between mutations at each marker site within approximately 2 h. For each target, the limit of detection (LOD) was lower than 10 copies/reaction (the LOD of N, G142D, R158G, Y505H, V213G, G446S, S413R, F486V, and S704L was 7.38, 9.72, 9.96, 9.96, 9.50, 7.80, 9.33, 8.25, and 8.25 copies/reaction, respectively). No cross-reactivity occurred with organisms of the specificity testing panel. In terms of variant detection, our results had a 97.9% (47/48) rate of agreement with standard Sanger sequencing. The multiplex HRM assay therefore offers a rapid and simple procedure for detecting SARS-CoV-2 variants. IMPORTANCE In the face of the current severe situation of increasing SARS-CoV-2 variants, we developed an upgraded multiplex HRM method for the predominant SARS-CoV-2 variants based on our original research. This method not only could identify the variants but also could be utilized in subsequent detection of novel variants since the assay has great performance in terms of flexibility. In summary, the upgraded multiplex HRM assay is a rapid, reliable, and economical detection method, which could better screen prevalent virus strains, monitor the epidemic situation, and help to develop measures for the prevention and control of SARS-CoV-2.

Rapid monitoring of SARS-CoV-2 variants through high resolution melt analysis

Since September 2020 the current global pandemic of COVID-19 caused by the SARS-CoV-2 coronavirus is characterized by a succession of waves of infection due to the emergence of new variants of the original virus, presenting various genomic mutations. Many mutations are present in the gene encoding the Spike protein, the main target of the nucleic acidbased vaccines. The Variants of Concern that have been reported since autumn 2020 include Alpha/ B.1.1.7 and sublineages, Beta/B.1.351, Gamma/P.1 and sublineages, Delta/B.1.617.2 and sublineages, Omicron/ B.1.1.529 and sublineages. The rapid and cheap variant monitoring in the population is pivotal for epidemiological studies and for the prompt detection of SARS-CoV-2 variants characterized by high transmissibility or reduced susceptibility to neutralizing antibodies induced by vaccination. Surveillance of genomic variants is currently based on viral whole genome sequencing (WGS) performed on a random fraction of samples positive to molecular tests. WGS involves high costs and extended analysis time compared to a PCR-based diagnostic test, as well as specialized staff and expensive instruments. To rapidly identify the variant in samples positive to SARS-CoV-2, different rapid tests based on real-time PCR and high-resolution melting (HRM) were designed and applied on 88 oropharyngeal swab samples collected from October 2020 to February 2022 (84 positive samples and 4 negative samples). The HRM results were confirmed by PCR product sequencing. Overall, the assays showed 100% specificity and sensitivity compared with commercial PCR assay for COVID-19 testing. Moreover, 83 samples out of 84 (98.8%) were correctly identified as follows: 8 Wuhan (wild type), 12 Alpha, 23 Delta, 37 Omicron BA.1, 1 Omicron BA.1.1, 2 Omicron BA.2. With our lab equipment, about 10 samples can be processed every 3 hours at the cost of 8.5 per sample, including RNA extraction. The identified variants overlapped with mutation and case prevalence over time in Italy (as reported in outbreak.info, which collects genomic data from the GISAID Initiative), accounting for the feasibility of this approach.

Rapid and qualitative identification of SARS-CoV-2 mutations associated with variants of concern using a multiplex RT-PCR assay coupled with melting analysis.

OBJECTIVES: Considering the spread of new genetic variants and their impact on public health, it is important to have assays that are able to rapidly detect SARS-CoV-2 variants. METHODS: We retrospectively examined 118 positive nasopharyngeal swabs, first characterized by the Sanger sequencing, using the Simplexa® SARS-CoV-2 Variants Direct assay, with the aim of evaluating the performance of the assay to detect N501Y, G496S, Q498R, Y505H, E484K, E484Q, E484A, and L452R mutations. RESULTS: A total of 111/118 nasopharyngeal swabs were in complete agreement with the Sanger sequencing, whereas the remaining seven samples were not amplified due to the low viral load. The evaluation of the ability of the assay to detect the E484Q mutation was performed using a viral isolate of the SARS-CoV-2 Kappa variant, showing concordance in 15/15 samples. Simplexa® SARS-CoV-2 Variant Direct assay was able to detect mutation pattern of Alpha, Beta, Gamma, Delta, and Omicron variants with 100% specificity and 94% sensitivity, whereas 100% sensitivity and specificity for the Kappa variant was observed. CONCLUSION: The assay can be useful to obtain faster results, contributing to a prompt surveillance of SARS-CoV-2 variants; however, it requires to be confirmed by the Sanger method, especially in the case of pattern of mutations that are different from those expected and also requires updates as new variants emerge.

Semi-nested RT-PCR enables sensitive and high-throughput detection of SARS-CoV-2 based on melting analysis.

BACKGROUND: Asymptomatic transmission was found to be the Achilles' heel of the symptom-based screening strategy, necessitating the implementation of mass testing to efficiently contain the transmission of COVID-19 pandemic. However, the global shortage of molecular reagents and the low throughput of available realtime PCR facilities were major limiting factors. METHODS: A novel semi-nested and heptaplex (7-plex) RT-PCR assay with melting analysis for detection of SARS-CoV-2 RNA has been established for either individual testing or 96-sample pooled testing. The complex melting spectrum collected from the heptaplex RT-PCR amplicons was interpreted with the support of an artificial intelligence algorithm for the detection of SARS-CoV-2 RNA. The analytical and clinical performance of the semi-nested RT-PCR assay was evaluated using RNAs synthesized in-vitro and those isolated from nasopharyngeal samples. RESULTS: The LOD of the assay for individual testing was estimated to be 7.2 copies/reaction. Clinical performance evaluation indicated a sensitivity of 100% (95% CI: 97.83-100) and a specificity of 99.87% (95% CI: 99.55-99.98). More importantly, the assay supports a breakthrough sample pooling method, which makes possible parallel screening of up to 96 samples in one real-time PCR well without loss of sensitivity. As a result, up to 8,820 individual pre-amplified samples could be screened for SARS-CoV-2 within each 96-well plate of realtime PCR using the pooled testing procedure. CONCLUSION: The novel semi-nested RT-PCR assay provides a solution for highly multiplex (7-plex) detection of SARS-CoV-2 and enables 96-sample pooled detection for increase of testing capacity. .

Development of qPCR tests for monitoring SARSCoV-2 variants through high resolution melting analysis

The current global pandemic (COVID-19) caused by the new Betacoronavirus SARS-CoV-2 is characterized by successive waves of infection due to new variants that include mutations in the gene encoding the Spike protein, the main target of the nucleic acidbased vaccines. In fact, as of autumn 2020, several countries have reported the detection of SARS-CoV-2 variants that have spread more efficiently (referred to as variants of concern by WHO). Such variants include the Alpha variant (English variant, B.1.1.7), the Beta variant (South African variant, B.1.351), the Gamma variant (Brazilian variant, P.1), and the more recent Delta variant (Indian variant, B.1.617. 2). Therefore, it is pivotal to monitor the virus and the onset of SARSCoV-2 variants characterized by high transmissibility or reduced susceptibility to neutralizing antibodies induced by vaccination.Surveillance of genomic variants is currently based on sequencing of viral genomes performed on a random fraction of samples positive by molecular test. The sequencing of 228 SARS-CoV-2 positive samples by ASUR Marche Area Vasta 1 (Fano-Pesaro-Urbino) from February to June 2021 highlighted the progressive increase of variants (mainly B.1.1.7 and to a lesser extent P.1) from early February until March 18th. From March 18th onwards, only variants B.1.1.7 and P.1 were detected. DNA sequencing involves high costs and extended analysis time compared to a PCR-based diagnostic test. To rapidly identify the samples containing virus variants to be sequenced for complete characterization, in synergy with the University of Urbino, five rapid tests based on real-time PCR and high-resolution melting (HRM) were designed on the gene encoding the Spike protein. Preliminary results indicated that the sensitivity of the assays was not significantly different from that of commercial molecular tests. Furthermore, through HRM analysis, it was possible to discriminate amplicons with mutation 1709 C > A causing the amino acid substitution A570D, specific for the alpha variant.