A Rapid and Consistent Method To Identify SARS-CoV-2 Variants By RT-PCR


 Background

Since 2020, the COVID-19 pandemic spread worldwide causing health, economic, and social distresses. Containment strategy relay on rapid and consistent methodology for molecular detection and characterization. The emerging variants of concern (VOCs) are currently associated with increased infectivity, and immune escape (natural defense mechanisms as well as a vaccine). Several VOCs has been detected and include lineage B.1.1.7 first identified in the UK, linage B.1.351 in South Africa, and lineage P.1 (B.1.1.28.1) in Brazil. Here we validated a rapid and low-cost technique to distinguish B.1.1.7, B.1.351 and P.1 SARS-CoV-2 variants by detecting Spike gene mutations using RT-PCR methodology.
Results

We recruited 77 positive patients affected by Coronavirus Disease-19 (COVID-19). Specific Real-time reverse transcription-polymerase chain reaction (RT-PCR) was employed targeting single nucleotide polymorphisms (SNPs) to screen Spike protein mutations. All data were validated by next generation sequencing (NGS) methodology and using sequence from a public database.Among 77 COVID-19 positive samples we could discriminate with 100% of concordance all the investigated SARS-CoV-2 variants when comparing with NGS method.
Conclusions

PCR-based assays for identification of circulating VOCs of SARS-CoV-2 resulted in a rapid method to identify the specific SARS-CoV-2 variants allowing a better survey of the spread of the virus and its transmissibility in the pandemic phase.


Frequency and Informativity of SNPs marker for SARS-Cov2 variants characterization by qPCR probe
To determine the technical and biological e ciency of each single TaqMan probe assay to discriminate between SARS-Cov2 variants, we considered the frequency and informativity of the single nucleotide polymorphism markers. We de ne "informativity" the percentage of difference between the frequency of a single SNP in one SARS-Cov2 variant respect his frequency in all other SARS-Cov2 variants ( Table 2). In Fig. 2, and Table 2 we showed the frequency and informativity of the single nucleotide marker for each variant group.

Discussion
The emergence of SARS-CoV-2 variants of concern (VOC) with the potential for increased transmission, disease severity, and resistance to vaccine induced immunity is of grave concern. A simple screening assay to monitor the emergence and spread of these strains, despite expensive and time-consuming sequencing the virus genome, may be helpful for implementing public health strategies to counter these and future strains. For early detection and prevalence calculation of VOCs (i.e., B. To date, SARS-CoV-2 variants are mostly screened by NGS methodology of positive samples. Our results show that qPCR methodology using TaqMan probes direct to speci c SNPs can be useful and rapid genotyping tool for SARS-CoV-2 positive samples at a low cost. Testing laboratories may also consider designing their own genotyping panel based on regional or national datasets to maximize the virus survey. The robustness of our panel is further increased by use of two probes in the same reaction, one complementary to the WT sequence and one complementary to the mutant sequence. All diagnostic structure quali ed to detect SARS-CoV-2 in oropharyngeal swab with this low-cost technique could further screen positive SARS-CoV-2 sample to identify variants, thereby providing valuable genomic data to investigate outbreaks, potentially identifying transmission pathways linking local and regional cases and helping to inform possible interventions. Notably, e cient methods for tracking transmission of certain lineages could be vital in situations where mutations are associated with increased transmission, severity of disease or vaccine failure. The limitation of this study is related to the limited number of samples, especially for P.1 variant counting only 6 patients positive and for B.1.351 variant counting only one sample. To overcome at this problem, we used an external sequenced dataset con rming the validity of our results.

Conclusion
Targeting speci c SARS-CoV-2 mutations by qPCR methodology using TaqMan probes is a rapid and consistent method to identify speci c SARS-CoV-2 variants. When used on large scale by a wide number of private or public laboratories this method would be helpful to minimize the spread of the recent emergence of several SARS-Cov2 variants allowing a better survey of the spread of the virus in the pandemic phase.

Sample Collection
Nasopharyngeal/Oropharyngeal swabs collected in Viral Transport Medium were processed in Altamedica laboratory (Rome) for the detection of SARS-CoV-2 virus using approved RT-PCR kits (KHB, Diagnostic kit for SARS-CoV-2). Among these, 77 Viral RNA samples that tested positive for SARS-CoV-2 were randomly selected for this study. The study was conducted with the consent of all the participants, and was approved by the internal Ethics Committee of Altamedica Laboratories, Artemisia S.p.A .

Detection of the SARS-CoV-2 virus by real-time polymerase chain reaction (RT-PCR)
Detection of the SARS-CoV-2 RNA was performed by RT-PCR (KHB, Diagnostic kit for SARS-CoV-2) in accordance with the manufacturer's instructions. The assay targets three genes of the SARS-CoV-2 (N, E and ORF1ab). All samples included in the study had a cycle threshold (Ct) value of the gene targets less than 30 for all genes.

RNA extraction and cDNA synthesis
Viral RNA was extracted from nasopharyngeal swab using automated systems PANA 9600s extractor according to manufacturer's instructions and stored at − 80°C until use. SuperScript 2 VILO cDNA Synthesis Kit (Thermo Fisher, USA) was used to reverse transcribe (RT) the SARS-CoV-2 RNA with the following protocol: 4 µL of 5X VILO™ Reaction Mix, 2 µL of 10X SuperScript™ Enzyme Mix, viral RNA (10 ng), and molecular water to a nal volume of 20 µL. Retrotrasciptase reaction was performed in Thermal Cycler with the following program: 25°C for 10 min, 42°C for 60 min and 85°C for 5 min. To ensure enough cDNA content for NGS work ow, RNA was quanti ed it with Qubit 3.0 uorometer (Thermo Fisher Scienti c, USA).
TaqMan RT-qPCR assay SARS-CoV-2 mutations was genotyping using TaqMan® Universal PCR Master Mix and speci c custom TaqMan probe. Each qPCR mixture is consisting of 10µL TaqMan Universal PCR Master Mix, 1ul of TaqMan probe 10x, 1µL cDNA template and 8µL of nuclease-free water. The thermal protocol was as follows: an initial enzyme activation step was included at 50°C for 2 min, an incubation at 95°C for 10 min followed by 40 cycles of 95°C for 15 s and 1 min annealing and elongation at 60°C. For the use of UMM, which contains Uracil-DNA Glycosylase (UNG), an initial enzyme activation step was included at 50°C for 2 min. All qPCR assays were performed on a QuantStudio 12K Flex Real-Time (Thermo Fisher, USA).

Libraries Preparation and Sequencing
For each sample target ampli cation reaction was set up using 10 µL of cDNA, 4.5 µL of 5X Ion AmpliSeq™ HiFi Mix, and 3.5 µL of water; this mixture was splitted into two different tubes and 2 µL of each of the 5X Ion AmpliSeq™ Primer Pool 1 and 2 were added to the corresponding tubes. Reaction of ampli cation was performed in Thermal Cycler with the following program: 98°C for 2 min, followed by 16 cycles at 98°C for 15 s and 60°C for 4 min. The previous reactions were then combined together and 2 µL FuPa Reagent were added to partially digest the primers (Thermo Fisher Scienti c), afterward the mixture was incubated in Thermal Cycler with the following program: 50°C for 10 min, 55°C for 10 min and 60°C for 20 min. Then, 2 µL of diluted Ion Xpress™ Barcode Adapters together with 4 µL of Switch Solution and 2 µL DNA Ligase were added to ligate the adapters to the ampli ed products, and the samples were incubated with the following program: 22°C for 30 min, 68°C for 5 min, and 72°C 5 min.
After ligation, each DNA library was puri ed with the magnetic beads (Agencourt™ AMPure™ XP Reagent, Beckman Coulter) and then ampli ed with 50 µL of Platinum™ PCR SuperMix HiFi and 2 µL of Library Ampli cation Primer Mix using the following conditions: 2 min at 98°C, 5 cycles of 15 s at 98°C and 1 min at 64°C. The ampli ed libraries were again puri ed with magnetic beads, and the nal concentration of each barcoded cDNA library was determined with

External Validation Dataset
All SARS-CoV-2 genomes sequences were retrieved from NCBI Virus (https://www.ncbi.nlm.nih.gov/labs/virus/vssi/#/). The criteria for download were the presence of all complete genome sequences and all Italian submitted sequences. The sequences met these criteria were 82. The FASTA le sequences was submitted to Pangolin tools for variant calling and to NextClade website for mutations calling.

Statistical analysis
Standard statistical analyses (average, standard deviation) were performed using Microsoft Excel and GraphPad Prism 8.4.3 for Windows. Visualization graphs in this paper are generated using Matplotlib in Python.

SARS-CoV-2
Severe Acute Respiratory Syndrome Coronavirus 2 VOCs variants of concern COVID-19 Coronavirus Disease-19 Declarations Ethics approval and consent to participate: The study was approved by the local ethical committee of Artemisia SPA. Consent for publication: Informed consent was obtained from all subjects involved in the study.
Availability of data and materials: Raw data are available in Supplementary materials, other datasets used and/or analysed during the current study are available from the corresponding author on reasonable

Supplementary Files
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