TITLE: A novel RT-qPCR assay for detection of SARS-CoV-2 variants based on RhAmp technology (IDT technologies)
AUTHORS: Victor Emmanuel Viana Geddes, Filipe Moreira, Diego Menezes Bonfim, Hugo José Alves, João Locke Ferreira de Araújo, Daniel Costa Queiroz, Rafael Marques de Souza, Rennan Garcias Moreira, Camila Zolini de Sá, Danielle Alves Gomes Zauli, Joice do Prado Silva, Aline Brito de Lima, Frederico Scott Varella Malta, Alessandro Clayton de Souza Ferreira, Victor Cavalcanti Pardini, atanuri , Renan Pedra Souza, Renato Santana Aguiar

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:justify">    Brazil was one of the most affected countries during the SARS-CoV-2 pandemic, with more than 10 million cases and more than 300.000 deaths reported. Together with the increasing rate of cases, the emergence of new variants of concern (VOCs) possessing mutations that affect immunogenicity and transmissibility poses a concerning burden in the healthcare system. In Brazil, three such lineages are currently circulating: P.1, a lineage firstly described in Manaus, P.2, firstly described in Rio de Janeiro, both already reported in other cities in the country, and B.1.1.7, a lineage initially identified in England and recently associated to increasing mortality rates.In this scenario, it urges the necessity of a rapid assessment methodology for identification of variants that could contribute to a better epidemiological surveillance and public healthcare administration decisions in dealing with the pandemic. Here, we describe a PCR genotyping approach using RhAmp technology (IDT Technologies) to correctly assess the circulating emergent variants P.1, P.2 and B.1.1.7 in Brazil. Genotyping is a quicker methodology requiring only a real time PCR machine available in most of laboratories preforming COVID-19 molecular diagnosis.</div><style>
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[STEPS]
?. Prepare the genotyping reaction mix as follows:Reagent1 reaction (µl)100 reactions (µl)Combined Master Mix5.3530SNP assay 20X (for K417T or N501Y assays)0.550Sample cDNA4.2-Total volume101000
Reagent1 reaction (µl)100 reactions (µl)Combined Master Mix5.3530SNP assay 20X (for K417T or N501Y assays)0.550Sample cDNA4.2-Total volume101000
?. Extract viral RNA using any commercially available kit according to manufacturer’s instructions. We used Quick-RNA Viral Kit (Zymo Research). Prepare cDNA using the maximum amount of RNA from the previous step allowed by your retrotranscriptase kit. We used High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific).
?. Thaw rhAmp® Reporter Mix with Reference and rhAmp® Genotyping Master Mix in ice and rhAmp® SNP assays (20X) (Single Nucleotide Polymorphism assays/primers) at room temperature. Primer sequences are available in Materials section. All reagents are stored at -20 °C. rhAmp® SNP assays are shipped as a resuspended solution but can be purchase in 20X or 80X formats. Working concentration is 20X, dilute 80X assays 4-fold in IDTE, pH 7.5 or RNase/DNase-free water. Briefly vortex and then spin down reagents before use.
?. Dilute rhAmp® Reporter Mix with Reference 1:20 in rhAmp® Genotyping Master Mix to make the Combined Master Mix. As an example, pippet 50 µl of rhAmp® Reporter Mix with Reference in 1 ml of rhAmp® Genotyping Master Mix. We recommend scale the Combined Master Mix for the number of reactions that will be performed, considering 1 negative control (no cDNA) and 2 positive controls (1 for each allele of each target SNPs).
?. Some modifications were performed specifically for the E484K SNP assay. Prepare the Genotyping Reaction Mix for E484K assay as follows: Reagent1 reaction (µl)100 reactions (µl)Combined Master Mix5.3530SNP assay 20X (for E484K assay)0.7575Sample cDNA3.95-Total volume101000
Reagent1 reaction (µl)100 reactions (µl)Combined Master Mix5.3530SNP assay 20X (for E484K assay)0.7575Sample cDNA3.95-Total volume101000
?. Add 5.8 µl of Genotyping Reaction Mix to each well and then add 4.2 µl of cDNA for K417T or N501Y assays, or 3.95 µl for E484K assay. After applying the cDNA, pipette the total volume up and down 10 times to mix. Each reaction is performed in final 10 µl volume. Seal the plate and briefly centrifuge to collect the liquid at the bottom of the plate.
?. Set your Real-Time PCR System for the “genotyping” module and checkbox the software to perform pre-amplification, amplification and post-amplification reads. Although only the post-amplification read is compulsory to perform in a Real-Time PCR System, we strongly recommend that the amplification also be performed in such systems (with data collection at each cycle) to validate the exponential amplification of the sample through multicomponent curves (exponential absolute fluorescence curves).
?. Set the fluorophore for the wild-type (WT) or mutant (Mut) alleles for each SNP assays in the software as follows:MutationFAM (WT) alleleHEX/VIC (Mut) alleleK417TACN501YATE484KGA
MutationFAM (WT) alleleHEX/VIC (Mut) alleleK417TACN501YATE484KGA
?. We recommend running only 1 SNP assay per plate for better visualization of the clustering results. The cycling can be performed with 55 cycles; however, 80 cycles are recommended for better visualization of results. Set the cycling parameters for K417T or N501Y as follows:StepRepetitionTemperatureTimeEnzyme activation195 °C10 minDenaturation55-8095 °C10 secAnnealing55-8060 °C30 sec*Extension55-8068 °C20 sec
StepRepetitionTemperatureTimeEnzyme activation195 °C10 minDenaturation55-8095 °C10 secAnnealing55-8060 °C30 sec*Extension55-8068 °C20 sec
?. For E484K SNP assay, annealing temperature and time were modified to improve the results. Set E484K SNP cycling parameters as follows:StepRepetitionTemperatureTimeEnzyme activation195 °C10 minDenaturation55-8095 °C10 secAnnealing55-8057 °C1 min*Extension55-8068 °C20 sec
StepRepetitionTemperatureTimeEnzyme activation195 °C10 minDenaturation55-8095 °C10 secAnnealing55-8057 °C1 min*Extension55-8068 °C20 sec
?. After the run is completed, the results should be displayed in a cartesian dot plot graph showing the samples clustering patterns in which the wild-type allele should be in one axis and the mutant allele in the other one. For interpretation of the genotypes, use the following chart:GenotypeK417TN501YE484KP.1Mut (C)Mut (T)Mut (A)P.2WT (A)WT (A)Mut (A)B.1.1.7WT (A)Mut (T)WT (G)B.1.351WT (A)Mut (T)Mut (A)OthersWT (A)WT (A)WT (G)OthersMut (C)WT (A)Mut (A)OthersMut (C)WT (A)WT (G)
GenotypeK417TN501YE484KP.1Mut (C)Mut (T)Mut (A)P.2WT (A)WT (A)Mut (A)B.1.1.7WT (A)Mut (T)WT (G)B.1.351WT (A)Mut (T)Mut (A)OthersWT (A)WT (A)WT (G)OthersMut (C)WT (A)Mut (A)OthersMut (C)WT (A)WT (G)