TITLE: TaqMan Genotyping Assay for Detection of B.1.1.7 SARS-CoV-2
AUTHORS: isaac.bell , Mark Adams

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">At the end of 2020, several new lineages of SARS-CoV-2 were reported that may be more transmissible, less responsive to antibody therapy, or able to “escape” the protection of recently approved vaccines, including B.1.1.7, first reported in the United Kingdom (1). Certain mutations affect functionally important parts of viral proteins and are termed “variants of concern” or VOC. The existing SARS-CoV-2 qPCR and antigen tests cannot identify these new viral variants. Advanced molecular diagnostic assays are needed to understand the spread of VOCs in the state. Viral genome sequencing is the gold standard for characterization mutations, lineage classification, and high-resolution genomic surveillance, but only a small proportion of isolates are currently analyzed by this approach in the USA. Faster and simpler approaches to identifying VOCs based on assays targeting specific mutations represent a complementary approach for monitoring known lineages using assays. </div><div class = "text-block">The B.1.1.7 strain exhibits 17 characteristic mutations including a del69-70 mutation in the S-gene binding domain. This mutation can be monitored using S-gene target of the Applied Biosystems TaqPath™ COVID-19 qPCR assay. Samples that are positive for the N-gene and Orf1ab targets of the multiplexed TaqPath™ assay with weak or no S-gene signal are classified as S-gene target failure (SGTF) samples. The del69-70 mutation has been observed in multiple variant lineages and is not unique to B.1.1.7. We report a set of TaqMan™ genotyping assays to identify B.1.1.7 cases based on discrimination between wild-type and mutant alleles that encode three variants in the spike protein. The genotyping assays described here can be used as follow-up assays to determine whether samples share other characteristic mutations that are more indicative of the B.1.1.7 strain – S.A570D and S.D1118H – as well as the S.N501Y mutation that is shared by many VOCs. The targeted genotyping approach can be performed in most labs that conduct SARS-CoV-2 testing using RT-PCR. Broad use of this and similar approaches can provide an expanded perspective on the prevalence of B.1.1.7 at lower cost and faster turn-around time than whole genome sequencing.</div><div class = "text-block"><span>(1) </span><a href="https://virological.org/t/preliminary-genomic-characterisation-of-an-emergent-sars-cov-2-lineage-in-the-uk-defined-by-a-novel-set-of-spike-mutations/563" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://virological.org/t/preliminary-genomic-characterisation-of-an-emergent-sars-cov-2-lineage-in-the-uk-defined-by-a-novel-set-of-spike-mutations/563</span></a></div></div>

[STEPS]
?. [Assay Preparation]
Gather Supplies Needed for the Assay as listed in the Materials tab and:384-Well PCR PlateOptical Grade PCR plate adhesive film seal384-Well cold blockIce Bucket (ice)Pipettes and Tips for p20 and p200 pipettes
?. [Sample Preparation]
Locate and thaw on ice the extracted RNA samples to be tested.
?. [Sample Preparation]
Label a sterile PCR Tube for each sample to be tested.
?. [Sample Preparation]
Aliquot 20uL of Molecular Grade water into each labeled tube.
?. [Sample Preparation]
Add 5uL of Extracted RNA into each labeled tube and triturate to mix. (avoid bubbles)
?. [Master Mix Preparation]
Label a sterile PCR Tube for each Allele of Interest. Keep on ice during preparation of Maser Mix.
?. [Master Mix Preparation]
For 10 ul reactions performed in a 384-well plate, calculate the number of reactions to be performed including the controls and prepare a Master Mix for each Allele using the following components
?. [Loading the PCR Plate]
Aliquot out 5.5uL of each Allele specific Master Mix into a Specific Quadrant of the 384 well plate for all samples.
?. [Loading the PCR Plate]
Aliquot 5.5uL of each Allele specific Master Mix into a Specific Quadrant of the 384 well plate for controls in Columns 23 and 24, leave columns 21 and 22 empty to avoid control contamination if possible.
?. [Loading the PCR Plate]
Add 4.5uL of Diluted Sample to each well containing Allele Specific Master Mix
?. [Loading the PCR Plate]
Add 4.5uL of Wuhan-Hu-1 control (WT), B.1.1.7 control (VAR), or a 50:50 mixture of the two controls to serve as a "heterozygous" equivalent control (100c/ul) for each Allele specific Master Mix. Use Molecular Grade Water as a no-template control.
?. [Loading the PCR Plate]
?. [Loading the PCR Plate]
Seal the PCR plate with an optical adhesive film Vortex the plate for 3 secondsSpin down the plate at 2000rpm for 1 minute
?. [Running on QS7]
Run the PCR with the following Cycling Conditions
?. [Analysis Settings]
Results are interpreted by the QuantStudio™ Real-Time PCR Software v1.3Open the run file and Navigate to Analysis/Analysis Settings/ CT SettingsEnsure the following analysis settings are appliedNavigate to Analysis/Analysis Settings/Call SettingsClick "Edit default Settings" and change the "Quality Value" to 90
?. [Interpreting Results]
?. [Assay Information]
Disclaimer - FOR INFORMATIONAL PURPOSES ONLY; USE AT YOUR OWN RISKThe protocol content here is for informational purposes only and does not constitute legal, medical, clinical, or safety advice, or otherwise; content added to protocols.io is not peer reviewed and may not have undergone a formal approval of any kind. Information presented in this protocol should not substitute for independent professional judgment, advice, diagnosis, or treatment. Any action you take or refrain from taking using or relying upon the information presented here is strictly at your own risk. You agree that neither the Company nor any of the authors, contributors, administrators, or anyone else associated with protocols.io, can be held responsible for your use of the information contained in or linked to this protocol or any of our Sites/Apps and Services.Compatible Systems Protocol has been tested using:Applied Biosystems Quantstudio™ 7 (384-well format)Applied Biosystems™ VIIA-7 (96-well format)Compatible SoftwareQuantStudio™ Real-Time PCR Software v1.3 or laterApplied Biosystems™ Design and Analysis v2.4.3 or later