In this current issue of Covid 19 Virus, we would like to introduce our self as the distributor for the LGC-Biosearch Technologies CDC ( CDC- Center for Disease Control and Prevention) approved Covid 2019 One step RT- qPCR Probe and Primer kit with EpiScript Reverse Transcriptase and the RapiDxFire qPCR Master Mix for Hyderbad Region.
https://www.cdc.gov/coronavirus/2019-ncov/downloads/List-of-Acceptable-Commercial-Primers-Probes.pdf
https://www.cdc.gov/coronavirus/2019-ncov/lab/tool-virus-requests.html
Assay preparation
The SARS-CoV-2 Probe and Primer kit includes 3 tubes. Each tube contains a lyophilised assay consisting of 1 probe and 2 primers (2.5 nmol probe and 10 nmol of each primer). Please see attached product insert if needed.
As explained in the CDC protocol (attached – Page 11), each assay should be resuspended in 1.5 mL of nuclease-free water. Please refer to the CDC protocol for recommendations on how to aliquot and store the assays.
Preparation of reaction mix
Use the recommendations of the CDC protocol for handling the RNA samples and organising the preparation of the reaction mix (although they are using different reagents).
I have attached the user manuals for the EpiScript RT and the RapiDxFire qPCR Master Mix for recommendations on storage and handling. You will receive them with the products you have ordered as well.
EpiScript comes with 10x RT Buffer and 100mM DTT. You can include DTT into the reaction optionally, but please don’t use 10x RT Buffer
Use the attach excel file to setup assay test (3 reactions per sample – N1, N2 and RP)
RT-qPCR reaction
The attached “Proof of concept” was run a Biorad CFX qPCR instrument, protocol is attached in the “One-Step RT-qPCR Methods_Episcript_COVID-19 v2” excel file.
You can use it as a start point on any qPCR / RT-PCR instrument and adapt it later if needed.
IMPORTANT NOTES
Please take the time to read both the CDC protocol and the Proof of Concept protocol.
Double check the information I have provided in the table above with your own calculations before starting with the tests.
We have never run this combination (2019-nCov Primer Probe set + EpiScript + RapiDxFire MM) but our R&D support is confident that this PCR set up should work (only the probe final concentration is changed) so hopefully you get expected results during your tests. However, we have done similar test using EpiScript + RapiDxFire MM + custom synthesize nCov primer probes in house. Also enclosed here is the data from this run (combining 2019-nCoV Probe and Primer, with RapiDxFire Master Mix and EpiScript RT). The result is promising, it suggest that it is possible to use this protocol for detecting presence of SARS-Cov Virus RNA.
However, since the result is just proof of concept, when the customer receive the 2019-nCoV Probe and Primer, with RapiDxFire Master Mix and EpiScript RT, customer will have to carry out own validation of assay and sensitivity determination. The customer has to determine in their own hands if the probe and primer concentration, reagent volume etc. work best for their RUO purposes. That is because we did not design the assay ourselves (CDC did), therefore we cannot make recommendations on how they can change the protocol.
Future Orders Information:
The 2019-nCov kit contains enough probes and primers for 1000 reactions per assay (without considering overage for pipetting)
There are 3 qPCR reactions to be run for each sample (N1, N2, Rp), so a total of 3000 reactions per kit.
If replicates is required, multiply reactions accordingly (e.g. 3 replicates x 3 qPCR reactions x number of samples)
If including 20% dead volume for pipetting, you should be able to run 800 samples per kit (so 2400 reactions in total).
Product: We are the supplier of ELISA, PCR, Real Time PCR, Rapid detection kits for Food Testing, Research, Diagnostics testing purpose.