Development of Multiplex Real-Time RT-qPCR Assays for the Detection of SARS-CoV-2, Influenza A/B, and MERS-CoV

The overall goal of this assay is to administrate a multiplex approach for the simultaneous detection of commonly circulating respiratory viruses such as SARS-CoV-2, MERS, influenza A, and influenza B.The prevailing technologies driving advancements in our field include CRISPR, lateral flow amino assays, paper-based biomolecular sensors, Sherlock testing in one pot, DNA aptamers, and multiplex RT-qPCR integrated with loop-mediated isothermal amplification, LAMP. However, multiplex RT-qPCR is the most sensitive, common, and is the gold standard for diagnosing various viruses. The current experimental challenge is to establish a protocol that can be used to detect RNAs copies as low as 10 copies.

We used in-house RT-qPCR kit for the assay, which is time and cost effective. To begin, prepare the 2x buffer for RT-qPCR in DNAs/RNAs-free water Store the buffer at minus 20 degrees Celsius until further use. Next, mix the primers and the probes in a 1.5 milliliter tube.

Make up the volume with elution buffer, then store the tube at minus 20 degrees Celsius. Prepare an enzyme mix with taq polymerase and MMLV-RT in a 1.5 milliliter tube on ice. Make up the volume with taq polymerase storage buffer.

Now resuspend the viral synthetic RNAs in separate tubes with 100 microliters of Tris-EDTA buffer to make stocks of 10 to the sixth RNA copies per microliter. To mix the virus stocks add five microliters Each of SARS-CoV-2, influenza A and B to 50 microliters of Tris-EDTA buffer. Similarly mix SARS-CoV-2 and MERS-CoV to obtain a second viral mix.

Dilute both mixtures tenfold to obtain a final dilution of 10 RNA copies per microliter. To each well of a 96 well plate, pipette 2x buffer, primer, enzyme, DNAs/RNAs-free water, and the corresponding RNA mixtures. Seal the plate with an adhesive plate seal.

Then centrifuge the plate for one minute to collect the liquid at the well bottom. Next program the PCR machine to accept the fast 96 well block type with the experimental type set to standard curve. Set the reagents to TaqMan reagents and select standard run properties.

Define the gene targets and the reporter dye. Then define the sample names for each reaction to be tested and assign targets and samples to the plate layout. Now input the cycle program into the instrument.

Transfer the plate to the real-time QPCR machine in the correct orientation and press run to initiate the cycle. Choose the file location to save the experimental data. Then examine the amplification plots provided by the QPCR program.

The two developed kits were able to successfully amplify all three target genes simultaneously, with as low as 10 RNA copies per reaction. The amplification efficiencies for all viral titrations were above 99%