A Molecular-Based Assay for Quantifying Influenza A and B Viruses

Take a molecular detection system containing a pre-filled sample receiver, a transfer cartridge, and a test base. 

The test base has reaction tubes containing T1 and T2 primers with nick sites and influenza  A and B recognition regions, an enzyme mix, and molecular beacons with a fluorophore and quencher.

Transfer the virus-containing nasopharyngeal samples in a lysis buffer from the sample receiver to the test base.

Viral lysis releases RNA, followed by the binding of T2 primer to the target region of RNA and extension by reverse transcriptase.

The other T2 primer binds to the same RNA — displacing the initially extended strand.

The T1 primer binds to the displaced strand, and DNA polymerase extends it. A nicking enzyme cuts its site, with polymerization and strand displacement, forming double-stranded DNA.

The process continues, amplifying duplexes with single-stranded overhangs.

Molecular beacons interact with overhangs, separating the fluorophore from the quencher and producing fluorescence to quantify Influenza A and B.

To begin the test process, touch Run Test on the instrument screen. Next, enter the patient ID by using the on-screen keyboard or barcode scanner, and press OK. After that, open the lid and insert the orange test base tube into the orange test base holder. Confirm that the correct test is displayed on the screen, and press OK. Then, insert the blue sample receiver into the blue sample receiver holder and wait for the sample receiver to warm up. Afterward, remove the foil seal by placing two fingers on the edge of the sample receiver to ensure that it stays in place.

Then, place the patient swab to be tested into the sample receiver. Vigorously mix the swab in the liquid for 10 seconds and press the swab head against the side of the sample receiver to help dislodge the specimen from the swab. If the testing swabs are stored in viral transport medium, vortex the viral transport media for 10 seconds, and add 200 microliters of it to the sample receiver. Then, press OK to proceed.

After that, press the white transfer cartridge into the blue sample receiver. Continue pressing on the sample receiver until the orange indicator rises. Then, lift and connect the transfer cartridge to the test base. The orange indicator should descend once the cartridge transfer is attached correctly. Close the lid and do not open until the result screen appears.

Now, touch Run QC Test on the home screen. Select the QC test to be run. Then, confirm the test type to match the QC sample intended for testing by clicking OK and following the on-screen prompts to complete testing. After the test run is complete, observe the results of the test displayed on the screen with interpretations for the presence of influenza A, B, or unknown subtypes and repeat the test for invalid results.