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

Overview

The video demonstrates an assay for quantifying influenza viruses A and B. This assay employs nicking endonuclease amplification with influenza-specific primers and target detection using molecular beacon probes.

Protocol

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.

1. Before Running the Assay

NOTE: The Influenza A and B assay is approved for nasopharyngeal swab specimens and for nasopharyngeal swabs stored in viral transport media. Swabs are included in the kit and should be used for optimal performance. However, rayon, foam, flocked swabs, or polyester nasal swabs can also be used to collect nasal swab samples.

1. To collect a nasal swab sample, insert the swab into the nostril that has the most visible drainage or that is most congested. With gentle rotation, push the swab into the nostril until resistance is met, and rotate several times against the nasal wall before slowly removing it from the nostril. Store the swabs and transport them in vials containing 3 ml of viral transport media (VTM).
   NOTE: Careful attention to appropriate specimen collection must be taken as inadequate specimen collection may yield erroneous results.        
   NOTE: Swab specimens should be tested as soon as possible after collection. However, they may be stored at room temperature for up to 2 hr or refrigerated at 2-8 °C for up to 24 hr if testing is not immediately available. Freshly collected specimens are ideal for optimal test performance. Improper sample handling, storage, and/or transport may yield erroneous results.        
   NOTE: As a precautionary measure, individuals performing this test should treat all specimens as potentially infectious by following universal precautions when handling specimens.
2. Bring all samples to room temperature prior to testing.
   NOTE: All test components are for a single use only and should not be used to perform multiple tests. Do not mix kit components from different lot numbers. Do not use kit reagents past their expiration date.
3. Bring the blue sample receiver to room temperature prior to testing. The orange test base can be tested without the need to warm to room temperature.
4. Leave all test pieces in the foil wrapping until immediately before use. Turn on the instrument by pressing the power button on the side of the instrument. Enter the user ID and press 'OK.

2. Running the Test

1. To begin the test process, touch 'Run Test' on the instrument screen. Enter the patient ID by using the on-screen keyboard or barcode scanner and press 'OK.' Open the lid and insert the Orange test base into the Orange test base holder.
2. Confirm that the correct test is displayed on the screen and press 'OK.' Insert the blue sample receiver into the blue sample receiver holder.
   NOTE: Do not open the sample receiver before placing it in the instrument as this will prevent the elution buffer from reaching the correct operating temperature and may impact test performance.
3. Wait for the sample receiver to warm up. When prompted by the instrument, remove the foil seal and place the patient swab to be tested into the sample receiver.
   NOTE: When removing the foil, place two fingers on the edge of the Sample Receiver to ensure that it stays in place.
4. Vigorously mix the swab in the liquid for 10 sec, pressing the swab head against the side of the sample receiver as you mix it to help dislodge the specimen from the swab. Press 'OK' to proceed. If testing swabs are stored in a viral transport medium, vortex the VTM for 10 sec then add 200 µl to the sample receiver.
5. Press the white transfer cartridge into the blue sample receiver. Continue pressing on the sample receiver until the orange indicator rises.
6. Lift and then connect the transfer cartridge to the test base. Observe the orange indicator descend once the cartridge transfer is attached correctly. Close the Lid and do not open until the "Test Complete" message appears on the screen.

3. Quality Control (QC)

1. Touch 'Run QC Test' on the home screen. Select the QC Test to be Run. Confirm the test type to match the QC sample intended for testing by touching 'OK' and following the on-screen prompts to complete testing.

4. Result Interpretation

1. 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, and unknown subtypes. For invalid results repeat the test.

5. Maintenance and Cleaning

1. Clean the instrument and the surrounding bench area daily with 70% ethanol or 10% bleach solution. Spray the cleaning solution on a damp, lint-free cloth to clean. Do not pour the solutions directly on the instrument.

Materials

Name	Company	Catalog Number	Comments
Alere i Instrument	Alere	NAT-000 (Global), NAT-024 (US)
Alere i Influenza A & B 24 Test Kit	Alere	425-000 (Global), 425-024 (US)
Alere i Barcode Scanner	Alere	EQ001001
Alere Universal Printer	Alere	55115 (Global), alereiprinter (US)
200 µL precision pipette
200 µL disposable pipette tips
Viral transport medium	Remel	M4-RT