Immunology and Infection
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Rapid Molecular Detection and Differentiation of Influenza Viruses A and B
Chapters
Summary January 30th, 2017
We describe a rapid, molecular-based Influenza A and B assay. The Influenza assay detects each target within 15 min by employing isothermal amplification with influenza-specific primers followed by target detection with molecular beacon probes. The Influenza A and B assay is user-friendly and required minimal hands-on time to perform.
Transcript
The overall goal of this assay is to diagnose or rule out influenza virus infection in patients presenting with respiratory systems. This method can help answer key questions in the clinical diagnostics field such as diagnosing key causes of respiratory infections. The main advantage of this technique is that it is a rapid method that can be used in appointive care setting.
Demonstrating the procedure will be Sam Kaplan, a technician from our laboratory. 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 from the nostril.
Subsequently, store the swab and transport it in a vial containing three milliliters of viral transport media. Next bring the sample and 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.
Leave all the test pieces in the foil until immediately before use. Then turn on the instrument, enter the user ID and press OK.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 foiled seal by placing two fingers on the edge of the sample receiver to ensure that it stays in place. Then place the patient's swab to be tested into the sample receiver.
Vigorously mix the swab and 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 Results 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.
After that clean the instrument and the surrounding bench area with 70%ethanol or 10%bleach solution. This figure shows the average CT values determined by the RP assay for the positive and negative samples for each influenza subtype. The results demonstrate that the false negative samples determined by the influenza A and B assay were due to low viral load samples.
Once mastered, this technique can be done in 15 minutes if it is performed properly. While attempting this procedure it's important to remember that when initially setting up the assay, the transfer cartridge must be connected to the test base until they click into place. If these two pieces are not secured properly, the run will fail and will need to be repeated from the beginning.
After watching this video you should have a good understanding of how to perform this diagnostic assay for influenza. Don't forget that working with patient samples potentially infected with influenza can be extremely hazardous and precautions, such as wearing appropriate personal protective equipment, should always be taken while performing this procedure.
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