Jan 23, 2024
Detection of Influenza A viruses and Avian H5 Subtype using a triplex qRT-PCR assay on the ABI Quantstudio 7 PCR system
- Frankie Tsang1,
- Kathleen Kolehmainen1,
- Natalie Prystajecky1,2,
- Agatha Jassem1,2,
- John Tyson1,2,
- Michael Chan1,
- Tracy Lee3
- 1BCCDC Public Health Laboratory;
- 2University of British Columbia;
- 3BCCDC
Protocol Citation: Frankie Tsang, Kathleen Kolehmainen, Natalie Prystajecky, Agatha Jassem, John Tyson, Michael Chan, Tracy Lee 2024. Detection of Influenza A viruses and Avian H5 Subtype using a triplex qRT-PCR assay on the ABI Quantstudio 7 PCR system. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvj81xxgk5/v1
Manuscript citation:
Tracy D. Lee, Frankie Tsang, Kathleen Kolehmainen, Natalie A. Prystajecky, Agatha N. Jassem, John R. Tyson. 2023. A multiplex qRT-PCR assay for detection of Influenza A and H5 subtype targeting new SNPs present in high pathogenicity avian influenza Canadian 2022 outbreak strains. medRxiv 2023.12.13.23298992; doi: https://doi.org/10.1101/2023.12.13.23298992
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 17, 2023
Last Modified: January 23, 2024
Protocol Integer ID: 78987
Funders Acknowledgement:
This study was funded through British Columbia Centre for Disease Control operational funding sources.
Abstract
This procedure provides instructions on how to perform a real-time PCR (qPCR) for the detection of Influenza A viruses and avian H5 subtypes from nucleic acid extracted from respiratory samples. This assay targets the avian influenza hemagglutinin (HA) gene of Influenza A subtype H5, primers and probes sequences are updated to enable detection of recent outbreak of Influenza A subtype H5N1. The matrix (M) gene of Influenza A virus is included in this triplex for detection of suspect avian influenza virus (AIV). This assay is not validated and does not contain an endogenous control for the human or avian samples.
Guidelines
This assay is not validated and does not contain an endogenous control for the human or avian samples.
Nucleic acid extracted from avian samples or patient respiratory samples according to validated procedures using the MagMax Express. All human samples for non-human Influenza A subtyping should be Influenza A positive by another method and be considered non-typable for H3 and pH1N1.
Quality Control:
A.Positive Extraction Control
Influenza A culture with expected Ct range at Ct 25 - 27
B.Negative Extraction Control
MEM or UltraPure DNase/RNase-Free Distilled water extracted alongside samples.
C.Positive PCR control (Synthetic)
Prepare synthetic control to act as a positive control for hemagglutinin H5, and the matrix gene (all subtypes). Examples can include gBlocks, GeneArt Strings, commerically prepared nucleic acids, or previous positive sample.
D.Negative PCR control (no template)
UltraPure DNase/RNase-Free Distilled water in PCR mastermix.
Materials
| A | B | C | |
| Reagents | Equipment | Supplies | |
| TaqMan™ Fast Virus 1-Step Master Mix (ThermoFisher Scientific, 444434) | ABI Quantstudio 7 Pro real-time PCR system | MicroAmp Optical Adhesive Film and Applicator | |
| IDTE 1x TE Buffer pH 8.0 | MicroAmp 96-Well Plate Holder | Applied Biosystems Fast Optical 96-Well Plates | |
| UltraPure™ DNase/RNase-Free Distilled Water | Biological safety cabinet- 6ft, Nuaire, Class II Type A/B3 | Combi tips | |
| Custom primers and probes from ThermoFisher Scientific and IDT | Appropriate volume dispensing pipettes (single and multi-channel) | Pipet tips; various sizes. Filter plugged and nuclease free | |
| DNA AWAY | Plate centrifuge | 1.7 ml microcentrifuge tube | |
| Vortex Mixer | Ziploc bags | ||
| Repeater pipet | Disposable Powder-free Gloves and Gowns | ||
| Discard pail | |||
| Waste bags |
Preparation of 20x Primer/Probe Mix
Preparation of 20x Primer/Probe Mix
In the reagent preparation clean room, take out the primers and probes listed in table 1 from the freezer and thaw in the refrigerator.
| A | B | C | D | |
| Gene Target | Oligo Name | Final Concentration (nM) | Sequence 5’ - 3’ | |
| H5-P3 | H5_22_F3 | 450 | GTTTATAGAGGGAGGATGGCAG | |
| H5_22_R3 | 450 | ATGATTGAGTTGACCTTATTGGTAACTC | ||
| H5_22_FAM_MGB_P3 | 150 | ATGGTTGATGGTTGGTATG | ||
| H5-P4 | H5_22_F4 | 450 | GACGTATGACTACCCTCAGTATTCAGA | |
| H5_22_R4 | 450 | CATTGGAGCACATCCATAAAGATAGA | ||
| H5_22_VIC_MGB_P4 | 150 | AGGAACTTACCAGATACTGTCAA | ||
| Influenza A (M Fouchier) (1) | FluA-M253R | 400 | AGGGCATTTTGGACAAAKCGTCTA | |
| FluA-M52C | 400 | CTTCTAACCGAGGTCGAAACG | ||
| FluA-M96C_taq ABY-QSY | 200 | CCGTCAGGCCCCCTCAAAGC |
Table 1. Primers and Probes used in this assay.
Once thawed, vortex well and centrifuge down all components
In a clean BSC, prepare the 20x primer/probe mix according to the recipe in Table 2
(Adjust volume accordingly based on the amount needed):
| A | B | C | D | E | F | |
| 20x H5 Triplex Fochier M Region, H5_P3, H5_P4 | ||||||
| Stock Reagent | Stock conc. (uM) | Final conc./rxn (nM) | 100 rxn (µl) | 500 rxn (µl) | 1000 rxn (µl) | |
| M52C primer | 100 | 400 | 8 | 40 | 80 | |
| M253R primer | 100 | 400 | 8 | 40 | 80 | |
| M96C (ABY QSY) | 100 | 200 | 4 | 20 | 40 | |
| H5_22_P4 Forward | 100 | 450 | 9 | 45 | 90 | |
| H5_22_P4 Forward | 100 | 450 | 9 | 45 | 90 | |
| H5_22_P4 (VIC MGB) | 100 | 150 | 3 | 15 | 30 | |
| H5_22_P3 Forward | 100 | 450 | 9 | 45 | 90 | |
| H5_22_P3 Forward | 100 | 450 | 9 | 45 | 90 | |
| H5_22_P3 (FAM MGB) | 100 | 150 | 3 | 15 | 30 | |
| IDTE 1x TE Buffer pH 8.0 | 1x | 1x | 38 | 190 | 380 | |
Table 2. 20X Primer/Probe mix Recipe. "Rxn": reaction.
Note: Note: Store the 20x mixes at -20°C in the dark. Excessive exposure to light may affect the fluorescent probes. Do not perform more than 10 freeze-thaw cycles. If you expect to freeze-thaw the 20x mix more than five times, consider aliquoting the 20x into smaller volume to minimize the number of freeze-thaw cycles
Setting up and Running qPCR on the ABI Quantstudio 7 Pro
Setting up and Running qPCR on the ABI Quantstudio 7 Pro
Prepare the qRT-PCR cocktail using the TaqMan® Fast Virus 1-Step Master Mix Kit, following the recipe. Calculate the volume needed based on number of reactions per run. Record lot number of reagents used.
| A | B | |
| Reagent | Vol./reaction (µl) | |
| PCR grade Water | 9 | |
| TaqMan® Fast Virus 1-Step Master Mix (4x) | 5 | |
| 20x primer/probe mix | 1 |
Table 3. Master Mix Cocktail Recipe
Vortex and spin down the mastermix cocktails.
Remove an Applied Biosystems Fast Optical 96-Well Plate from the box, inside the BSC, and place it in a MicroAmp 96-Well Plate Holder to ensure that the bottom of the plate has no contact with anything that could fluoresce.
Note: Do not use permanent marker on optical plates, the ink will fluoresce.
Aliquot 15µL of each cocktail to the wells being used for the assays in the ABI Fast Optical 96-Well plate. Seal with an adhesive film aseptically.
Place the plate in a clean plastic bag and transport it to the genomic preparation room. Place the plate in a MicroAmp 96-Well Plate Holder.
In the genomic room, add 5µL of sample template and controls to the appropriate wells of the 96-well plate.
Seal the plate with a MicroAmp optical plate film using the plate film applicator.
Note: Be careful not to contaminate the plate film optical surface with anything that could fluoresce such as ink, dust or fingerprints.
Place the sealed 96-well plate back in the clean plastic bag. Centrifuge in a plate centrifuge and ensure there are no bubbles in the wells before proceeding. Transport the plate to the ABI QuantStudio 7 Pro.
Note: Centrifugation and transportation in the clean plastic bag prevents contamination with dust prior to loading on the instrument.
Load the PCR plate into the ABI Quantstudio 7 Pro.
Load the following run method on the ABI Quantstudio 7 Pro:
Thermalcycling conditions:
| A | B | C | D | |
| Step | Temperature (°C) | Time (min:sec) | Cycles | |
| Reverse Transcription | 50 | 5:00 | 1 | |
| Initial Denaturation | 95 | 0:20 | 1 | |
| Denaturation | 95 | 0:03 | 40 | |
| Annealing/Extension | 60 | 0:30 |
Table 4. ABI Quantstudio 7 Pro Thermalcycling Program
Experimental Properties:
- Analysis Module: Presence Absence
- Run Mode: Fast
- Heat Cover Temperature: 105.0 °C
- Reaction Vol. per Well: 20µL
- Template: 5µL
- Passive Reference: ROX
Confirm that the cycling conditions are correct before starting the instrument.
Enter the samples under "Plate Set-up" with the following Target properties:
| Target | Reporter | Quencher | |
| H5-P3 | FAM | NFQ-MGB | |
| H5-P4 | VIC | NFQ-MGB | |
| Flu A-M96C | ABY | QSY |
Table 5. Target properties used in the Plate Set-up
Save then start the run. Confirm that the instrument is running before you leave.
Results Analysis
Results Analysis
Remove your plate from the QS 7, place it back in the clean plastic bag, seal, and discard in biohazardous bin.
Ensure the following thresholds are set for each target:
| A | B | |
| Target | Threshold | |
| Influenza A (M-Fouchier) | 0.05 | |
| H5-P3 | 0.06 | |
| H5-P4 | 0.1 |
Table 6. Threshold settings
Analyze the qRT-PCR results of the controls. Verify that the results fall within the acceptable range.
Protocol references
1. Fouchier, R., et al. (2000). Detection of Influenza A Viruses from Different Species by PCR Amplification of Conserved Sequences in the Matrix Gene. Journal of Clinical Microbiology. 38(11): 4096-4101