TITLE: Detection of Influenza A viruses and Avian H5 Subtype using a triplex qRT-PCR assay on the ABI Quantstudio 7 PCR system
AUTHORS: Tracy Lee, frankie.tsang, Kathleen Kolehmainen, Natalie Prystajecky, Agatha N. Jassem, John R. Tyson, Michael Chan

[DESCRIPTION]
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.

[STEPS]
SECTION: Preparation of 20x Primer/Probe Mix
1. In the reagent preparation clean room, take out the primers and probes listed in table 1 from the freezer and thaw in the refrigerator.

 

SECTION: Preparation of 20x Primer/Probe Mix
2. Once thawed, vortex well and centrifuge down all components
SECTION: Preparation of 20x Primer/Probe Mix
3. 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):

 
     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 probe)      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   
 
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
SECTION: Setting up and Running qPCR on the ABI Quantstudio 7 Pro
4. 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.
 
       Reagent         Vol./reaction (µl)            PCR grade Water         9            TaqMan® Fast Virus   1-Step Master Mix (4x)         5            20x primer/probe mix          1      

Vortex and spin down the mastermix cocktails.
 
SECTION: Setting up and Running qPCR on the ABI Quantstudio 7 Pro
5. 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.
SECTION: Setting up and Running qPCR on the ABI Quantstudio 7 Pro
6. 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.
 

 
SECTION: Setting up and Running qPCR on the ABI Quantstudio 7 Pro
7. 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.
SECTION: Setting up and Running qPCR on the ABI Quantstudio 7 Pro
8. In the genomic room, add 5µL of sample template and controls to the appropriate wells of the 96-well plate.
SECTION: Setting up and Running qPCR on the ABI Quantstudio 7 Pro
9. 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.
SECTION: Setting up and Running qPCR on the ABI Quantstudio 7 Pro
10. 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.
SECTION: Setting up and Running qPCR on the ABI Quantstudio 7 Pro
11. Load the PCR plate into the ABI Quantstudio 7 Pro.

SECTION: Setting up and Running qPCR on the ABI Quantstudio 7 Pro
12. Load the following run method on the ABI Quantstudio 7 Pro:

Thermalcycling conditions:
 
     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   

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.

SECTION: Setting up and Running qPCR on the ABI Quantstudio 7 Pro
13. 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   

SECTION: Setting up and Running qPCR on the ABI Quantstudio 7 Pro
14. Save then start the run. Confirm that the instrument is running before you leave.
SECTION: Results Analysis
15. Remove your plate from the QS 7, place it back in the clean plastic bag, seal, and discard in biohazardous bin.
SECTION: Results Analysis
16. Ensure the following thresholds are set for each target:
 
     Target      Threshold        Influenza A (M-Fouchier)      0.05        H5-P3      0.06        H5-P4      0.1   

 
 
SECTION: Results Analysis
17. Analyze the qRT-PCR results of the controls. Verify that the results fall within the acceptable range.