Oct 23, 2023

Public workspaceCanine Influenza A Subtype Identification Assay

DOI
dx.doi.org/10.17504/protocols.io.14egn2o9pg5d/v1
  • 1Louisiana Animal Disease Diagnostic Laboratory and Department of Pathobiological Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA, USA;
  • 2Louisiana Animal Disease Diagnostic Laboratory, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA, USA
Come J Thieulent
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DOI: dx.doi.org/10.17504/protocols.io.14egn2o9pg5d/v1
Protocol Citation: Come J Thieulent, Mariano Carossino, Laura Peak, Udeni B. R. Balasuriya 2023. Canine Influenza A Subtype Identification Assay. protocols.io https://dx.doi.org/10.17504/protocols.io.14egn2o9pg5d/v1
Manuscript citation:
Development and validation of a panel of one-step 4-plex qPCR/RT-qPCR assays for simultaneous detection SARS-CoV-2 and other pathogens associated with canine infectious respiratory disease complex. Thieulent C. J., Carossino M., Peak L., Strother K., Wolfson W., Balasuriya U. B. R. Applied Microbiology and Biotechnology.
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 29, 2023
Last Modified: October 23, 2023
Protocol Integer ID: 79703
Funders Acknowledgement:
Vet-LIRN
Grant ID: 1 U18 FD007514
Disclaimer
Reference to any commercial materials, equipment, or process does not in any way constitute approval, endorsement, or recommendation by the Food and Drug Administration.
Abstract
The Canine Influenza A Subtype Identification Assay is intended as an in vitro veterinary reagent set, based on Reverse Transcription quantitative PCR (RT-qPCR), for the detection of canine influenza A virus (CIV) and identification of CIV H3N2, CIV H3N8 and CIV H1N1 in nasal and pharyngeal swab samples.



Guidelines
Storage and Shipping
The Canine Influenza A Subtype Identification Assay is shipped on dry ice. Reagents should arrive frozen. The Reagents in the purple and red tubes may arrive liquid, this will not result in a reduction in performance.

All reagents should be stored at -20°C upon arrival. All reagents can be stored for a minimum of one year (from the date of shipment) at -20°C without showing a reduction in performance. Positive controls should be stored at -80°C

Limitation:
  1. Strict compliance with the instructions is required for optimal results.
  2. Appropriate specimen collection, transport, storage, and processing procedures are required for the optimal performance of this test.
  3. The presence of RT-PCR inhibitors may cause false negatives.
  4. Results of Canine Influenza A Subtype Identification Assay need to be interpreted in consideration of all clinical and laboratory findings.

Quality Control:
  1. The specificity of each test was validated using a panel of reference and related canine respiratory pathogens.
  2. The analytical sensitivity of each assay was determined using ten-fold dilution of in vitro transcribed RNA. All assays have a limit of detection (LOD95) 12 copies/l.

Materials
Assay description and components
The reagents are assembled for 60 reactions (+ 10% extra).


Probe dye setting
TaqMan QSYTM Probe sets are used as follows:
Table 2. TaqMan probe set
Material and equipment required but NOT provided.

  • Appropriate nucleic acid extraction instrument and kits
  • Appropriate real-time PCR instrument calibrated for ABYTM, FAMTM, JUNTM and VICTM dyes (e.g., Applied Biosystems 7500 Fast Real-time PCR machine)
  • Vortex and benchtop centrifuge
  • Appropriate 96-well reaction plate or reaction tubes with corresponding closing tape or caps
  • Pipettes & tips
  • Personal Protective Equipment (PPE)

Reaction Setup
Reaction Setup
Thaw all reagents on ice.
Centrifuge all reagents on a benchtop centrifuge to ensure no liquid is in the cap and keep on ice

Note
The Canine Influenza A Subtypes Identification Assay does not include an internal control, but positive controls are provided. A positive and a negative control should be run simultaneously with each sample setup.

Setup the Master Mix according to the following table 1:


Table 1.

Programming the Thermocycler
Programming the Thermocycler
The following fluorescence channels should be selected: ABYTM, FAMTM, JUNTM, and VICTM.
ROXTM should be used as a passive reference dye.
The standard mode should be selected. Setup cycling condition following table 2:
Table 2. Thermal profile

Results interpretation
Results interpretation
Before analysis of results, the threshold value of each fluorescent dye must be manually set in the region of exponential amplification, typically 0.1 × ΔRn value at the plateau phase.
Each assay is considered valid if the following criteria are met:
Table 3. Assays criteria

The results are qualitative (Positive or Negative). A specimen is considered positive if the Ct value obtained is below the following Ct cut-off values:
Table 4. Ct cut-off values

Protocol references
Molecular Virology Laboratory
Louisiana Animal Disease Diagnostic Laboratory (LADDL)
River Road 1043, Baton Rouge, LA 70803
Cat. No. CMD-002 Version 1.0