Nov 21, 2023

Public workspaceFeline Respiratory Pathogen Detection Assays

DOI
dx.doi.org/10.17504/protocols.io.ewov1q4z2gr2/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
Open access
DOI: dx.doi.org/10.17504/protocols.io.ewov1q4z2gr2/v1
Protocol Citation: Come J Thieulent, Mariano Carossino, Laura Peak, Udeni B. R. Balasuriya 2023. Feline Respiratory Pathogen Detection Assays. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov1q4z2gr2/v1
Manuscript citation:

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: July 12, 2023
Last Modified: November 21, 2023
Protocol Integer ID: 84921
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 Feline Respiratory Pathogen (FRP) Detection Assays is intended as an in vitro veterinary reagent set, based on quantitative PCR (qPCR) and Reverse Transcription qPCR (RT-qPCR), for the detection of feline calicivirus (FCV), feline herpesvirus type 1 (FHV-1), influenza A virus (IAV), SARS-CoV-2, Bordetella bronchiseptica, Mycoplasma felis and Chlamydia felis in nasal and pharyngeal swab samples.
Guidelines
SHIPPING & STORAGE INFORMATION
The FRP Detection Assays are 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.

LIMITATIONS:
  • Strict compliance with the instructions is required for optimal results.
  • Appropriate specimen collection, transport, storage, and processing procedures are required for the optimal performance of this test.
  • The presence of RT-PCR inhibitors may cause false negatives.
  • Results of FRP Detection Reagents need to be interpreted in consideration of all clinical and laboratory findings.

QUALITY CONTROL
  • The specificity of each test was validated using a panel of reference and related canine respiratory pathogens.
  • The analytical sensitivity of each assay was determined using ten-fold dilution of in vitro transcribed RNA or plasmid copies number. All assays have a limit of detection (LOD95) 15 copies/l.
Materials
ASSAY DESCRPTION & COMPONENTS

The reagents are assembled for 60 reactions (+ 10% extra).
Table 1. Kit description.
* 2 tubes of primers & probes and positive controls are provided and correspond to the feline respiratory assay 1 (FRA_1) and FRA_2.
PROBE DYE SETTING
TaqMan Probe settings should be as follows:
Table 2. TaqMan probe set
OTHER MATERIALS:
  • Appropriate nucleic acid extraction instrument and kits
  • Appropriate real-time PCR instrument calibrated for ABYTM, Cy5, FAMTM 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)
Thaw all reagents on ice.
Centrifuge all reagents on a benchtop centrifuge to ensure no liquid is in cap and keep on ice
Note
The FRP Detection Reagents do not include an internal control, but positive controls are provided for each of the two assays (FRA_1 and FRA_2). 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. Reaction mix preparation

PROGRAMMING THE THERMOCYCLER
PROGRAMMING THE THERMOCYCLER
Select the following fluorescence channels: ABYTM, Cy5, FAMTM, and VICTM.

Note
ROXTM should be used as a passive reference dye.

The standard mode should be selected, following the table below:
Table 2. Thermo 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. Positive control validation 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. FMD-001 Version 1.1