Oct 23, 2023
Canine Respiratory Pathogen Detection Assays
- Come J Thieulent1,
- Mariano Carossino1,
- Laura Peak2,
- Udeni B. R. Balasuriya1
- 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
Protocol Citation: Come J Thieulent, Mariano Carossino, Laura Peak, Udeni B. R. Balasuriya 2023. Canine Respiratory Pathogen Detection Assays. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygx9x7zg8j/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: March 29, 2023
Last Modified: October 23, 2023
Protocol Integer ID: 79708
Funders Acknowledgement:
Vet-LIRN
Grant ID: 1 U18 FD007514
Disclaimer
Reference to any commercial materials, equipment, or processes do not in any way constitute approval, endorsement, or recommendation by the Food and Drug Administration.
Abstract
The Canine Respiratory Pathogen (CRP) Detection Assays is intended as an in vitro veterinary reagent set, based on Reverse Transcription quantitative PCR (RT-qPCR), for the detection of canine adenovirus-2 (CAdV-2), canine distemper (CDV), canine herpesvirus type 1 (CHV-1), canine influenza A virus (CIV), canine parainfluenza (CPiV), canine pneumovirus (CPnV), canine respiratory coronavirus (CRCoV), SARS-CoV-2, Bordetella bronchiseptica, Streptococcus equi subsp. zooepidemicus, Mycoplasma cynos and Mycoplasma canis in nasal and pharyngeal swab samples.
Guidelines
Shipping and Storage
The CRP 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.
Limitation:
- 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 Canine Influenza A Subtypes Identification Assay 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) 60 copies/l.
Materials
Assay description and components
The reagents are assembled for 60 reactions (+ 10% extra).
* 3 tubes of primers & probes and positive controls are provided and correspond to the canine respiratory assays CRA_1, CRA_2, and CRA_3.
Probe dye setting
TaqMan QSYTM Probe set 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 cap and keep on ice
Note
The CRP Detection Reagents do not include an internal control, but positive controls are provided for each of the three assays (CRA_1, CRA_2 and CRA_3). 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:
| A | B | C | D | |
| Step | Number of cycles | Temp. (°C) | Time (min:sec) | |
| Reverse transcriptase | 1 | 50 | 20:00 | |
| Initial activation | 1 | 95 | 15:00 | |
| Denaturation | 40 | 94 | 00:45 | |
| Annealing/extension | 60 | 00:75 |
Table 2. Thermal profile. The data acquisition is performed during the annealing/extension step.
Results interpretation
Results interpretation
Before results analysis, 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-001 Version 1.0