Canine Respiratory Pathogen Detection Assays

Come J Thieulent, Mariano Carossino, Laura Peak, Udeni B. R. Balasuriya

Published: 2023-10-24 DOI: 10.17504/protocols.io.kxygx9x7zg8j/v1

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.

Steps

Reaction Setup

1.

Thaw all reagents on ice.

2.

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.

3.

Setup the Master Mix according to the following table 1:

Programming the Thermocycler

4.

The following fluorescence channels should be selected: ABY^TM, FAM^TM, JUN^TM, and VIC^TM.

5.

ROX^TM should be used as a passive reference dye.

6.

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

7.

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.

8.

Each assay is considered valid if the following criteria are met:

9.

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: