Canine Enteric Virus Detection Assays

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

Published: 2023-10-24 DOI: 10.17504/protocols.io.e6nvwd5z9lmk/v1

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 Enteric Virus 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 type 1 (CAdV-1), canine distemper virus (CDV), canine enteric coronavirus (CECoV), canine parvovirus (CPV), canine rotavirus A (CRV) and SARS-CoV-2 in rectal swabs and fecal samples.

Steps

Reaction Set Up

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 Canine Enteric Virus Detection Assays do not include an internal control, but positive controls are provided for each of the three assays (CEA_1 and CEA_2). 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.

Select the following fluorescence channels: ABY^TM, FAM^TM, JUN^TM and VIC^TM.

Note

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

5.

The standard mode should be selected, following the table below:

Table 2. Thermo profile

RESULTS INTERPRETATION

6.

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.

7.

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

Table 3. Positive control validation criteria

8.

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: