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    Feline Respiratory Pathogen Detection Assays

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

    Published: 2023-11-21 DOI: 10.17504/protocols.io.ewov1q4z2gr2/v1
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    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.

    Steps

    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 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.

    3.

    Setup the Master Mix according to the following table 1:

    Table 1. Reaction mix preparation
    Table 1. Reaction mix preparation

    PROGRAMMING THE THERMOCYCLER

    4.

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

    Note
    ROXTM should be used as a passive reference dye.

    5.

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

    Table 2. Thermo profile
    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
    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:

    Table 4. Ct cut-off values
    Table 4. Ct cut-off values

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