ORIGINAL RESEARCH article
Front. Cell. Infect. Microbiol.
Sec. Veterinary and Zoonotic Infection
Development of Multiplex PCR assay for detection of Canine Infectious Respiratory Disease (CIRDC) pathogens in dogs
Provisionally accepted
- 1Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar, India
- 2Department of Animal Biotechnology, College of Veterinary Sciences, Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar, India
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Canine Infectious Respiratory Disease Complex (CIRDC) is a highly contagious, multifactorial syndrome that primarily affects dogs in congested environments such as shelters, kennels, and breeding facilities. This study reports the development and optimization of a multiplex PCR (mPCR) assay for the simultaneous detection of three major CIRDC-associated pathogens: Canine distemper virus (CDV), Canine adenovirus type 2 (CAV-2) and Bordetella bronchiseptica (Bb), circulating in Indian population of canines. The multiplex assay was designed targeting three genes H, E3 and bfrZ of CDV, CAV-2 and Bb respectively. This multiplex assay was optimized in singlex as well as multiplex format for various parameters of PCR such as primer concentration, annealing temperature, incubation time in order to achieve distinct and reproducible amplification of all three targets. It demonstrated high analytical sensitivity, detecting 1,060 copies /μL for CDV, 11,403 copies/μL for CAV-2, and 11,016 copies/μL for Bb, with 100% specificity and no cross-reactivity to non-target organisms. The assay was validated on 55 clinical samples of dogs suspected with CIRDC, the assay detected pathogens in 32.2% of cases, with CDV being the most prevalent (25%). Compared to previous published singleplex PCR methods, the mPCR exhibited excellent diagnostic performance, achieving 94.12% sensitivity, 94.74% specificity and 94.55% overall accuracy. These results establish the mPCR assay as a rapid, specific and cost-effective diagnostic tool capable of efficiently identifying key CIRDC pathogens in a single reaction, making it highly suitable for both diagnostics of disease at molecular level and large-scale field surveillance.
Keywords: canine infectious respiratory disease complex (CIRDC), multiplex PCR, Sensitivity, specificity, Dogs
Received: 07 Jul 2025; Accepted: 07 Nov 2025.
Copyright: © 2025 Kaul, Bhagwan, BATRA, Kumar and Agnihotri. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
* Correspondence:
Jai Bhagwan, jaivety.com@gmail.com
KANISHT BATRA, drkanishtbatra@gmail.com
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