Table 1_Development of Multiplex PCR assay for detection of Canine Infectious Respiratory Disease Complex (CIRDC) pathogens in dogs.docx

BackgroundCanine Infectious Respiratory Disease Complex (CIRDC) is a highly contagious, multifactorial syndrome that primarily affects dogs in crowded environments such as shelters, kennels, and breeding facilities. Three major CIRDC-associated pathogens: Canine distemper virus (CDV), Canine adenovirus type 2 (CAV-2), and Bordetella bronchiseptica (Bb) have been reported in the canine population of India.
Materials and MethodsIn this study, a multiplex PCR (mPCR) assay was developed and optimized for the simultaneous detection of these three pathogens. 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 both singleplex and multiplex formats by adjusting key PCR parameters such as primer concentration, annealing temperature, and incubation time to achieve distinct and reproducible amplification of all three targets.
ResultsThe developed assay 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 with 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 with previously published singleplex PCR methods, the mPCR showed excellent diagnostic performance, achieving 94.12% sensitivity, 94.74% specificity, and 94.55% overall accuracy.
ConclusionThis study demonstrates a rapid, specific, and cost-effective diagnostic mPCR assay capable of efficiently identifying key CIRDC pathogens in a single reaction. The assay is highly suitable for molecular diagnosis as well as large-scale field surveillance.