For the development of mPCR assay targeting Hemagglutinin gene (H gene) of CDV, Early region gene (E3 gene) of CAV-2 and Bordetella filamentous hemagglutinin related gene Z (bfrZ gene) of Bordetella bronchiseptica were selected, respectively. Primers were designed using ClustalW and Mega 10.2 version softwares, with conserved regions identified via sequence alignment of reported GenBank accessions no. such as KC479140, KC479141, LC011103, KC479138, MF964181 (CDV), OP618116, OP644981, MT892837, S38212, U77082 (CAV-2) and AJ251793, CP132332, BX640451, LR134326 (Bb) (Supplementary Table S1). Primer properties were checked in silico using NCBI BLAST and commercially synthesized by IDT, India (Table 1).

CDV and CAV-2 were obtained from live attenuated freeze-dried vaccine Canishot^® DHPPL (Intas Pharmaceuticals, India) and Bb from live freeze-dried vaccine Nobivac^® KC (MSD Animal Health, India).

The extraction of DNA from vaccine was using the DNA extraction kit (HiPurA^® mammalian Genomic DNA Purification Kit, Kennett Square, PA, USA) according to manufacturer’s recommendation and TRIzol method was used for RNA extraction. Briefly, DNA extraction was done by adding 200 μL of vaccine, 200 μL HiPurA^® Genomic lysis/binding buffer, 20 μL of Proteinase K. This mixture was incubated at 55 °C for 1 hour in water bath. After incubation, 200 μL of 100% ethanol was added and homogenous mixture was carefully poured in the HiPurA^® spin column in a 2 ml collection tube and centrifuged at 12000 rpm for 1 min. The spin column was washed with 500 μL of prewash buffer and 500 μL of wash buffer by successive centrifugation at 12000 rpm for one min. After centrifugation, the DNA was eluted by adding 20 μL of elution buffer in spin column and subsequently extracted DNA was stored at -20 °C for future use. For RNA extraction, 400 μL of vaccine was mixed with 1 mL of TRIzol reagent and vortexed vigorously. In this mixture, 200 μL of chloroform was added followed by vigorous vortexing to avoid formation of insoluble aggregates. This mixture was centrifuged for 15 min at 12,000 rpm at 4°C for phase separation. The aqueous phase was then mixed with equal volume of chilled isopropanol and kept at -20°C overnight. RNA was pelleted by spinning at 12,000 rpm for 20 min at 4°C and the supernatant was discarded. The washing of pellet was done by adding 1 mL of 70% ethanol (chilled) and operated at a speed of 12,000 revolutions per minute continuously at 4°C for 10 minutes. After centrifugation, the pellet was air dried for 2 to 3 hours followed by addition of nuclease free water (NFW). Nucleic acids were quantified using Nanodrop spectrophotometer (Thermo Fisher Scientific) at an absorbance of 260 and 280 nm to derive the A₂₆₀/A₂₈₀ ratio.

The cDNA of the isolated RNA was prepared using iScript cDNA Synthesis kit (BioRad, California, USA) as per the manufacturer’s protocol. Briefly, reaction mixture was made using 4 μL of 5X Buffer, 1 μL of Reverse Transcriptase (RT) enzyme, 5 μL of NFW and 10 μL of extracted RNA (50ng) as template. The PCR conditions for the reaction were 25°C for 5 min, followed by 46°C for 1 hour and inactivation at 95°C for 1 min. The cDNA and DNA were stored at −20 °C until used for further PCR amplification.

The extracted DNA (CAV-2 and Bb) and synthesized cDNA (CDV) were subjected to conventional PCR amplification using self-designed primers specific to each target. The PCR for DNA was performed in Thermal cycler (Sure Cycler 8800, Agilent Technologies) in 12.5 μL reaction containing 3 μL of template DNA, 6.25 μL of 2X GoTaq^® Green Master Mix (Promega, Wisconsin, US), 0.4 μM (0.5 μL of 10 μM concentration) each of forward and reverse primers and 2.25 μL of NFW. The PCR for cDNA includes 12.5 μL reaction containing 5 μL of template cDNA, 6.25 μL of 2X GoTaq^® Green Master Mix (Promega, Wisconsin, US), 0.4 μM (0.5 μL of 10 μM concentration) each of forward and reverse primers and 0.25 μL of NFW. The cyclic conditions for all the three PCR (CDV, CAV-2 and Bb) were initial denaturation at 95 °C for 2 minutes, followed by 40 cycles of denaturation at 95 °C for 1 minute, annealing at temperatures ranging from 45 °C to 60 °C for 30 seconds (depending on the primer set), and extension at 72 °C for 30 seconds. A final extension step was carried out at 72 °C for 10 minutes to ensure complete amplification of the target sequences. The PCR products generated were then purified using the Monarch^® DNA Gel Extraction Kit (New England Biolabs Inc., Massachusetts, US), following the manufacturer’s protocol, to obtain clean gel-extracted amplicons suitable for downstream applications.

For generation of positive controls, PCR amplicons of CDV, CAV-2 and Bb were cloned into the pJET1.2/blunt vector (CloneJET™ Kit, Thermo Scientific, Waltham, MA USA) and transformed into E. coli DH5α prepared via calcium chloride method. Briefly, a single colony of E. coli DH5α was inoculated in 5mL LB broth and incubated overnight at 37°C in shaking incubator at 160 rpm. From the overnight grown culture, culture was inoculated in ratio of 1:100 and incubated at 37°C in shaking incubator until the OD₆₀₀ was between 0.4-0.6. After incubation, the cells were centrifuged at 5000 rpm for 10 min at 4°C for pelleting. This pellet was resuspended in 0.6 volume of ice cold 0.1M MgCl2 (4.8 mL) + 0.1M CaCl2 (1.2 mL) in the ratio of 4:1 followed by incubation on ice for 15 min. Cells were again centrifuged at 5000 rpm for 10 min at 4°C. Supernatant was discarded and cell pellet was again resuspended in 500 μL of ice cold 0.1M CaCl2 and stored at 4°C for further use. The PCR products are treated with blunt-end enzyme and ligated in pJET1.2 vector using T4 DNA ligase. The transformants were selected on LB agar with ampicillin. Recombinant clones were identified by colony touch PCR using gene-specific primers under standardized conditions (95°C for a period of 2 min, denaturation at 95°C for a duration of 1 minute, followed by annealing at 55°C for 30 sec, extension at 72°C for 30 sec and final extension at 72°C for 10 minutes).Plasmids were subsequently isolated using the Monarch^® Plasmid Miniprep Kit (NEB, Massachusetts, US) and quantified using Nanodrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA USA). Plasmids were confirmed for presence of targeted genes using automated sanger sequencing (Biokart Pvt LTD).

Simplex PCR assays were optimized using the generated plasmid for CDV, CAV-2, and Bb by adjusting annealing temperature, extension/annealing times, and primer concentrations. Reactions were performed in a 12.5 µL reaction volume using 3 µL of simplex plasmid DNA as template, 6.25 µL of 2X GoTaq^® Green Master Mix, and 10 µM each of forward and reverse primers, 2.25 µL of NFW. Annealing temperature optimization was conducted using a gradient of 50–60 °C. Time optimization involved three combinations of annealing (15 s, 30 s, 45 s) and extension (15 s, 30 s, 45 s) durations. Primer concentrations were optimized using a 4×4 checkerboard matrix ranging from 0.2 µM-0.8 µM for both forward and reverse primers.

Multiplex PCR assays were developed and optimized for CDV, CAV-2, and Bb by adjusting annealing temperature, extension/annealing times, and primer concentrations. Reactions were performed in a 12.5 µL reaction volume using 3 µL of multiplex plasmid DNA as template, 6.25 µL of 2X GoTaq^® Green Master Mix, and 10 µM each of forward and reverse primers, 2.25 µL of NFW for all three targets. Annealing temperature optimization was carried out using a gradient of 45–60 °C. Time optimization involved testing three combinations of annealing (15s, 30s, 45 s) and extension (15s, 30s, 45 s) durations. Primer concentrations were optimized using a checkerboard matrix ranging from 0.1-0.4 µM for both forward and reverse primers.

Analytical sensitivity of the multiplex PCR assay was assessed using ten-fold serial dilutions of plasmid DNA, and the lowest dilution consistently yielding a detectable band was used to determine the detection limit. Plasmid copy number was calculated using the formula: Copy number = [AxNo]/ [length (plasmid+ insert size) ×1×10⁹×660], in this formula, A represents the DNA concentration in ng/μl No. is Avogadro’s number (6.022X10²³) the average weight of a nucleotide base pair (bp) is assumed to be 660 Daltons, and the number of template copies in the sample can be estimated by multiplying the DNA concentration by 1x10⁹ (conversion factor for ng). Analytical specificity was evaluated by testing the assay against target plasmids for CDV, CAV-2, and Bb, as well as the commercial DHPPiL vaccine. Amplification products were analyzed on a 1.5% agarose gel, confirming specific amplification of target sequences without cross-reactivity. Reproducibility was evaluated by measuring both intra-assay and inter-assay variations using positive controls and sequenced clinical samples. Intra-assay variation was assessed by performing triplicate amplifications of templates at 10⁻¹⁰ ng and 10⁻¹ ng per reaction within a single multiplex PCR assay. Inter-assay variation was determined by conducting the same multiplex PCR assay in three independent experimental runs using the same template concentrations.

Oropharyngeal swabs were collected from dogs presented to the Canine Section of the Veterinary Clinical Complex, Lala Lajpat Rai University of Veterinary and Animal Sciences (LUVAS), Hisar between January, 2024 and January, 2025. A total of 55 dogs exhibiting clinical signs of respiratory illness were included in the study, comprising 38 males and 17 females. The majority of the dogs were between 1 and 3 years of age (47.27%). Coughing was the most commonly observed clinical sign (100%), followed by nasal discharge (60%), inappetence to anorexia (45.45%), and sneezing (40%).The majority of dogs were presented with incomplete vaccination status (43.63%). Clinical profile of dogs, sex- wise occurrence, age-wise occurrence and vaccination status of dogs in study is summarized in Tables 2–5. Dogs exhibiting respiratory signs such as coughing, nasal discharge or clinical evidence of bronchopneumonia were included in the study. Cases with respiratory symptoms attributable to cardiovascular disorders, tracheal dysfunction or allergic conditions were excluded. The extraction of DNA from swab samples were also done using the DNA extraction kit (HiPurA^® mammalian Genomic DNA Purification Kit, Kennett Square, PA, USA) according to manufacturer’s recommendation and TRIzol method was used for RNA extraction. Briefly, swab samples were dissolved in 400 μL of PBS for 15 minutes. The supernatant was centrifuged at 10,000 rpm for 10 minutes to remove any debris. For DNA extraction, 200 μL HiPurA^® Genomic lysis/binding buffer was added in 200 μL of sample supernatant along with 20 μL of Proteinase K. For RNA extraction, 200 μL of same sample supernatant was mixed with 300 μL of TRIzol reagent and vortexed vigorously. In this mixture, 120 μL of chloroform was added followed by vigorous vortexing to avoid formation of insoluble aggregates. Rest of the procedure for DNA and RNA extraction was same as mentioned in section 2.3. To assess the reliability of the developed multiplex PCR for clinical application, its performance was compared with a previously established PCR assay for CDV, for CAV-2 and for Bb (Hozbor et al., 1999; Agnihotri et al., 2017; Raja et al., 2021). The key diagnostic parameters including sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated. A chi-square test (with Yates’ continuity correction) was conducted using SPSS software (version 23.0) to assess the statistical significance of differences in pathogen detection rates between the two assays.

Simplex PCR assays were optimized individually for CDV, CAV-2 and Bb by systematically adjusting annealing temperature, extension/annealing time, and primer concentration. For CDV, optimal conditions included an annealing temperature of 55°C, 30 s annealing/extension time, and 0.4 µM primer concentration, yielding a 274 bp amplicon. For CAV-2, the ideal parameters were 60°C annealing temperature, 30 s annealing/extension, and 0.4  µM primers, producing a 451 bp product. Similarly, the Bb assay was optimized at 60°C with 30 s annealing/extension time and 0.4  µM primers, amplifying a 672 bp fragment. All optimizations were confirmed via 1.5% agarose gel electrophoresis.

Optimization of the assay resulted in the successful amplification of all target fragments: 274 bp for CDV, 451 bp for CAV-2 and 672 bp for Bb. Gradient PCR conducted across a temperature range of 45°C to 60°C demonstrated the most distinct and intense amplification at 55°C, which was subsequently selected as the optimal annealing temperature (Supplementary Figure S1). To refine incubation conditions, amplification was assessed at 55°C for 15, 30, and 45 seconds. Although all expected amplicons were detected at all time points, the strongest signal intensities were observed at 30 seconds, which was chosen as the optimal incubation time (Supplementary Figure S2). Primer concentration was further optimized through systematic evaluation. Initial multiplex reactions using various primer combinations identified 0.4 µM as the most effective concentration for each primer set. (Supplementary Figures S3-S5). Consistently, this concentration produced the most robust and specific amplification of all three targets, and was therefore selected as the final optimized primer concentration.

For the detection of the sensitivity of the assay, copy number of all the three pathogens were calculated. The initial undiluted plasmid concentration of CDV, CAV-2 and Bb was 38.2 ng/μL, 61.4 ng/μL and 49.1 ng/μL. For multiplex assay, the copy number of all the three plasmids was calculated as per formula described in section 2.8. The copy number of CDV, CAV-2 and Bb in undiluted sample were 10.6 x 10⁹copies/μL, 16.29x 10⁹copies/μL and Bb were 12.24 x 10⁹copies/μL. Theses plasmids were further mixed in equivalent copy number to avoid the effect of size of different products. To make the copy number same the following concentrations were used 10 μL of CDV plasmid= 106x 10⁹copies/μL, 7 μL of CAV 2 plasmid= 114.03 x 10⁹copies/μL and 9 μL of Bb plasmid= 110.16x 10⁹copies/μL The sensitivity of the multiplex PCR was tested by detection of the three plasmids in serial dilutions expressed in copy number and was calculated as 1,060 copies/µL for CDV, 11,403 copies/µL for CAV-2 and 11,016 copies/µL for Bb based on standardized input plasmid concentrations (Figure 1). These sensitivity experiments were done in triplicate and were repeated with different dilutions prepared at different times as well as by different persons. The intra and inter-assay evaluations, both demonstrated consistent and comparable results across replicates at different intervals. Intra- and inter-assay evaluations, performed using three dilutions near the detection limit, exhibited a coefficient of variation (CV) below 5%, confirming the assay’s precision and robustness.

Specificity of the assay was evaluated using individual plasmids for CDV, CAV-2 and Bb each yielding amplification exclusively at the expected product sizes with no cross-reactivity observed, thereby confirming 100% analytical specificity of the multiplex PCR assay (Figure 2).

A total of 55 oropharyngeal swab samples from dogs suspected of CIRDC were screened using the developed multiplex PCR assay for the simultaneous detection of CDV, CAV-2 and Bb. The samples were tested initially individually with standardized singlex PCR methods to confirm the reliability of assay (Hozbor et al., 1999; Agnihotri et al., 2017; Raja et al., 2021). For each swab sample 3 µL (10–50 ng/µL) of extracted DNA/cDNA was added to each multiplex PCR reaction. Out of the 55 samples tested, 18 (32.7%) tested positive using the developed multiplex PCR assay while 17 (30.9%) were found positive with singlex assay. CDV was the most frequently detected pathogen, identified in 14 samples (25%) as a single infection. Dual infections were observed in 2 samples (3.6%) with CDV and Bb, and in 1 sample (1.8%) with CDV and CAV-2. Additionally, Bb was detected alone in 1 sample (1.8%) (Table 6). The amplification of representative samples is shown in Figure 3. The previously established singlex PCR assay identified CDV DNA in 14 samples (25%) as a single infection, 2 samples (3.63%) with CAV-2 and 1 sample (1.8%) was detected for Bb. The 2x2 contingency table used for evaluation of diagnostic assay is given in Supplementary Tables S2-S4. The key diagnostic parameters are given in Table 7. Application of the chi-square test revealed a statistically significant association between the two assays, with chi-square values of 41.762 for CDV, 13.245 for CAV-2 and 37.318 for Bb (critical χ² = 3.84, df = 1). The high chi-square value indicating a highly significant association (p < 0.0001) between the two variables. These findings suggested that both assays exhibited comparable performance in detecting CIRDC pathogens.