Multiplex Digital Droplet PCR for Simultaneous Detection of Canine Parvovirus Type 2, Canine Distemper Virus, and Canine Adenovirus Type 2 in Fecal Samples from Shelter Dogs

Introduction

Canine viral enteric and respiratory infections remain a leading cause of morbidity and mortality in shelter populations worldwide [1, 2]. Three viruses frequently implicated in outbreaks are canine parvovirus type 2 (CPV-2), canine distemper virus (CDV), and canine adenovirus type 2 (CAdV-2) [3, 4]. CPV-2 causes hemorrhagic gastroenteritis and panleukopenia, especially in young puppies [5]. CDV is a morbillivirus that produces multisystemic disease affecting respiratory, gastrointestinal, and central nervous systems, with high fatality rates in unvaccinated shelters [6, 7]. CAdV-2 is primarily a respiratory pathogen and a component of the infectious tracheobronchitis complex, yet it is also shed in feces [8].

Traditional diagnosis relies on point-of-care antigen tests and quantitative real-time PCR (qPCR), but these methods have limitations. qPCR requires standard curves for quantification and is susceptible to inhibition in fecal matrices [9]. Digital droplet PCR (ddPCR) achieves absolute quantification without standard curves by partitioning the sample into thousands of nanoliter droplets and counting positive droplets after endpoint PCR [10]. Multiplex ddPCR can detect several targets simultaneously, conserving sample volume and reagents [11].

We describe the development and validation of a multiplex ddPCR assay targeting conserved genes of CPV-2 (VP2 gene), CDV (hemagglutinin H gene), and CAdV-2 (hexon gene) in fecal samples from shelter dogs. Assay performance is compared to singleplex qPCR, and field data from shelter populations are presented.

Materials and Methods

Primer and Probe Design

Conserved regions were identified using alignments of available sequences. For CPV-2, primers targeting the VP2 gene were selected based on published designs [12, 13]. For CDV, the hemagglutinin (H) gene was targeted owing to its sequence conservation among circulating strains [14, 15]. For CAdV-2, the hexon gene was chosen [16]. Dual-labeled hydrolysis probes (FAM for CPV-2, HEX for CDV, Cy5 for CAdV-2) were designed using standard algorithms. All oligonucleotides were synthesized using conventional phosphoramidite chemistry and purified by high-performance liquid chromatography.

Sample Collection and Processing

Fecal samples (n=150) were collected from dogs housed in three shelter facilities. Ethical approval was obtained from institutional committees [17]. Samples were placed in sterile containers and transported on ice. Viral nucleic acid was extracted using a commercial silica-membrane kit following the manufacturer’s protocol. Eluates were stored at -80 degrees Celsius until analysis.

Multiplex ddPCR Optimization

Reactions were prepared using a generic ddPCR supermix with a final volume of 20 microliters. Primer and probe concentrations were optimized using checkerboard titrations. Optimum concentrations were: CPV-2 forward/reverse 900 nM each, probe 250 nM; CDV forward/reverse 600 nM each, probe 200 nM; CAdV-2 forward/reverse 900 nM each, probe 250 nM. Droplets were generated using a fully automated droplet generator. Thermal cycling conditions were: 95 degrees Celsius for 10 minutes (enzyme activation), followed by 40 cycles of 94 degrees Celsius for 30 seconds and 58 degrees Celsius for 60 seconds, then 98 degrees Celsius for 10 minutes (droplet stabilization), and a 4 degrees Celsius hold. Ramp rate was set at 2 degrees Celsius per second.

Droplets were read using a dedicated droplet reader. Thresholds were set manually based on fluorescence amplitude of negative droplets and positive controls.

Singleplex qPCR

For comparison, singleplex qPCR assays were performed using the same primer and probe sets on a real-time PCR platform. Standard curves were generated using tenfold serial dilutions of plasmid clones containing each target. Amplification efficiencies between 90% and 110% were accepted.

Analytical Sensitivity and Specificity

Analytical sensitivity was determined by serially diluting quantified synthetic RNA (CDV) or DNA (CPV-2, CAdV-2) standards spiked into negative fecal matrix. Limit of detection (LoD) was defined as the lowest concentration at which 95% of replicates were positive [18]. Specificity was assessed against nucleic acid extracts from canine adenovirus type 1, canine herpesvirus, canine coronavirus, canine parainfluenza virus, and Bordetella bronchiseptica. Cross-reactivity was evaluated by testing each target individually and in combinations.

Field Sample Testing

All 150 fecal samples were tested in triplicate using the multiplex ddPCR assay and singleplex qPCR. Discordant results were resolved by sequencing amplicons. Viral loads were expressed as copies per gram of feces.

Results

Analytical Performance

The multiplex ddPCR assay showed linearity across five log10 dilutions for each target. The LoD for CPV-2 was 10 copies per reaction, for CDV was 8 copies per reaction, and for CAdV-2 was 12 copies per reaction. No cross-reactivity was observed with any of the non-target pathogens tested. Intra-assay and inter-assay coefficients of variation were below 15% for all targets.

Comparison of ddPCR against qPCR showed strong correlation (R squared > 0.96) for all three viruses, but ddPCR detected positive samples at lower viral loads that were undetectable by qPCR. Specifically, 12 samples were positive by ddPCR for CDV but negative by qPCR (confirmed by sequencing). Absolute quantification by ddPCR eliminated the need for standard curves and reduced quantification variability.

Field Sample Results

Of 150 samples, 45 (30%) were positive for at least one virus. CPV-2 was detected in 28 samples (18.7%), CDV in 18 samples (12%), and CAdV-2 in 10 samples (6.7%). Co-infections were observed in 8 samples (5.3%), with the most common combination being CPV-2 and CDV (5 samples). Viral loads ranged from 1.2 x 10^2 to 4.7 x 10^8 copies per gram for CPV-2, 5.0 x 10^1 to 2.1 x 10^7 copies per gram for CDV, and 8.0 x 10^1 to 6.3 x 10^6 copies per gram for CAdV-2.

Comparison with qPCR

Overall agreement between ddPCR and qPCR was 94.7%. Discordant results were predominantly low-copy samples positive only by ddPCR. The absolute quantification feature of ddPCR provided accurate viral load measurements without the bias inherent in standard curve-based methods.

flowchart TD
    A[Fecal sample collection], > B[Nucleic acid extraction]
    B, > C[Multiplex ddPCR reaction setup]
    C, > D[Droplet generation]
    D, > E[PCR amplification in droplets]
    E, > F[Droplet reading and fluorescence detection]
    F, > G[Poisson statistical analysis]
    G, > H[Absolute quantification (copies/μL)]
    H, > I[Result interpretation]
    I, > J[Positive for CPV-2, CDV, and/or CAdV-2]
    I, > K[Negative for all targets]
    J, > L[Report viral loads]
    K, > M[Confirm with alternative assay if clinically indicated]

Figure 1: Workflow of the multiplex ddPCR assay for simultaneous detection of CPV-2, CDV, and CAdV-2 in canine fecal samples.

Discussion

Multiplex ddPCR offers several advantages for shelter diagnostics. The ability to quantify three pathogens simultaneously in a single reaction reduces cost and sample volume, which is critical when sampling young puppies or when fecal material is limited [19, 20]. The absolute quantification obtained from Poisson statistics eliminates the variability introduced by standard curves, improving inter-laboratory reproducibility [21, 22].

Detection of low-copy viral genomes by ddPCR in samples that were qPCR-negative suggests that ddPCR may improve sensitivity for early infections or samples with inhibitors [23]. This is particularly relevant for CDV, which can be shed at low levels in the early febrile stage before clinical signs appear [24, 25]. Shelters that can identify infected animals earlier can implement isolation protocols more rapidly, potentially reducing outbreak size [26, 27].

The assay targets the H gene of CDV, which is conserved across lineages including Arctic-like strains [4] and those circulating in wildlife reservoirs [13, 28]. Similarly, the VP2 gene of CPV-2 is stable across variants CPV-2a, 2b, and 2c [29]. The hexon gene of CAdV-2 is distinct from CAdV-1, allowing differential detection without cross-reactivity [30].

Shelter biosecurity benefits from highly sensitive multiplex detection. Co-infections were found in 5.3% of samples, underscoring the need for a panel approach [31]. Vaccination strategies against CPV-2 and CDV are widely used, but breakthrough infections occur due to maternal antibody interference or insufficient coverage [32, 33]. The ddPCR assay can complement serological monitoring by providing direct viral detection in fecal samples [34, 35].

Limitations include the cost of ddPCR instrumentation and reagents compared to qPCR, though the reduction in consumables for multiplexing partially offsets this. The assay requires a dedicated droplet generator and reader, which may not be available in all diagnostic laboratories.

Conclusion

A multiplex ddPCR assay for simultaneous detection and absolute quantification of CPV-2, CDV, and CAdV-2 in canine fecal samples has been developed and validated. The assay demonstrates high analytical sensitivity, no cross-reactivity, and superior performance for low-copy detection compared to qPCR. Implementation in shelter diagnostic workflows can improve early outbreak detection and support evidence-based biosecurity decisions.

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