Validation of a real-time PCR panel for detection and quantification of nine pathogens commonly associated with canine infectious respiratory disease.

Canine infectious respiratory disease (CIRD) is a complicated respiratory syndrome in dogs [1], [2], [3]. A panel PCR was developed [4] to detect nine pathogens commonly associated with CIRD: Mycoplasma cynos, Mycoplasma canis, Bordetella bronchiseptica; canine adenovirus type 2, canine herpesvirus 1, canine parainfluenza virus, canine distemper virus, canine influenza virus and canine respiratory coronavirus [5], [6], [7], [8], [9], [10], [11], [12], [13], [14], [15], [16]. To evaluate diagnostic performance of the assay, 740 nasal swab and lung tissue samples were collected and tested with the new assay, and compared to an older version of the assay detecting the same pathogens except that it does not differentiate the two Mycoplasma species. Results indicated that the new assay had the same level of specificity, but with higher diagnostic sensitivity and had identified additional samples with potential co-infections. To confirm the new assay is detecting the correct pathogens, samples with discrepant results between the two assays were sequence-confirmed. Spiking a high concertation target to samples carrying lower concentrations of other targets was carried out and the results demonstrated that there was no apparent interference among targets in the same PCR reaction. Another spike-in experiment was used to determine detection sensitivity between nasal swab and lung tissue samples, and similar results were obtained.•A nine-pathogen CIRD PCR panel assay had identified 139 positives from 740 clinical samples with 60 co-infections;•High-concentration target does not have apparent effect on detecting low-concentration targets;•Detection sensitivity were similar between nasal swab and lung tissue samples.

Development of a three-panel multiplex real-time PCR assay for simultaneous detection of nine canine respiratory pathogens.

Infectious respiratory disease is one of the most common diseases in dogs worldwide. Several bacterial and viral pathogens can serve as causative agents of canine infectious respiratory disease (CIRD), including Mycoplasma cynos, Mycoplasma canis, Bordetella bronchiseptica, canine adenovirus type 2 (CAdV-2), canine herpesvirus 1 (CHV-1), canine parainfluenza virus (CPIV), canine distemper virus (CDV), canine influenza virus (CIA) and canine respiratory coronavirus (CRCoV). Since these organisms cause similar clinical symptoms, disease diagnosis based on symptoms alone can be difficult. Therefore, a quick and accurate test is necessary to rapidly identify the presence and relative concentrations of causative CIRD agents. In this study, a multiplex real-time PCR panel assay was developed and composed of three subpanels for detection of the aforementioned pathogens. Correlation coefficients (R2) were >0.993 for all singleplex and multiplex real-time PCR assays with the exception of one that was 0.988; PCR amplification efficiencies (E) were between 92.1% and 107.8% for plasmid DNA, and 90.6-103.9% for RNA templates. In comparing singular and multiplex PCR assays, the three multiplex reactions generated similar R2 and E values to those by corresponding singular reactions, suggesting that multiplexing did not interfere with the detection sensitivities. The limit of detection (LOD) of the multiplex real-time PCR for DNA templates was 5, 2, 3, 1, 1, 1, 4, 24 and 10 copies per microliter for M. cynos, M. canis, B. brochiseptica, CAdV-2, CHV-1, CPIV, CDV, CIA and CRCoV, respectively; and 3, 2, 6, 17, 4 and 8 copies per microliter for CAdV-2, CHV-1, CPIV, CDV, CIA and CRCoV, respectively, when RNA templates were used for the four RNA viruses. No cross-detection was observed among the nine pathogens. For the 740 clinical samples tested, the newly designed PCR assay showed higher diagnostic sensitivity compared to an older panel assay; pathogen identities from selected samples positive by the new assay but undetected by the older assay were confirmed by Sanger sequencing. Our data showed that the new assay has higher diagnostic sensitivity while maintaining the assay's specificity, as compared to the older version of the panel assay.