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. 2023 Nov 11:11:102476.
doi: 10.1016/j.mex.2023.102476. eCollection 2023 Dec.

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

Junsheng Dong  1   2 , Wai Ning Tiffany Tsui  1 , Xue Leng  1   3 , Jinping Fu  1 , Molly Lohman  1 , Joseph Anderson  1 , Vaughn Hamill  1 , Nanyan Lu  1   4 , Elizabeth Poulsen Porter  1 , Mark Gray  1 , Tesfaalem Sebhatu  1 , Susan Brown  4 , Roman Pogranichniy  1   5 , Heng Wang  2 , Lance Noll  1   5 , Jianfa Bai  1   5
Affiliations

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

Junsheng Dong et al. MethodsX. .

Abstract

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.

Keywords: CIRD; Canine infectious respiratory disease; Diagnostic validation; PCR assay; Validation method for a canine respiratory PCR assay.

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Conflict of interest statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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