A triplex real-time PCR assay for simultaneous and differential detection of Bordetella bronchiseptica, Mycoplasma cynos, and Mycoplasma canis in respiratory diseased dogs
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Bordetella (B .) bronchiseptica , Mycoplasma (M .) cynos , and M. canis are the major bacterial patho-gens that cause canine infectious respiratory disease complex (CIRDC). In this study, we developed a triplex real-time polymerase chain reaction (tqPCR) assay for the differential detection of these bacteria in a single reaction. The assay specifically amplified three bacterial genes with a detection limit of below 10 copies/reaction. The assay showed high repeatability and reproducibility, with coef-ficients of intra- and inter-assay variations of less than 1%. The diagnostic results of the assay using 94 clinical samples from household dogs with CIRDC clinical signs, the prevalence of B. bronchisep-tica , M. cynos , and M. canis was 22.3%, 18.1%, and 20.2%, respectively, indicating that the diagnostic sensitivity was comparable to those of previously reported qPCR assays. The dual infection rate of B. bronchiseptica and M. cynos , B. bronchiseptica and M. canis , and M. cynos and M. canis was 5.3%, 7.4%, and 3.1%, respectively. Moreover, the triple infection rate of B. bronchiseptica , M. cynos , and M. canis was 2.1%. These results indicate that coinfections with B. bronchiseptica , M. cynos , and M. ca-nis have frequently occurred in the Korean dog population. The newly developed tqPCR assay in the present study will be a useful tool for etiological and epidemiological studies on these three CIRDC-associated bacterial pathogens. The prevalence and coinfection data revealed through this study will contribute to expanding knowledge on the epidemiology of CIRDC in the recent Korean dog popula-tion.
Abstract
Bordetella (B .) bronchiseptica , Mycoplasma (M .) cynos , and M. canis are the major bacterial patho-gens that cause canine infectious respiratory disease complex (CIRDC). In this study, we developed a triplex real-time polymerase chain reaction (tqPCR) assay for the differential detection of these bacteria in a single reaction. The assay specifically amplified three bacterial genes with a detection limit of below 10 copies/reaction. The assay showed high repeatability and reproducibility, with coef-ficients of intra- and inter-assay variations of less than 1%. The diagnostic results of the assay using 94 clinical samples from household dogs with CIRDC clinical signs, the prevalence of B. bronchisep-tica , M. cynos , and M. canis was 22.3%, 18.1%, and 20.2%, respectively, indicating that the diagnostic sensitivity was comparable to those of previously reported qPCR assays. The dual infection rate of B. bronchiseptica and M. cynos , B. bronchiseptica and M. canis , and M. cynos and M. canis was 5.3%, 7.4%, and 3.1%, respectively. Moreover, the triple infection rate of B. bronchiseptica , M. cynos , and M. canis was 2.1%. These results indicate that coinfections with B. bronchiseptica , M. cynos , and M. ca-nis have frequently occurred in the Korean dog population. The newly developed tqPCR assay in the present study will be a useful tool for etiological and epidemiological studies on these three CIRDC-associated bacterial pathogens. The prevalence and coinfection data revealed through this study will contribute to expanding knowledge on the epidemiology of CIRDC in the recent Korean dog popula-tion.
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