Simone Scherrer, Sarah Schmitt, Fenja Rademacher, Peter Kuhnert, Giovanni Ghielmetti, Sophie Peterhans, Roger Stephan
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The development of the qPCR was based on reference strains of 15 known serovars of <i>G. parasuis</i>, as well as on the type strains <i>M. hyorhinis</i> ATCC 17981<sup>T</sup> and <i>M. hyosynoviae</i> NCTC 10167<sup>T</sup>. The new qPCR was further evaluated using 21 <i>G. parasuis</i>, 26 <i>M. hyorhinis</i>, and 3 <i>M. hyosynoviae</i> field isolates. Moreover, a pilot study including different clinical specimens of 42 diseased pigs was performed. The specificity of the assay was 100% without cross-reactivity or detection of other bacterial swine pathogens. The sensitivity of the new qPCR was demonstrated to be between 11–180 genome equivalents (GE) of DNA for <i>M. hyosynoviae</i> and <i>M. hyorhinis</i>, and 140–1200 GE for <i>G. parasuis</i> and <i>vtaA</i>. The cut-off threshold cycle was found to be at 35. The developed sensitive and specific qPCR assay has the potential to become a useful molecular tool, which could be implemented in veterinary diagnostic laboratories for the detection and identification of <i>G. parasuis</i>, its virulence marker <i>vtaA</i>, <i>M. hyorhinis</i>, and <i>M. hyosynoviae</i>.</p>","PeriodicalId":18573,"journal":{"name":"MicrobiologyOpen","volume":null,"pages":null},"PeriodicalIF":3.9000,"publicationDate":"2023-05-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/mbo3.1353","citationCount":"1","resultStr":"{\"title\":\"Development of a new multiplex quantitative PCR for the detection of Glaesserella parasuis, Mycoplasma hyorhinis, and Mycoplasma hyosynoviae\",\"authors\":\"Simone Scherrer, Sarah Schmitt, Fenja Rademacher, Peter Kuhnert, Giovanni Ghielmetti, Sophie Peterhans, Roger Stephan\",\"doi\":\"10.1002/mbo3.1353\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><i>Glaesserella parasuis</i>, <i>Mycoplasma hyorhinis</i>, and <i>Mycoplasma hyosynoviae</i> are important porcine pathogens responsible for polyserositis, polyarthritis, meningitis, pneumonia, and septicemia causing significant economic losses in the swine industry. A new multiplex quantitative polymerase chain reaction (qPCR) was designed on one hand for the detection of <i>G. parasuis</i> and the virulence marker <i>vtaA</i> to distinguish between highly virulent and non-virulent strains. On the other hand, fluorescent probes were established for the detection and identification of both <i>M. hyorhinis</i> and <i>M. hyosynoviae</i> targeting 16S ribosomal RNA genes. The development of the qPCR was based on reference strains of 15 known serovars of <i>G. parasuis</i>, as well as on the type strains <i>M. hyorhinis</i> ATCC 17981<sup>T</sup> and <i>M. hyosynoviae</i> NCTC 10167<sup>T</sup>. The new qPCR was further evaluated using 21 <i>G. parasuis</i>, 26 <i>M. hyorhinis</i>, and 3 <i>M. hyosynoviae</i> field isolates. Moreover, a pilot study including different clinical specimens of 42 diseased pigs was performed. The specificity of the assay was 100% without cross-reactivity or detection of other bacterial swine pathogens. 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Development of a new multiplex quantitative PCR for the detection of Glaesserella parasuis, Mycoplasma hyorhinis, and Mycoplasma hyosynoviae
Glaesserella parasuis, Mycoplasma hyorhinis, and Mycoplasma hyosynoviae are important porcine pathogens responsible for polyserositis, polyarthritis, meningitis, pneumonia, and septicemia causing significant economic losses in the swine industry. A new multiplex quantitative polymerase chain reaction (qPCR) was designed on one hand for the detection of G. parasuis and the virulence marker vtaA to distinguish between highly virulent and non-virulent strains. On the other hand, fluorescent probes were established for the detection and identification of both M. hyorhinis and M. hyosynoviae targeting 16S ribosomal RNA genes. The development of the qPCR was based on reference strains of 15 known serovars of G. parasuis, as well as on the type strains M. hyorhinis ATCC 17981T and M. hyosynoviae NCTC 10167T. The new qPCR was further evaluated using 21 G. parasuis, 26 M. hyorhinis, and 3 M. hyosynoviae field isolates. Moreover, a pilot study including different clinical specimens of 42 diseased pigs was performed. The specificity of the assay was 100% without cross-reactivity or detection of other bacterial swine pathogens. The sensitivity of the new qPCR was demonstrated to be between 11–180 genome equivalents (GE) of DNA for M. hyosynoviae and M. hyorhinis, and 140–1200 GE for G. parasuis and vtaA. The cut-off threshold cycle was found to be at 35. The developed sensitive and specific qPCR assay has the potential to become a useful molecular tool, which could be implemented in veterinary diagnostic laboratories for the detection and identification of G. parasuis, its virulence marker vtaA, M. hyorhinis, and M. hyosynoviae.
期刊介绍:
MicrobiologyOpen is a peer reviewed, fully open access, broad-scope, and interdisciplinary journal delivering rapid decisions and fast publication of microbial science, a field which is undergoing a profound and exciting evolution in this post-genomic era.
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