Mycoplasma cynos is recognized as an emerging causative pathogen of canine infectious respiratory disease (CIRD) worldwide. We developed a new open-source real-time PCR (rtPCR) assay for M. cynos that performs well under standard rtPCR conditions. Primers and probes were designed to target the M. cynos tuf gene. Reaction efficiencies for the M. cynos tuf gene assay on 2 platforms were based on amplification of standard curves spanning 8 orders of magnitude: ABI 7500 platform, 94.3-97.9% (r2 ≥ 0.9935); QuantStudio OpenArray platform, 119.1-122.5% (r2 = 0.9784). The assay performed very well over a range of template input, from 109 copies to the lower limit of quantification at 4 copies of the M. cynos genome on the ABI 7500 platform. Diagnostic performance was estimated by comparison with an in-house legacy assay on clinical specimens as well as testing isolates that were characterized previously by intergenic spacer region (ISR) sequencing. Exclusivity was established by testing 12 other Mycoplasma species. To substantiate the high specificity of the M. cynos tuf gene assay, sequence confirmation was performed on ISR PCR amplicons obtained from clinical specimens. One ISR amplicon sequence revealed M. mucosicanis rather than M. cynos. The complete protocol of the newly developed M. cynos tuf assay is provided to facilitate assay harmonization.

Characterization of a novel Mycoplasma cynos real-time PCR assay / R.L. Tallmadge, R. Anderson, P.K. Mitchell, Z.C. Forbes, B. Werner, G. Gioia, P. Moroni, A. Glaser, A.J. Thachil, L.B. Goodman. - In: JOURNAL OF VETERINARY DIAGNOSTIC INVESTIGATION. - ISSN 1040-6387. - (2019 Nov 21). [Epub ahead of print] [10.1177/1040638719890858]

Characterization of a novel Mycoplasma cynos real-time PCR assay

G. Gioia;P. Moroni;
2019

Abstract

Mycoplasma cynos is recognized as an emerging causative pathogen of canine infectious respiratory disease (CIRD) worldwide. We developed a new open-source real-time PCR (rtPCR) assay for M. cynos that performs well under standard rtPCR conditions. Primers and probes were designed to target the M. cynos tuf gene. Reaction efficiencies for the M. cynos tuf gene assay on 2 platforms were based on amplification of standard curves spanning 8 orders of magnitude: ABI 7500 platform, 94.3-97.9% (r2 ≥ 0.9935); QuantStudio OpenArray platform, 119.1-122.5% (r2 = 0.9784). The assay performed very well over a range of template input, from 109 copies to the lower limit of quantification at 4 copies of the M. cynos genome on the ABI 7500 platform. Diagnostic performance was estimated by comparison with an in-house legacy assay on clinical specimens as well as testing isolates that were characterized previously by intergenic spacer region (ISR) sequencing. Exclusivity was established by testing 12 other Mycoplasma species. To substantiate the high specificity of the M. cynos tuf gene assay, sequence confirmation was performed on ISR PCR amplicons obtained from clinical specimens. One ISR amplicon sequence revealed M. mucosicanis rather than M. cynos. The complete protocol of the newly developed M. cynos tuf assay is provided to facilitate assay harmonization.
Mycoplasma cynos; canine infectious respiratory disease; tuf gene;
Settore VET/05 - Malattie Infettive degli Animali Domestici
21-nov-2019
Article (author)
File in questo prodotto:
File Dimensione Formato  
1040638719890858.pdf

accesso riservato

Tipologia: Publisher's version/PDF
Dimensione 800.23 kB
Formato Adobe PDF
800.23 kB Adobe PDF   Visualizza/Apri   Richiedi una copia
Mcynos manuscript v3 02272019 pm.pdf

accesso aperto

Tipologia: Post-print, accepted manuscript ecc. (versione accettata dall'editore)
Dimensione 728.61 kB
Formato Adobe PDF
728.61 kB Adobe PDF Visualizza/Apri
Pubblicazioni consigliate

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2434/692381
Citazioni
  • ???jsp.display-item.citation.pmc??? 4
  • Scopus 7
  • ???jsp.display-item.citation.isi??? 5
social impact