Development and application of two multiplex real-time PCR assays for detection and speciation of bacterial pathogens in the koala

Hulse, Lyndal S., Hickey, Danica, Mitchell, Jessica M., Beagley, Kenneth W., Ellis, William and Johnston, Stephen D. (2018) Development and application of two multiplex real-time PCR assays for detection and speciation of bacterial pathogens in the koala. Journal of Veterinary Diagnostic Investigation, 1040638718770490. doi:10.1177/1040638718770490


Author Hulse, Lyndal S.
Hickey, Danica
Mitchell, Jessica M.
Beagley, Kenneth W.
Ellis, William
Johnston, Stephen D.
Title Development and application of two multiplex real-time PCR assays for detection and speciation of bacterial pathogens in the koala
Journal name Journal of Veterinary Diagnostic Investigation   Check publisher's open access policy
ISSN 1943-4936
1040-6387
Publication date 2018-04-09
Sub-type Article (original research)
DOI 10.1177/1040638718770490
Open Access Status Not yet assessed
Start page 1040638718770490
Total pages 7
Place of publication Thousand Oaks, CA, United States
Publisher Sage Publications
Language eng
Abstract Infectious diseases have contributed to the decline in the health of koala ( Phascolarctos cinereus) populations in the wild in some regions of Australia. Herein we report the development and validation of 2 multiplex real-time PCR (rtPCR) panels for the simultaneous detection of Mycoplasma spp., Ureaplasma spp., Bordetella bronchiseptica, and Chlamydia, including speciation and quantification of Chlamydia, in ocular, reproductive, and nasal swab samples in addition to semen and male urogenital and reproductive tissues, from koalas. Each rtPCR panel was developed for use as a single-tube reaction using pathogen-specific primers and fluorescently labeled probe sets. DNA extracted from reference strains and isolates was used for validation of sequence gene targets for the multiplex rtPCR panels. Each panel was shown to be sensitive and specific in detecting and differentiating the bacterial pathogens. The multiplex rtPCR panels were used to screen clinical samples from free-ranging and hospitalized koalas for multiple pathogens simultaneously. The multiplex rtPCR will improve turnaround time compared to individual-pathogen rtPCR methods used, to date, for confirmation of diagnosis and will provide the wildlife clinician with the ability to make treatment decisions more rapidly.
Formatted abstract
Infectious diseases have contributed to the decline in the health of koala (Phascolarctos cinereus) populations in the wild in some regions of Australia. Herein we report the development and validation of 2 multiplex real-time PCR (rtPCR) panels for the simultaneous detection of Mycoplasma spp., Ureaplasma spp., Bordetella bronchiseptica, and Chlamydia, including speciation and quantification of Chlamydia, in ocular, reproductive, and nasal swab samples in addition to semen and male urogenital and reproductive tissues, from koalas. Each rtPCR panel was developed for use as a single-tube reaction using pathogen-specific primers and fluorescently labeled probe sets. DNA extracted from reference strains and isolates was used for validation of sequence gene targets for the multiplex rtPCR panels. Each panel was shown to be sensitive and specific in detecting and differentiating the bacterial pathogens. The multiplex rtPCR panels were used to screen clinical samples from free-ranging and hospitalized koalas for multiple pathogens simultaneously. The multiplex rtPCR will improve turnaround time compared to individual-pathogen rtPCR methods used, to date, for confirmation of diagnosis and will provide the wildlife clinician with the ability to make treatment decisions more rapidly.
Keyword Bordetella bronchiseptica
Chlamydia
Mycoplasma
Ureaplasma
Koalas
Multiplex real-time PCR
Q-Index Code C1
Q-Index Status Provisional Code
Institutional Status UQ

Document type: Journal Article
Sub-type: Article (original research)
Collections: HERDC Pre-Audit
School of Agriculture and Food Sciences
 
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Created: Wed, 11 Apr 2018, 10:01:32 EST