Hulse Lyndal S, Hickey Danica, Mitchell Jessica M, Beagley Kenneth W, Ellis William, Johnston Stephen D
School of Agriculture and Food Sciences, University of Queensland, Gatton, Queensland, Australia (Hulse, Mitchell, Ellis, Johnston).
Institute of Health and Biomedical Innovation (Hickey, Beagley), Faculty of Health, Queensland University of Technology, Brisbane, Queensland, Australia.
J Vet Diagn Invest. 2018 Jul;30(4):523-529. doi: 10.1177/1040638718770490. Epub 2018 Apr 9.
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.
在澳大利亚的一些地区，传染病导致了野生考拉（Phascolarctos cinereus）种群健康状况的下降。在此，我们报告了2种多重实时荧光定量PCR（rtPCR）检测方法的开发与验证，用于同时检测考拉眼部、生殖器官、鼻腔拭子样本以及精液、雄性泌尿生殖和生殖组织中的支原体属、脲原体属、支气管败血波氏杆菌和衣原体，包括衣原体的分型和定量。每种rtPCR检测方法均采用病原体特异性引物和荧光标记探针组，开发为单管反应。从参考菌株和分离株中提取的DNA用于验证多重rtPCR检测方法的序列基因靶点。结果表明，每种检测方法在检测和区分细菌病原体方面均具有敏感性和特异性。多重rtPCR检测方法用于同时筛查自由放养和住院考拉的临床样本中的多种病原体。与迄今为止用于确诊的单个病原体rtPCR方法相比，多重rtPCR将缩短周转时间，并使野生动物临床医生能够更快地做出治疗决策。
