Kishimoto Mai, Tsuchiaka Shinobu, Rahpaya Sayed Samim, Hasebe Ayako, Otsu Keiko, Sugimura Satoshi, Kobayashi Suguru, Komatsu Natsumi, Nagai Makoto, Omatsu Tsutomu, Naoi Yuki, Sano Kaori, Okazaki-Terashima Sachiko, Oba Mami, Katayama Yukie, Sato Reiichiro, Asai Tetsuo, Mizutani Tetsuya
Research and Education Center for Prevention of Global Infectious Diseases of Animals, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai, Fuchu, Tokyo 183-8509, Japan.
J Vet Med Sci. 2017 Mar 18;79(3):517-523. doi: 10.1292/jvms.16-0489. Epub 2017 Jan 8.
Bovine respiratory disease complex (BRDC) is frequently found in cattle worldwide. The etiology of BRDC is complicated by infections with multiple pathogens, making identification of the causal pathogen difficult. Here, we developed a detection system by applying TaqMan real-time PCR (Dembo respiratory-PCR) to screen a broad range of microbes associated with BRDC in a single run. We selected 16 bovine respiratory pathogens (bovine viral diarrhea virus, bovine coronavirus, bovine parainfluenza virus 3, bovine respiratory syncytial virus, influenza D virus, bovine rhinitis A virus, bovine rhinitis B virus, bovine herpesvirus 1, bovine adenovirus 3, bovine adenovirus 7, Mannheimia haemolytica, Pasteurella multocida, Histophilus somni, Trueperella pyogenes, Mycoplasma bovis and Ureaplasma diversum) as detection targets and designed novel specific primer-probe sets for nine of them. The assay performance was assessed using standard curves from synthesized DNA. In addition, the sensitivity of the assay was evaluated by spiking solutions extracted from nasal swabs that were negative by Dembo respiratory-PCR for nucleic acids of pathogens or synthesized DNA. All primer-probe sets showed high sensitivity. In this study, a total of 40 nasal swab samples from cattle on six farms were tested by Dembo respiratory-PCR. Dembo respiratory-PCR can be applied as a screening system with wide detection targets.
牛呼吸道疾病综合征（BRDC）在全球范围内的牛群中屡见不鲜。BRDC的病因因多种病原体感染而变得复杂，这使得确定致病病原体变得困难。在此，我们开发了一种检测系统，通过应用TaqMan实时PCR（登博呼吸道-PCR）在一次检测中筛选与BRDC相关的多种微生物。我们选择了16种牛呼吸道病原体（牛病毒性腹泻病毒、牛冠状病毒、牛副流感病毒3型、牛呼吸道合胞病毒、丁型流感病毒、牛鼻炎A病毒、牛鼻炎B病毒、牛疱疹病毒1型、牛腺病毒3型、牛腺病毒7型、溶血曼氏杆菌、多杀性巴氏杆菌、睡眠嗜组织菌、化脓隐秘杆菌、牛支原体和多样脲原体）作为检测靶点，并为其中9种设计了新型特异性引物-探针组。使用合成DNA的标准曲线评估检测性能。此外，通过向登博呼吸道-PCR检测病原体核酸或合成DNA呈阴性的鼻拭子提取物中添加溶液来评估该检测方法的灵敏度。所有引物-探针组均显示出高灵敏度。在本研究中，通过登博呼吸道-PCR对来自六个农场的40份牛鼻拭子样本进行了检测。登博呼吸道-PCR可作为一种具有广泛检测靶点的筛选系统应用。
