Goecke Nicole B, Nielsen Bodil H, Petersen Mette B, Larsen Lars E
Centre for Diagnostics, Technical University of Denmark, Lyngby, Denmark.
Department of Veterinary and Animal Sciences, University of Copenhagen, Copenhagen, Denmark.
Front Vet Sci. 2021 Jun 24;8:677993. doi: 10.3389/fvets.2021.677993. eCollection 2021.
Bovine respiratory and enteric diseases have a profound negative impact on animal, health, welfare, and productivity. A vast number of viruses and bacteria are associated with the diseases. Pathogen detection using real-time PCR (rtPCR) assays performed on traditional rtPCR platforms are costly and time consuming and by that limit the use of diagnostics in bovine medicine. To diminish these limitations, we have developed a high-throughput rtPCR system (BioMark HD; Fluidigm) for simultaneous detection of the 11 most important respiratory and enteric viral and bacterial pathogens. The sensitivity and specificity of the rtPCR assays on the high-throughput platform was comparable with that of the traditional rtPCR platform. Pools consisting of positive and negative individual field samples were tested in the high-throughput rtPCR system in order to investigate the effect of an individual sample in a pool. The pool tests showed that irrespective of the size of the pool, a high-range positive individual sample had a high influence on the cycle quantification value of the pool compared with the influence of a low-range positive individual sample. To validate the test on field samples, 2,393 nasal swab and 2,379 fecal samples were tested on the high-throughput rtPCR system as pools in order to determine the occurrence of the 11 pathogens in 100 Danish herds (83 dairy and 17 veal herds). In the dairy calves, (38.4%), rotavirus A (27.4%), spp. (26.2%), and (25.5%) were the most prevalent pathogens, while (71.4%), spp. (58.9%), (53.6%), and (42.9%) were the most often detected pathogens in the veal calves. The established high-throughput system provides new possibilities for analysis of bovine samples, since the system enables testing of multiple samples for the presence of different pathogens in the same analysis test even with reduced costs and turnover time.
牛呼吸道和肠道疾病对动物健康、福利及生产力有着深远的负面影响。大量病毒和细菌与这些疾病相关。在传统实时荧光定量PCR（rtPCR）平台上进行病原体检测成本高且耗时，这限制了其在牛医学诊断中的应用。为减少这些限制，我们开发了一种高通量rtPCR系统（BioMark HD；Fluidigm），用于同时检测11种最重要的呼吸道和肠道病毒及细菌病原体。高通量平台上rtPCR检测的灵敏度和特异性与传统rtPCR平台相当。为研究混合样本中单个样本的影响，将由阳性和阴性个体现场样本组成的混合样本在高通量rtPCR系统中进行检测。混合样本检测表明，无论混合样本大小，高浓度阳性个体样本对混合样本的循环定量值影响比低浓度阳性个体样本大。为验证该检测方法对现场样本的有效性。将2393份鼻拭子和2379份粪便样本作为混合样本在高通量rtPCR系统上进行检测，以确定100个丹麦牛群（83个奶牛群和17个犊牛群）中11种病原体的感染情况。在奶牛犊牛中，冠状病毒（38.4%）、A组轮状病毒（27.4%）、弯曲杆菌属（26.2%）和大肠杆菌（25.5%）是最常见的病原体，而在犊牛中，隐孢子虫（71.4%）、弯曲杆菌属（58.9%）、产气荚膜梭菌（53.6%）和大肠杆菌（42.9%）是最常检测到的病原体。所建立的高通量系统为牛样本分析提供了新的可能性，因为该系统能够在同一分析测试中对多个样本进行不同病原体的检测，同时降低成本和缩短周转时间。
