Ding Tian, Suo Yuanjie, Zhang Zhaohuan, Liu Donghong, Ye Xingqian, Chen Shiguo, Zhao Yong
Department of Food Science and Nutrition, Zhejiang Key Laboratory for Agro-Food Processing, Zhejiang UniversityHangzhou, China.
College of Food Science and Technology, Shanghai Ocean UniversityShanghai, China.
Front Microbiol. 2017 May 31;8:989. doi: 10.3389/fmicb.2017.00989. eCollection 2017.
This study firstly developed a multiplex real-time PCR (RT-PCR) technique combined with a pre-enrichment step to simultaneously detect (), () and spp. in raw milk and the dairy farm environment (feces, soil, feed, water) in one reaction. Brain heart infusion (BHI) broth was selected for the enrichment step to increase the density of the target bacteria by using an incubation of 4 h before multiplex RT-PCR. The results showed that the detection limit of the multiplex real-time assay was approximately 10 CFU/mL for pure cultures and artificially contaminated milk without enrichment, while 12, 14, and 10 CFU/25 mL, respectively, for , and spp. after pre-enrichment. The newly developed multiplex RT-PCR assay was applied to 46 dairy farm environmental samples and raw milk samples covering a wide variety of sample types. The results demonstrated that the multiplex RT-PCR assay coupled with the BHI enrichment broth was suitable for the simultaneous screening of , and spp. in the pasture environment and in raw milk. The multiplex RT-PCR assay clearly and successfully shortened the total detection time and reduced labor compared to conventional culture-based methods for testing natural samples.
本研究首先开发了一种多重实时荧光定量聚合酶链反应（RT-PCR）技术，并结合预富集步骤，以便在一个反应中同时检测原料奶以及奶牛场环境（粪便、土壤、饲料、水）中的（）、（）和 菌属。选择脑心浸液（BHI）肉汤进行富集步骤，通过在多重RT-PCR之前进行4小时的培养来增加目标细菌的密度。结果表明，对于纯培养物和未经富集的人工污染牛奶，多重实时检测的检测限约为10 CFU/mL，而对于经预富集后的（）、（）和 菌属，检测限分别为12、14和10 CFU/25 mL。新开发的多重RT-PCR检测方法应用于46份涵盖多种样本类型的奶牛场环境样本和原料奶样本。结果表明，多重RT-PCR检测方法与BHI富集肉汤相结合适用于同时筛查牧场环境和原料奶中的（）、（）和 菌属。与传统的基于培养的天然样本检测方法相比，多重RT-PCR检测方法明显成功地缩短了总检测时间并减少了劳动力。
