Ruan Jinghua, Wang Wujun, Zhang Tiyin, Zheng Teng, Zheng Jing, Yu Shiyu, Yu Daojin, Huang Yifan
Fujian Key Laboratory of Traditional Chinese Veterinary Medicine and Animal Health, Fujian Agriculture and Forestry University, Fuzhou, 350002, Fujian, People's Republic of China.
College of Animal Science, Zhejiang University, Hangzhou, 310058, Zhejiang, People's Republic of China.
AMB Express. 2020 Nov 24;10(1):207. doi: 10.1186/s13568-020-01144-x.
Salmonella spp. is a high-risk bacterial pathogen that is monitored in imported animal-derived feedstuffs. Serratia fonticola is the bacterial species most frequently confused with Salmonella spp. in traditional identification methods based on biochemical characteristics, which are time-consuming and labor-intensive, and thus unsuitable for daily inspection and quarantine work. In this study, we established a duplex real-time qPCR method with invA- and gyrB-specific primers and probes corresponding to Salmonella spp. and S. fonticola. The method could simultaneously detect both pathogens in imported feedstuffs, with a minimum limit of detection for Salmonella spp. and S. fonticola of 197 copies/μL and 145 copies/μL, respectively (correlation coefficient R = 0.999 in both cases). The amplification efficiency for Salmonella spp. and S. fonticola was 98.346% and 96.49%, respectively. Detection of fishmeal was consistent with method GB/T 13091-2018, and all seven artificially contaminated imported feed samples were positively identified. Thus, the developed duplex real-time qPCR assay displays high specificity and sensitivity, and can be used for the rapid and accurate detection of genomic DNA from Salmonella spp. and S. fonticola within hours. This represents a significant improvement in the efficiency of detection of both pathogens in imported feedstuffs.
沙门氏菌属是一种高风险的细菌病原体，在进口动物源性饲料中受到监测。丰氏沙雷氏菌是在基于生化特性的传统鉴定方法中最常与沙门氏菌属混淆的细菌物种，这些方法既耗时又费力，因此不适用于日常检验检疫工作。在本研究中，我们建立了一种双重实时荧光定量PCR方法，使用与沙门氏菌属和丰氏沙雷氏菌对应的invA和gyrB特异性引物及探针。该方法能够同时检测进口饲料中的这两种病原体，沙门氏菌属和丰氏沙雷氏菌的最低检测限分别为197拷贝/μL和145拷贝/μL（两种情况下相关系数R均为0.999）。沙门氏菌属和丰氏沙雷氏菌的扩增效率分别为98.346%和96.49%。鱼粉检测结果与GB/T 13091-2018方法一致，所有7份人工污染的进口饲料样本均被阳性鉴定。因此，所建立的双重实时荧光定量PCR检测方法具有高特异性和高灵敏度，能够在数小时内快速准确地检测出沙门氏菌属和丰氏沙雷氏菌的基因组DNA。这代表了进口饲料中这两种病原体检测效率的显著提高。
