基于实时荧光PCR技术快速检测饲料中的蜡样芽孢杆菌  

Rapid Detection of Bacillus cereus in Feed by Real-time PCR

作　　者：包海燕 岳喜庆[1] 武俊瑞[1] 乔宇[2] 彭晴[2] 石波[2] 纪善琴 王治颖 陈旭[1] 乌日娜[1] BAO Hai-yan;YUE Xi-qing;WU Jun-rui;QIAO Yu;PENG Qing;SHI Bo;JI Shan-qin;WANG Zhi-ying;CHEN Xu;WU Ri-na(College of Food Science,Shenyang Agricultural University,Shenyang 110161,China;Feed Research Institute,Chinese Academy of Agricultural Sciences,Beijing 100081,China)

机构地区：[1]沈阳农业大学食品学院,沈阳110161 [2]中国农业科学院饲料研究所,北京100081

出　　处：《沈阳农业大学学报》2020年第3期349-354,共6页Journal of Shenyang Agricultural University

基　　金：国家重点研发计划项目(2018YFD0501400);辽宁省青年科技人才“育苗”项目(LSNQ201911)。

摘　　要：蜡样芽孢杆菌(Bacillus cereus)在自然界中广泛存在,而且大部分菌株能产生肠毒素和呕吐毒素,是引起养殖动物中毒的常见致病菌。在饲料生产过程中,为对该病原菌进行有效监控,以蜡样芽孢杆菌肠毒素FM基因(entFM)和肠毒素T基因(bceT)保守区域片段为靶序列,利用DNAMAN设计两对特异性引物,建立一种基于实时荧光PCR(real-time PCR)技术的蜡样芽孢杆菌快速检测方法,并对所建立方法的重复性、特异性及灵敏度进行分析。结果表明:所设计的引物可以实现对蜡样芽孢杆菌的快速检测而且具有较高的重复性;对8株蜡样芽孢杆菌和8株非蜡样芽孢杆菌共16株菌株进行特异性检测,8株蜡样芽孢杆菌检测全部为阳性,8株非蜡样芽孢杆菌检测全部为阴性,无漏检和误检现象;对纯培养条件下蜡样芽孢杆菌提取的DNA模板进行10倍梯度稀释并进行灵敏度检测,两种肠毒素基因的检测限均为1.0×104CFU·mL^-1;对人工染菌的饲料样品进行检测,在增菌18~20h后进行实时荧光PCR检测,检测限均可达到10CFU·mL^-1,证明该方法具有良好的特异性和灵敏度,是快速检测饲料中蜡样芽孢杆菌的有效方法。Bacillus cereus(B.cereus)is widespread in nature and considered a common pathogenic bacterium that causes poisoning of cultured animals.And most strains of B.cereus can produce enterotoxin and vomitoxin.In order to effectively monitor this pathogen during feed production,a Real-time PCR assay for rapid detection of B.cereus was developed in this study.In this system,two pairs of primers were designed by DNAMAN based on entFM gene and bcet gene.The repeatability,specificity and sensitivity of the method were respectively analyzed.The results showed that the primers designed in this study can detect B.cereus rapidly and have high repeatability;and all 8 strains of B.cereus yielded positive results and showed no cross reaction with 8 tested non-B.cereus strains;The detection limit of the method was 1.0×104 CFU·mL^-1 for pure bacterial.Additionally,the method with the enrichment step could reliably detect B.cereus in artificially contaminated feed samples at concentration as low as 10 CFU·mL^-1.These results indicate the developed Real-time PCR method is a potential useful method for rapid detection of B.cereus in feed samples.

关 键 词：蜡样芽孢杆菌 实时荧光定量PCR 饲料 

分 类 号：TS207.4[轻工技术与工程—食品科学] O657.3[轻工技术与工程—食品科学与工程]