牛奶样品布氏杆菌套式PCR检测方法的建立  

Nested-PCR for detection of Brucella in milk

作　　者：杜振昆[1] 郭军庆[1] 张妙仙 项玉燕 周继勇[1] 

机构地区：[1]浙江大学动物预防医学研究所 [2]金华市畜牧兽医局,浙江金华321017

出　　处：《浙江大学学报（农业与生命科学版）》2008年第2期169-174,共6页Journal of Zhejiang University:Agriculture and Life Sciences

基　　金：浙江省重大科技攻关资助项目(2005C12009-02).

摘　　要：为建立从奶品中监控布氏杆菌感染的方法,根据牛布氏杆菌omp25、omp31和16SrRNA基因序列设计内、外向引物,优化牛奶样品中布氏杆菌基因组DNA提取方法,建立牛奶样品布氏杆菌套式PCR方法.结果显示,使用人工合成的两对F4/R2和F2D/R1U套式引物,通过两次扩增,可有效扩增出牛奶中污染布氏杆菌的16S rRNA的特异性DNA片段,其对牛奶样品中布氏杆菌检测下限达3·3CFU·mL-1.该套式PCR与大肠杆菌、溶血性链球菌、沙门氏菌、克雷伯氏杆菌、金黄色葡萄球菌、绿脓杆菌和螺旋杆菌等相关细菌DNA无交叉反应,显示高度的布氏杆菌特异性,其与布氏杆菌分离鉴定的符合率达100%.本试验建立的布氏杆菌套式PCR检测方法具有较好的特异性和敏感性,适用于牛奶样品中布氏杆菌的实验室快速检测.To detect Brucella inflection in milk, a nested PCR assay was developed by designing outwarddirected and inward-directed primers for omp25, omp31 and 16S rRNA genes of Brucella abortus, and optimizing the extraction of Brucella genomic DNA. The results show that two rounds of amplification with the two pairs of synthesized primers F4/R2 and F2D/R1U can effectively detect the specific DNA fragment of 16S rRNA from the Brucella contaminated milk, of which the detection limit was as low as 3.3 CFU·mL^-1. This PCR assay showed a high specificity for Brucella with no cross-reaction of the related bacteria DNA, including Escherichia coli, Hemolytic streptococci, Salmonella, Klebsiella, Staphylococcus aureus, Pseudomonas aeruginosa, and Helicobacter pylori. The developed nested PCR assay has 100% identity to Brucella isolation. Therefore, it is suggested that the nested-PCR assay for Brucella with high specificity and sensitivity is potential to detect Brucella infection of milk in laboratory.

关 键 词：布氏杆菌 套式PCR 牛奶 

分 类 号：Q93-332[生物学—微生物学] S852.61[农业科学—基础兽医学]