啤酒有害菌的PCR检测和SYBR Green实时PCR定量

PCR Detection and SYBR Green Real-time PCR Quantification of Beer Spoilage Bacteria

啤酒有害菌是一些能在啤酒中存活并使啤酒的外观和风味发生改变的细菌，对其进行快速检测和定量是啤酒生产急待解决的问题。我们从华润雪花啤酒（中国）有限公司各工厂提供的样品中分离到28株啤酒有害菌，16S rDNA序列的系统进化分析表明，其中26个菌株属于乳杆菌属（Lactobacillus spp．）、1个菌株为明串珠菌属（Leuconostoc spp．），1个菌株为片球菌属（Pediococcu sp．）。根据酒花（hop）抗性基因horA、horB和horC的保守序列设计了扩增这3个基因的PCR引物，用这些引物对28株啤酒有害菌进行了常规PCR检测，检出率分别为89％、79％和75％，用hor A—horC双引物进行检测，检出率为100％。用SYBR Green实时定量PCR技术，以horA基因为靶序列，建立了对啤酒有害菌的细胞数进行快速定量的新方法，用该方法测定的污染啤酒样品中有害菌的浓度与平板培养法相近。

Beer spoilage bacteria are able to survive in beer and to transform the appearance and flavour of beer. Therefore rapid detection and quantification of these bacteria possess important significance for brewers. Twenty-eight beer spoilage bacterial strains were isolated from the samples of beer brewing factories of China Resources Snow Breweries. Among them, 26 strains are identified as Lactobacillus spp., I strains as Leuconostoc spp., and 1 strain as Pediococcu sp.. On the basis of the conserved sequences of hop-resistance genes, three primer pais for amplification of horA, horB and horC genes were designed. Twenty-eight beer spoilage strains were detected by conventional PCR using these primers. The experimental results indicated that positive reaction rates for horA, horB and horC genes were 89%, 79% and 75%, respectively. When these strains were detected using horA - horC primers, positive reaction rates were 100%. A new methord for rapid quantification of beer spoilage bacteria was developed in which SYBR Green quantitative real-time PCR （qRT-PCR） technique and horA primers were applied. The concentration of spoilage bacteria in contaminated beer sample detected by qRT-PCR was similar to that detected by plate cultivation.