摘要
根据嗜冷菌中占多数的是假单胞菌属,选择假单胞菌属特异性引物进行PCR扩增代表原料乳中的嗜冷菌;建立一种可行的原料乳中收集微生物的前处理方法(三氯乙酸前处理法);筛选出DNA模板制备方法,优化PCR扩增条件,在PCR产物中加入荧光染料SYBR Green I进行荧光分析,建立原料乳中快速PCR计数嗜冷菌的新方法。利用已知标准菌液浓度复原乳的嗜冷菌平板计数结果与荧光测定结果得到相应的标准曲线,结果表明,检测的检出限低于103cfu/mL,线性范围为(103～108)cfu/mL,检测时间为10h,检测结果与IDF平板计算法结果一致。由此建立的原料乳嗜冷菌PCR计数方法比较国际标准计数方法并无显著差异,但大大缩短计数时间。
To establish a rapid detection technique for psychrophile in raw milk, one set of the specific primers were designed, according to the Pseudomonas spp. that are in the majority of the psychrophile in raw milk. A feasible raw milk pre-treatment that collection of bacteria from raw milk was achieved by trichloroacetic acid. Quantitative analysis was achieved by optimizing DNA extraction method, PCR assay conditions and adding fluorescence- intensifier SYBR Green I dye to samples of post-PCR for fluorescence analysis. A standard curve was made by relating plate colony count of psychrophile in known concentration reconstituted milk to fluorescence intensity obtained by fluorescence analysis. A rapid PCR method for enumeration of psychrotrophic bacteria in raw milk was established therefore. The analysis results showed that the detection limit of the established method was less than 10^3 cfu/mL, linear range was （10^3-10^8） cfu/mL and analysis time was less than 10 h. The counting results obtained from PCR method were same to that obtained from standard method of IDF （IDF 132A）. There were no significant differences between the IDF method and this new method, but the advantage of shorten in time and lower experimental cost were existed.
出处
《食品工业》
北大核心
2009年第5期63-66,共4页
The Food Industry
基金
十一五奶业重大专项(2006DAD04A09)研究工作一部分
关键词
PCR
嗜冷菌
原料乳
检测
polymerase chain reaction
psychrophile
raw milk
detection
