摘要
目的利用荧光定量PCR技术,建立食品中单核细胞增生李斯特菌污染的快速敏感特异的检测方法。方法以单核细胞增生李斯特菌的-hlyA基因作为靶序列,设计一对引物和探针,以单核细胞增生李斯特菌菌株,提取核酸DNA作为模板,优化引物和探针的浓度比和退火温度,以单核细胞增生李斯特菌和10种相关细菌考核检测体系的灵敏性、稳定性和特异性。并初步应用于样品的检测。结果本研究建立的反应体系在引物和探针的浓度为0.8μmol/L,0.6μmol/L,退火温度为60℃时,具有良好的特异性和敏感性。在10株相关菌株的检测中,除单核细胞增生李斯特菌出现很好的阳性外,其余菌株均为阴性。在纯菌条件下,定量检测低限19cfu/ml。稳定性分析表明同一样品重复检测3次Ct值的变异系数均小于5%。检测样品结果显示Real-timePCR方法较传统方法敏感、快捷、简便。结论该方法特异性强,稳定性高,操作简便快捷,适应食品微生物检验发展需要,具有较大的推广及应用价值。
To establish a rapid sensitive and specific detection A pair of primers and probe depending on hlyA gene was desiged by method of L. monocytogenes with Real-time PCR in food. way of target sequence of L. monocytogenes, and then apply L. monocytogeneces of standard bacterium strain for template to appraise L. monocytogenes. The best annealing tempera ture, primer and probe ratio were optimized. Specificity,sensitivity and stability analysis test were performed by L. monocytogeneees and 10 other strains associated bacteria,the constructed real-time PCR method was primarily used to mosquito samples test for L. monocytogenes. It was proved by the results that the best annealing temperature was 60℃. Ratio of primers and probe was 0.8μmol/L, 0. 6μmol/L. Test showed that the probe were highly conservative and specific. The results of all 10 strains bacteria were negative except of strains of L. monocytogenes. The quantitative detection Limit of the method was 19cfu/ ml in pure cultured broth, stability test showed that Co-efficient Variables were all less than 5 % in 4 different samples, primary application that real-time PCR method is more sensitive, more easier and more faster than conventional culture method for detection of L. monocytogenes in food. In conclusion, the real-time PCR method has high sensitivity and specialty. The method is handily and rapid, which might have adaptated food microorganism examination development need, and have great value in practical work.
出处
《中国人兽共患病学报》
CAS
CSCD
北大核心
2007年第4期380-383,共4页
Chinese Journal of Zoonoses
基金
湖州市医药卫生科学研究计划项目
关键词
食品
李斯特菌
快速PCR
Food
Listeria monoeytogenes
Real-time PCR
