PCR检测乳粉中肺炎克雷伯氏菌

Detection of Klebsiella pneumoniae in Emulsifiable Dust by Polymerase Chain Reaction Assay

利用PCR技术直接检测乳粉中的肺炎克雷伯氏菌，无需增菌．通过滤膜法成功地从人工污染肺炎克雷伯氏菌的乳粉中提取了肺炎克雷伯氏菌的DNA．以肺炎克雷伯氏菌的间区序列（ITS）为靶基因，经过PCR扩增得到158bp的产物，经过DNA测序证实该产物为目的扩增产物．该方法灵敏度高，乳粉中检测的灵敏度为10CFU／mL，可在6h内完成对乳品中肺炎克雷伯氏菌的检测，比目前普遍采用的先增菌再进行PCR检测的方法缩短了12～24h．

A uniplex polymerase chain reaction（PCR） assay was developed for direct detection Klebsiella pneumoniae without enrichment in emulsifiable dust. A solvent extraction procedure was successfully modified for extraction of Klebsiella pneumoniae DNA from artifically by contaminated whole milk, skim milk and cheese. Primer targeting the thermostable nuclease gene（ITS） was used in the uniplex PCR. A DNA fragment of 158 bp was amplified. PCR product was confirmed by DNA sequencing. The sensitivity of the uniplex PCR is 10 CFU/mL. The developed methodology allows detection of Klebsiella pneumoniae in emulsifiable dust in less than 6 h,the time of this developed PCR assay is 12--24 h less than the time of general PCR assay with enrichment method.