食品科学 ›› 2008, Vol. 29 ›› Issue (4): 225-229.

• 生物工程 • 上一篇    下一篇

PCR-DGGE技术在乳酸菌检测中的条件研究

 马俊孝, 孔健   

  1. 山东大学微生物技术国家重点实验室; 山东大学微生物技术国家重点实验室
  • 出版日期:2008-04-15 发布日期:2011-08-24

Study on Detection Conditions of Lactic Acid Bacteria by PCR-DGGE

 MA  Jun-Xiao, KONG  Jian   

  1. State Key Laboratory of Microbial Technology, Shandong University
  • Online:2008-04-15 Published:2011-08-24

摘要: 变性梯度凝胶电泳技术广泛应用于微生物生态学的研究,最近也渗透到食品微生物学领域。为了检测发酵乳制品中乳酸菌的组成及动态变化,本实验对PCR-DGGE技术在乳酸菌检测中所需要的条件如变性剂梯度、电泳时间进行研究。结果表明,变性剂梯度在30%~55%,电泳时间为200min时,能够有效分离乳酸菌的16SrRNA基因的V3区域。在此基础上,就PCR技术对染色体DNA混合模板的扩增效率进行分析,结果发现,常规PCR技术与降落PCR技术对乳酸菌16SrRNA基因V3区域的扩增没有差异,而细菌通用引物对乳酸菌模板DNA的识别和扩增具有优先和偏爱性,预示PCR-DGGE技术用于微生物群体检测时还应选用乳酸菌引物进行佐证。

关键词: 变性梯度凝胶电泳, 聚合酶链式反应, 乳酸菌, V3可变区域

Abstract: Denaturing gradient gel electrophoresis (DGGE) analysis was recently applied in the detection of food microbial diversities as well as of environmental microbial communities widely. To analyze the component and structure of lactic acid bacteria in food samples and dairy fermented products, DGGE conditions such as gradient of denaturant and run time for electrophoresis were firstly optimized. The results showed that 30%~55% was a suitable denaturant range of gradient for partially melting DNA fragments, and about 200 min was a proper time for the variable V3 region separation of 16S rRNA genes of lactic acid bacteria. Five chromosomes DNA mixture of lactic acid bacteria was used as template DNA for traditional PCR and touch down PCR to amplify the V3 region, respectively. The PCR production was subjected to DGGE analysis as described above. The different efficiencies of amplification during PCR by universal bacteria primer set were also observed. This suggested that the limit in the detection potential of PCR-DGGE is a consequence of DNA template competition during the amplification reaction.

Key words: denaturing gradient gel electrophoresis, polymerase chain reaction, lactic acid bacteria, variable V3 region