设计乳杆菌特异性引物并运用菌落PCR技术快速检出和鉴定四川泡菜中的乳杆菌

Rapid detection and identification Lactobacillus from Sichuan pickle by colony PCR using Lactobacillus-specific primers

摘要:

乳杆菌(Lactobacillus)是益生菌, 也是当前的研究热点之一。研究泡菜等样品中的乳杆菌需要快速的检出方法。根据已完成全基因组测序的14种乳杆菌的16S rDNA序列, 设计一对乳杆菌特异性引物。PCR检测结果表明该引物对乳杆菌和明串珠菌能扩增出800 bp的片段, 对表皮葡萄球菌、乳酸乳球菌和枯草芽胞杆菌却没有扩增条带, 具有一定的乳杆菌特异性。结合MRS乳杆菌半选择培养基和革兰氏染色, 运用菌落PCR技术, 可以快速高效地检出四川泡菜中的乳杆菌。再通过对PCR扩增片段测序, 可以将乳杆菌鉴定到种。从16份四川泡菜样品中检出了15株乳杆菌, 其中14株被鉴定为植物乳杆菌, 1株需进一步鉴定才能确定种。该方法可以检出乳杆菌新种。

Abstract:

Lactobacillus is used as probiotics and there has been much interest. The detection and identification of Lactobacillus from pickle need a rapid method. We designed a pair Lactobacillus-specific primer by analyzing 16S rDNA of 14 Lactobacillus species whose complete genomes had been sequenced. The results of colony PCR showed that an 800 bp band was generated when Lactobacillus and Leuconostoc strains used as template but no band appeared when Staphylococcus epidermidis, Bacillus subtilis, or Lactococcus lactis used as template. Lactobacillus of Sichuan pickle was rapidly detected by a procedure including selection of MRS culture, Gram stain and colony PCR. By sequencing the 800 bp fragment, the detected strain was identified at the species level. 15 Lactobacillus strains were detected from 16 samples of Sichuan pickle. 14 Lactobacillus strains were identified as L. plantarum and a Lactobacillus strain need more identification. The method would detect new Lactobacillus species.