聚合酶链反应检测乳酸菌酪氨酸脱羧酶基因

Detection of tyrosine decarboxylase gene of lactic acid bacteria by PCR

酪氨酸脱羧酶与乳酸菌发酵食品中酪胺的产生密切相关。根据从GenBank中检索到的酪氨酸脱羧酶基因序列设计一对特异性引物,采用PCR技术对乳酸菌的基因组DNA片段进行扩增,以此建立以酪氨酸脱羧酶基因为靶的产酪胺乳酸菌的分子生物学检测方法。结果表明,供试12株乳酸菌中有9株菌扩增出1133bpDNA片段。对其中3株菌的扩增产物进行DNA序列测定,测序结果采用国际互联网上NCBI的BLAST工具进行同源性检索分析,发现它与已知的Enterococcusfaecalis,Enterococcusfaecium,Carnobacteriumdivergens的酪氨酸脱羧酶基因序列均高度同源,其中PLP(5'-磷酸吡哆醛)的结合位点高度保守,证明该扩增产物是酪氨酸脱羧酶的基因片段。应用该法与常规平板检测法比较显示,二者检测结果基本一致,表明本研究建立的PCR方法可作为一种快速、高度特异的检测产酪胺乳酸菌菌株的新方法。

The tyrosine decarboxylase is close correlated to the tyramine pro du ction in the fermented foods. In this paper, based on the nucleotide sequence of tyrosine decarboxylase(TyrDC) in GenBank, a pair of primers were designed. Usin g the primers PCR experiments were performed to amplify the genomic DNA of lacti c acid bacteria , and the molecular tool for the detection of the tyramine-prod ucing bacteria was developed. The results of the experiments showed that 1133bp fragments were amplified from the DNA of nine strains in twelve supplied strains . The PCR productions of three strains were sequenced. The sequence similarity b y BLAST showed that the sequence is highly homology with the known sequence of t yrdc gene(Enterococcus faecalis、Enterococcus.faecium、Carnobacterium divergens) and the PLP attachment site is highly conserved. This proved that the PCR produ ctions were fragments of tyrdc gene. Compared with the method of detection plate , the two methods have high consistency. So the PCR method can be used as a fast and special method to detect the tyramine-production lactic acid bacteria.