食源性单核增生李斯特氏菌重复序列-PCR分型与鉴定研究

Typing and identification of food-borne Listeria monocytogenesis by repetitive element sequence PCR assay

目的探索我国北方部分省市单核增生李斯特氏菌食品分离株与标准菌株间的亲缘关系,筛选食品中该菌的鉴定用标识序列。方法利用REP-PCR和ERIC-PCR技术,对单核增生李斯特氏菌的基因组进行分型研究,并以相似性系数构建进化树。回收、克隆该菌共有的ERIC-PCR扩增DNA条带,通过序列测定、BLAST分析及PCR验证,确定使用该序列鉴定该菌的可行性。结果本研究所用的单核增生李斯特氏菌分别被ERIC-PCR和REP-PCR扩增产生约1 800bp的共有扩增带;当欧式距离的平方为20时,ERIC-PCR和REP-PCR均将25株菌分为3个群,当欧式距离的平方为5时,ERIC-PCR将25株菌分为16个型,REP-PCR将25株菌分为20个型。ERIC-PCR得到的1 800bp共有扩增片段测序后的长度为1 816bp,为李斯特氏菌属共有的精氨酸生物合成双功能argJ基因。结论重复序列-PCR可将本研究的单核增生李斯特氏菌食品分离株分为3个群;筛选获得的ERIC-PCR特异DNA扩增片段可用于李斯特氏菌属的鉴定。

To understand the phylogenesis of food-borne Listeria monocytogenesis in northern China in order to screen the identifier sequence used in the identification of L. monocytogenesis, the repetitive extragenic palindromic PCR（REP-PCR） and entero-bacterial repetitive intergenic consensus PCR（ERIC-PCR） were used for the genetic typing of 38 strains of L. mono-cytogenesis. The cluster dendrograms were made according to the fingerprinting and the DNA fragment from ERIC-PCR was retrieved, cloned, sequenced and then blasted in Genbank. In addition, the specificity for the identification was analyzed by PCR. It was found that all the strains of L. monocytogenesis could be amplified by means of REP-PCR or ERIC-PCR, to produee the coneensus sequence of 1800 bp DNA fragment, in which 25 strains were grouped into 3 clusters both by REP-PCR and ERIC-PCR when the squared Euclidean distance was 20; and could be grouped into 16 and 20 patterns when it was 5. The length of the concensus sequence of 1800 bp amplified by ERIC-PCR was 1816 bp and it was proved to be the argJ gene with the double functions in the arginine biosynthesis of all L. monocytogenesis. It is evident that the specific DNA fragment amplified by ERIC-PCR can be used as the identifier sequence for the identification of listeria spp. with PCR assay.