食源性金黄色葡萄球菌溶血素基因的多重PCR检测和聚类分析

Genotypic and phenotypic analysis of hemolysis in foodborne Staphylococcus aureus

目的建立多重PCR方法检测金黄色葡萄球菌α-溶血素、β-溶血素、h-溶血素基因以及16S rDNA,了解食源性金黄色葡萄球菌中3种溶血素基因的分布情况和溶血表型,并对菌株进行聚类分析。方法建立多重PCR方法检测148株食源性金黄色葡萄球菌的hla、hlb和hemolysin基因,并研究其分布情况,同时用血平板法检验其溶血表型,对数据进行汇总分析。结果148株菌中131株检出hla基因(88.51%),90株菌检出hlb基因(60.81%),28株菌检出hemolysin基因(18.92%);表型溶血的有131株(88.51%),不溶血的有17株(11.49%);以溶血素基因型和溶血表型为聚类因素,将148株菌以100%相似度为界分为A～L共12个型,其中以A型为主,有58株(39.19%),B型37株(25.00%),C型18株(12.16%)。结论该四重PCR方法特异性高,快速简便,能满足高通量菌株筛选的要求;食源性金黄色葡萄球菌普遍携带hla基因,聚类分析以A型和B型为主。

Objective To establish a multiplex PCR method for detecting genes of α-hemolysin（hla）,β-hemolysin（hlb）,hemolysin and 16S rDNA,and to learn the distribution of three hemolysin genes and the characteristics of hemolytic phenotype in 148 foodborne Staphylococcus aureus strains,and to classify the strains with cluster analysis.Methods The multiplex PCR method was established and used to detect the genes of α-hemolysin,β-hemolysin,hemolysin and 16S rDNA.The blood agar method was used to detect the characteristics of hemolytic phenotype.The experiment data was analyed with SPSS16.0.Results 131 strains were positive for hla gene（88.51%）,90 hlb gene（60.81%）,28 hemolysin gene（18.92%）.131 strains had the characteristics of hemolysis（88.51%）,while the hemolysis were negative in 17 strains（11.49%）.With the clustering factors of the hemolysin genotype and hemolytic phenotype,148 strains were classified into 12 types from type A to type L with 100% similarity.Among them,type A contained 58 strains（39.19%）,type B 37（25.00%）,type C 18（12.16%）.Conclusion This multiplex PCR method is fast,convenient and specific,and could be used for high-throughput screening of hemolysin genes in S.aureus.Most of the foodborne Staphylococcus aureus strains carrying the hla gene mainly belong to type A and type B.