葡萄球菌属不同靶基因序列种水平鉴定能力的比较评价研究

Evaluation on molecular identification capacity of different target gene sequences for Staphylococcus species

目的:比较评价靶基因16S rRNA、tuf A、fem A、rpo B和gap序列对葡萄球菌种水平的鉴定能力。方法:选择60株菌种鉴定明确的葡萄球菌(涵盖18个种),比较评价16S rRNA基因序列及VITEK 2全自动微生物生化鉴定系统对常见葡萄球菌鉴定的准确性;采用PCR方法特异扩增60株菌tuf A、fem A、rpo B和gap的基因序列,并对阳性扩增产物进行核酸测序,利用Mega 7.0和DNASP 5.0软件分析靶基因序列多样性,通过序列聚类关系分析,进一步评价不同靶基因序列对葡萄球菌种水平的分子鉴定能力。结果:16S rRNA基因序列比对不能有效区分山羊葡萄球菌和头状葡萄球菌,而VITEK方法则将巴氏葡萄球菌错误鉴定为沃氏葡萄球菌。序列多样性分析表明,尽管靶基因tuf A、fem A、rpo B和gap序列比16S rRNA基因序列多样性更强,但基于tuf A、fem A、rpo B和gap序列的聚类分析在种水平仍会错误鉴定部分葡萄球菌,如表皮葡萄球菌、头状葡萄球菌、腐生葡萄球菌和科氏葡萄球菌等。进一步评价16S rRNA基因序列分别与靶基因tuf A、fem A、rpo B和gap的串联序列对葡萄球菌种水平的鉴定能力,结果表明只有靶基因fem A与16S rRNA基因的串联组合能够实现对18种葡萄球菌的准确鉴定,而其他靶基因与16S rRNA基因的串联组合对于葡萄球菌种水平的鉴定能力没有明显改善。结论:基于单个靶基因的序列分析会造成部分葡萄球菌种水平的错误鉴定,而基于16S rRNA与femA基因的串联序列分析则能够显著提高对葡萄球菌种水平鉴定的准确性。

Objective：To evaluate the molecular identification capacity of different target gene sequences including 16 S rRNA,tuf A,fem A,rpo B and gap for Staphylococcus species.Methods：60 Staphylococcus isolates covering 18 different species were collected,then the accuracy of 16 S rRNA sequence analysis and VITEK 2 system method in species-level identification was evaluated;the target gene sequences of 60 Staphylococcus isolates were amplified by PCR method,and the positive PCR products were sequenced commercially,nucleotide diversity of target genes were analyzed by Mega 7.0 and DNASP 5.0,molecular identification capacity of different target gene sequences for 18 different Staphylococcus species was evaluated by phylogenetic relationship analysis. Results：Staphylococcus caprae and Staphylococcus capitis strains could not be distinguished by 16 S rRNA sequence,and Staphylococcus pasteuri strains were falsely identified as Staphylococcus warneri by VITEK 2 system.Sequence diversity analysis demonstrated that tuf A,fem A,rpo B and gap gene sequence showed a stronger divergence than 16 S rRNA sequence,but some species still could not be distinguished based on these target gene sequences,such as Staphylococcus epidermidis,Staphylococcus capitis,Staphylococcus saprophyticus and Staphylococcus cohnii.Furthermore,the compilation of 16 S rRNA with different housekeeping gene sequences was evaluated for molecular identification of Staphylococcus species,respectively.It was shown that only the compilation of 16 S rRNA with fem A gene sequence could accurately identify all 18 Staphylococcus species.The compilation of 16 S rRNA with other housekeeping gene sequences did not obviously improve the molecular identification capacity.Conclusion：Some Staphylococcus species could not be distinguished based on the single target gene sequence.However,the concatenated sequence of 16 S rRNA with fem A could improve the identification accuracy of Staphylococcus species.