金氏金杆菌特异性靶基因的选择及健康人群带菌率调查

Selection of specific target genes of Kingella kingae and investigation of carrier rate in healthy people

目的对现有金氏金杆菌特异性PCR检测的相关靶基因进行特异性分析,选择适宜检测方法进行国内健康人群带菌率调查。方法利用生物信息学技术比较美国国立生物技术信息中心(NCBI)数据库中已公布的金氏金杆菌16S rDNA、groEL、rtxA基因以及其他近缘基因序列,探讨3种靶基因在金氏金杆菌中的保守性。对所选择的金氏金杆菌采用实时荧光定量PCR(real-time PCR)方法进行特异性、敏感性、稳定性等验证。应用已建立的方法对513份咽拭子样品进行检测。结果金氏金杆菌rtxA仅与牛莫拉菌近缘基因有72%相似度,金氏金杆菌不同菌株间相似度均达99%以上,rtxA基因保守性和特异性均优于16S rDNA与groEL基因,理论上更适合金氏金杆菌检测。基于rtxA基因的金氏金杆菌real-time PCR检测方法敏感性好,最低检测下限1 copies/μl,可用于金氏金杆菌检测。513份咽拭子,共检出阳性标本107份,阳性率为20.86%。结论 rtxA基因在金氏金杆菌中高度保守,可作为核酸检测的靶基因。基于rtxA的real-time PCR检测显示金氏金杆菌在我国健康人群中存在较高携带率且具有一定的年龄分布趋势。

Objective To analyze the specificity of target genes used in specific PCR detection of Kingella kingae and select suitable assay for the K.kingae carriage investigation in healthy population in China.Methods By comparing the relationship of 16 S rDNA,groEL and rtxA genes with other closely related gene sequences published in National Center for Biotechnology Information(NCBI)database,the conservation of three target genes in K.kingae was analyzed.The specificity,sensitivity and stability of the selected real-time PCR were verified.By using the established assay,513 pharyngeal swabs were detected.Results Phylogenetic tree based on rtxA gene showed high homology among different strains of K.kingae,and the similarity of rtxA gene with Moraxella bovis was 72%.rtxA gene was more conservative and specific than 16 S rDNA and groEL genes,which is more suitable for the detection of K.kingae.The real-time quantitative PCR assay for K.kingae was proved to be rapid,specific,sensitive and reproducible,the detection sensitivity was 1 copies/μl.For 513 pharynx swabs,107 were detected positive(20.86%).Conclusion rtxA gene is highly conserved in K.kingae and can be used as a target gene for nucleic acid detection.Real-time PCR based on rtxA showed that K.kingae had a high carriage rate in healthy population with certain age distribution characteritics in China.