反向斑点杂交法检测肺炎支原体耐大环内酯类抗生素耐药基因

Detection of macrolid resistant genes of Mycoplasma pneumoniae by reverse dot blot hybridization

目的:建立反向斑点杂交法快速检测肺炎支原体(MP)耐大环内酯类抗生素耐药基因。方法:根据MP 23SrRNAⅤ区域常见的耐药突变位点设计检测探针,将其分别点样在尼龙膜上制备成反向斑点杂交平台。以该区域序列已知的菌株和标准均株来扩增靶基因,扩增过程中掺入地高辛标记的dNTPs。扩增产物与尼龙膜上点样阵列杂交后,用地高辛检测试剂盒来检测,根据显色结果来判定相应位点突变情况。结果:斑点杂交法检测了24株临床菌株,结果显示A2063G或A2064G突变,与序列测定结果完全相同,同时未检测到A2063C和C2617G突变。结论:应用本方法可以快速、准确地预测MP对常见大环内酯类抗生素的耐药情况。

Objective：To establish a reverse dot blot hybridization assay to detect macrolid resistant gene in Mycoplasma pneumoniae.Methods：The detecting probes were designed for the hot mutations in 23S rRNAⅤ region of M.pneumoniae,which spotted into the nylon membrane.The target genes were amplified using polymerase chain reaction from the strains sequenced and the standard strain,and digoxigenin labeled dNTPs were mixed into genes in the amplification process.The digoxigenin detection kit was used to detect hybridization results of PCR products and probes array in membrane.The mutations were determined by whether the color appeared.Results： Twenty-four clinical strains by dot blot hybridization assay showed A2063G or A2064G mutation,and the results were identical by sequencing formerly,but no A2063C and C2617G mutation were detected.Conclusion： Application of this method can quickly and accurately predict whether M.pneumoniae resistance to the common macrolids.