建立一种反向斑点杂交法检测肺炎支原体23S rRNAV区A2063G基因突变的方法

目的建立检测肺炎支原体（Mycoplasma pneumoniae,MP）23S rRNA V区2063基因型的反向斑点杂交方法,并与PCR产物直接测序法的结果进行比较分析。方法19例自临床咽拭子标本分离培养的MP,用自行设计的反向斑点杂交法检测23S rRNA V区2063基因型,同时对相应序列测序分析。抽提标准菌株FH和127份经PCR测定MP阳性的临床标本基因组DNA,用MP阴性的基因组DNA抽提物5份作阴性对照,用反向斑点杂交法检测23S rRNA V区2063基因型。结果19例分离培养的MP,经反向斑点杂交法检测15株有23S rRNA V区基因A2063G位点突变;4株为2063A,与测序结果一致（Kappa一致性检验,P〉0.05）。经反向斑点杂交法检测,127份MP阳性的基因组DNA抽提物中有122份标本为A2063G位点突变,标准菌株FH和2份DNA抽提物的2063位点碱基为A,还有3份抽提物标本的2063位点碱基既有A又有G,5份阴性对照均无显色。结论反向斑点杂交方法能快速、准确检测临床MP 23S rRNA V区2063基因型。

Detection of A2063G gene mutation in 23S rRNA domain V of Mycoplasma pneumoniae by a reverse dot-blot hybridization

Objective To establish a reverse dot-blot hybridization in detecting the genotypes of 23S rRNA domain V of Mycoplasma pneumoniae and compare it with the direct sequencing method.Method A self-designed reverse dot-blot hybridization was applied to detect the genotypes of 23S rRNA domain V of Mycoplasma pneumoniae in 19 clinical isolates from oropharyngeal swabs,and the corresponding sequence was sequenced meanwhile.Reference strains FH and 127 Mycoplasma pneumoniae DNA extractions from patient specimens tested positive by PCR, with 5 Mycoplasma pneumoniae negative DNA extractions as controls, were detected macrolide resistant genotypes of 23S rRNA domain V by the reverse dot-blot hybridization method. Result 15 strains of the 19 clinical isolates were found to possess point mutations of A2063G within domain V of the 23S rRNA gene by the reverse dot-blot hybridization while the other 4 strains were 2063A in domain V of the 23S rRNA gene. These findings were concordant with sequencing data （ Kappa consistency test, P 〉 0.05 ）. Among the 127 Mycoplasma pneumoniae positive DNA extractions from patient specimens, 122 extractions were found to possess point mutations of A2063G by the reverse dot-blot hybridization;2 extractions only contained A base and 3 extractions possessed both A and G base in domain V of the 23S rRNA gene;no positive result was found in 5 negative controls. Conclusion The reverse dot-blot hybridization was capable of detecting the A2063G point mutation in domain V of 23S rRNA of Mycoplasma pneumoniae from isolates and patient specimens.