运用变性高效液相色谱对肺炎克雷伯菌产ESBL进行基因分型

目的 通过运用变性高效液相色谱（DHPLC）技术对前期研究已确认产超广谱β-内酰胺酶（ESBL）的肺炎克雷伯菌临床分离株TEM型质粒进行基因分型,试图建立一种方便快捷的用于ESBL分子诊断及其流行病学监测的新方法.方法 利用PCR技术从肺炎克雷伯菌临床分离株中扩增出TEM型质粒的编码序列,扩增产物运用DHPLC技术进行分析,分析提示,异常的样本通过测序确定其基因突变的类型,最后通过比对确定其基因型.结果 共分析了101例肺炎克雷伯菌临床分离株,全部样本均扩增出TEM型质粒的编码序列,经过DHPLC分析,52例（51.4%）样本表现为单一的洗脱峰,其形态与TEM-1标准菌株的峰型相一致,测序确定它们的碱基序列亦相一致,不存在变异,为TEM-1型;49例（48.6%）样本表现为异常的洗脱峰,它们均为双峰,形态一致,但异源双链峰的高度有差异,测序结果表明它们均存在四种相同的基因突变,在NCBI网站比对后确定为TEM-116;测序结果还提示,部分样本中TEM-1和TEM-116混合存在,其比例的不同表现为DHPLC时异源双链峰高度的差异;文献检索表明,本次确定的TEM-116为一新的基因亚型,为国内首次报道.结论 DHPLC具有简便快捷、高通量和自动化的特点,重复性好,不仅可对已知突变作出即时诊断,还可发现新的基因亚型,不失为一种较好的ESBL分子诊断方法及其流行病学监测手段.

Study on genotyping profile of Klebsiella pneumoniae's ESBL by denaturing high-performance liquid chromatography

Objective To investigate the genotype of Klebsiella pneumoniae(ESBL strains) by denaturing high-performance liquid chromatography(DHPLC), to establish a quick and convenient method for ESBL molecular diagnosis and epidemiology monitoring. Methods After PCR amplifying the coding rank of TEM-type plasmid from clinic isolated Klebsiella pneumoniae strains, we used DHPLC to analyze the PCR production for sequencing and identifing gene mutation. The genotypes were eventually determined by sequence analysis. Results One hundred and one strains of Klebsiella pneumoniae were studied, all the strains had the coding rank of TEM-type plasmid as shown by PCR analysis. By DHPLC analysis, 52 strains (51.4%) had a single elution peak, which was identical to the morphology of TEM-1-type standard strain. By sequencing analysis, these strains were determined as TEM-1-type, which had the same base sequence to TEM-1-type strain and had no dissociation. Forty-nine strains(48.6%) were found abnormal elution peak, they all had double peaks and the same peak morphology, but there were difference in the height of heteroduplex peak. The sequencing results showed that there were four same gene mutations in these strains. The TEM-116-type was approved a new genotype which was the first report in our country, by bibliographic retrieval. Conclusion DHPLC analysis is a good method for molecular diagnosis and epidemiology monitoring of ESBL.