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目的建立沙门菌聚合酶链反应-变性高效液相色谱(PCR-dHPLC)基因分型方法。方法采用16S～23S rRNA内转录间隔(ITS)作为沙门菌分型目的基因,确定特异性扩增引物,进行PCR扩增,扩增产物经dHPLC分离,根据dHPLC图谱峰型差异进行分型,并与血清学和生化分型结果比较。结果 89株沙门菌共分为12个dHPLC型(D型);所有沙门菌均有1个相同色谱峰,克隆测序结果表明,其片断大小为600 bp,其他8种食源性致病菌对照株无此色谱峰,应为沙门菌16S～23S rRNA基因序列的特征性条带,分型结果与血清学差异较大,与生化分型结果有一定一致性。结论所建立的沙门菌PCR-dHPLC基因分型方法具快速、操作方便、重复性好、成本低廉、高通量和自动化的特点。
Objective To establish a method for genotyping Salmonella by polymerase chain reaction-denaturing high performance liquid chromatography (PCR-dHPLC). Methods 16S ~ 23S rRNA internal transcribed spacer (ITS) was used as the target gene for Salmonella typing, specific primers were identified and amplified by PCR. The amplified products were separated by dHPLC and classified according to the difference of dHPLC patterns Compared with serological and biochemical typing results. Results Eighty-nine Salmonella strains were divided into 12 dHPLC types (D type). All Salmonella strains had the same chromatographic peak. Cloning and sequencing showed that the size of the fragment was 600 bp. The other eight kinds of food-borne pathogenic bacteria Strain without this peak should be characteristic bands of Salmonella 16S ~ 23S rRNA gene sequences, typing results and serological differences are large, and biochemical typing results have a certain consistency. Conclusion The established Salmonella PCR-dHPLC genotyping method has the advantages of fast, convenient operation, good repeatability, low cost, high throughput and automation.