空肠弯曲杆菌ERIC-PCR分型技术研究

摘要

目的：建立空肠弯曲杆菌肠杆菌科基因间重复一致序列PCR（ERIC-PCR）的分子分型技术，快速分析空肠弯曲杆菌指纹谱。方法本研究提取空肠弯曲杆菌ATCC33560的基因组DNA作为模板，采用对优化项目设置梯度其它条件不变的方法，对ERIC-PCR反应中的模板量、引物浓度和退火温度等进行优化，建立空肠弯曲杆菌的ERIC-PCR分型技术，并采用该技术对20株空肠弯曲杆菌的指纹图谱进行了分析。结果 ERIC-PCR反应体系中DNA模板量96ng/25μL，ERIC1、ERIC2引物浓度各0.8μmol/L，退火温度40℃时，指纹图谱条带清晰、明亮；20株空肠弯曲杆菌临床分离株ERIC-PCR指纹图谱差异较大，可在160bp~32000bp范围内出现2条~13条条带，分为14型。结论研究显示成功建立了空肠弯曲杆菌ERIC-PCR指纹图谱分型技术，应用该方法可从分子水平上对空肠弯曲杆菌基因组DNA进行快速指纹图谱分析，具有简便和快速等优点，能够为空肠弯曲杆菌流行病学调查提供科学依据。%Objective To generate an efifcient enterobacterial repetitive intergenic consensus sequence (ERIC-PCR) typing and analyze molecular type of different food-borne Campylobacter jejuni.Method Genomic DNA of Campylo-bacter jejuni reference strain ATCC33560 was used as the template for PCR. Target sequences in Campylobacter jejuni genomic DNA were amplified with the primers designed according to the reference. To obtain optimum fingerprint maps,template and primer concentration of the reaction system and annealing temperature of PCR,the factors to be optimized were designed in different concentration grads and other factors were ifxed. Then 20 Campylobacter jejuni isolates were classiifed by PCR.Result The optimal concentration of template DNA was 96ng/25mL,the concentration of primers was 0.8mmol/L each primer and annealing temperature of PCR was 40 ℃.The ERIC-PCR ifngerprint maps of different isolates were different. The ampliifcation products contained 2 to 13 bands in length were ranging between 160bp and 32000bp. The 20 Campylobacter jejuni isolates were classiifed into 14 types according to ifngerprint maps. Conclusion The optimum ERIC-PCR molecular classiifcation method of Campylobacter jejuni was established. ERIC-PCR system is an efficient method for identification,typing and tracking analysis. It will be a promising method in studying molecular epidemiology of. Campylobacter jejuni.