应用PCR方法检测鼠疫现场标本的研究

Study on application of PCR to detect field samples of plague

目的用PCR方法检测疑似鼠疫现场标本,以建立准确、简便和快速的诊断技术。方法对采自疫源地的自毙鼠标本用两种方法提取DNA,进行caf1、pla基因检测和染色体标识基因检测。结果 2份样本均能检测到caf1、pla基因(与Yersinia pestis caf1、pla基因序列比对同源性达到99%以上)和4个染色体标识基因ypo 2087、ypo 2090、ypo2094、ypo 2102,试剂盒提取方式敏感性高于煮沸法。结论优化模板提取方式、提高扩增效率、质粒和染色体多基因检测,可以提高鼠疫PCR诊断技术的检出率。

Objective To detect suspected field samples of plague by PCR to establish accurate,simple and rapid diagnostic technique.Methods DNA of dead rodents found in epidemic focus was extracted by two ways to detect caf1,pla and several tag sequences in chromosome.Results Caf1,pla（their homology with Yersinia pestis caf1,pla from GenBank was more than 99%） and four tag sequences in chromosome ypo 2087,ypo 2090,ypo 2094 and ypo 2102 were found from the two samples in gene amplification,and sensitivity was higher used by DNA extract kits than by boiling.Conclusions Positive rate of diagnosis of plague by PCR could be increased by optimization of templet collection,improvement of amplification efficiency,and multiple genes test in both plasmids and chromosome.