摘要
目的应用基于非对称发夹探针的杂交链反应技术,检测肺炎克雷伯菌不对称PCR产物,建立一种新的病原菌快速检测方法。方法采用Array Designer 4.0软件,针对肺炎克雷伯菌的16SrDNA可变区域设计非对称发夹式DNA探针,优化探针浓度,以不对称PCR扩增肺炎克雷伯菌16rDNA可变区所制备的单链DNA为靶序列,采用HCR进行检测和分析。结果 HCR体系中发夹探针最佳浓度为1μmol/L;当靶序列初始浓度≥0.05μmol/L时,通过琼脂糖凝胶电泳可观察到明显的HCR聚合物;不对称PCR法制备的单链DNA可以启动杂交反应。结论应用基于非对称发夹探针的杂交链反应技术检测病原菌具有反应体系简单,无需酶促反应等优点,可对血流感染病原菌基因组进行简便、快速、特异的检测。
OBJECTIVE To optimize the hybridization chain reaction technique based on dissymmetry hairpin probe to detect the dissymmetry PCR products,and to develop a rapid method to detect the pathogenic bacteria.METHODS The Array Designer 4.0 was used to design dissymmetry hairpin DNA probe for the detection of 16S rDNA variable region of Klebsiella pneumoniae.The probe concentration in hybridization chain reaction system was optimized.The single strand DNA produced by asymmetric PCR acting as an initiator was analyzed by hybridization chain reaction.RESULTS The optimal final hairpin probe concentration was 1 μmol/L in hybridization chain reaction system.Initiator I at concentrations equal to or greater than 0.05 μmol/L could induce the hybridization chain reaction polymers observed via traditional agarose gel electrophoresis.The single strand DNA amplified by asymmetric PCR could also trigger hybridization chain reaction.CONCLUSION The dissymmetry hairpin probe based hybridization chain reaction has such advantages as the reaction system is quite simple and enzyme-free,which can be used for convenient,rapid and specific detection of pathogenic bacteria.
出处
《中华医院感染学杂志》
CAS
CSCD
北大核心
2012年第20期4452-4455,共4页
Chinese Journal of Nosocomiology
基金
国家"863"项目(2011AA02A100)
国家自然科学基金(30927002
81071429
30901388)
国家
军队重大专项(2012ZX10004801-003-006
AWS11C0012011)
第三军医大学青年人才创新基金(2009XQN25)
关键词
肺炎克雷伯菌
发夹DNA探针
杂交链反应
Klebsiella pneumoniae
Hairpin DNA probe
Hybridization chain reaction
