检验科通知-中国病原生物学杂志.docVIP

荧光定量PCR检测志贺菌方法的建立 高春燕 高庆双 刘树平 许林骥 王晶晶 郑建霞 （河北省唐山市妇幼保健院 检验科 063000 河北 唐山） 注：高春燕为本刊通讯员 摘要：目的　建立一种检测志贺菌属快速敏感的荧光定量PCR方法。方法　根据志贺菌属侵袭性质粒抗原H ( invasive p lasmid, ipaH)编码基因序列,设计1对PCR引物特异性靶基因片段，通过检测3株志贺菌属菌株和9株非志贺菌属菌株来评价该方法的特异性；对福氏志贺菌菌液做一系列10倍稀释后进行荧光定量PCR分析敏感性，PCR扩增产物经电泳和测序鉴定。结果 3株志贺菌属菌株出现特定大小的目的条带，9株非志贺菌属菌株未出现目的条带；测序也证实了PCR产物的特异性;荧光定量PCR检测福氏志贺菌ipaH基因的下限为200拷贝/ ml。结论　本研究建立的方法快速、特异、敏感。 关键词 荧光定量PCR;志贺菌属;ipaH Establishment and application of fluorescence quantitative PCR for detection of Shigella Gao Chun-yan, Gao qing-shuang, Liu Shu-ping,XU Lin-ji,Wang Jing-jing ZhengJian-xia (Tangshan Municipal Maternal and Child Care Hospital,Tangshan 063000,Hebei,P.R.China) Abstract: Objective To establish a testing shigella is rapid sensitive fluorescence quantitative polymerase chain reaction (PCR). Methods: According to shigella belong to invade plasmid antigen H (invasive p lasmid, ipaH) coding gene sequence, design an PCR primers specific target gene fragment, by detecting 3 strains of shigella strains and 9 strains not shigella strains of this method to evaluate the specificity, For mans shigella bacteria liquid do a series of ten times dilution after fluorescence quantitative PCR analysis sensitivity, PCR amplification products by electrophoresis and sequencing appraisal. Results: 3 strains of shigella strains in the size of the specific purpose banding, 9 strains not shigella strains of does not appear purpose banding; Sequencing also confirmed the specificity of the PCR products; Fluorescence quantitative PCR detections shigella ipaH gene lower limit for 200 copies/ml. Conclusion:The established method is rapid, specific and sensitive. Key words Fluorescence quantitative PCR; Shigella;ipaH 志贺菌是引起细菌性痢疾的常见致病菌，， 1 材料和方法 1.1 仪器和试剂　PE7700PCR扩增仪（美国PE公司）；水平式电泳仪(北京东方仪器厂)； GDS7600凝胶扫描系统（DF-23B）(英国UVP公司);台式高速离心机（TGL-16G）和水平离心机（上海安亭科学仪器厂）；主要试剂为PCR试剂（北京索奥公司产品）；DNA分子标准100bp DNA Ladder(大连宝生物公司)；DNA提取试剂和Taq酶系（北京索奥公司产品）；dNTPs（上海生物工程公司产品）；引物和探针（北京索奥生物有限公司合成）。 1.2 实验菌株　鲍氏志贺菌（为卫生部质控菌株）、福氏志贺菌、宋内志贺菌;