聚合酶链反应扩增伤寒患者粒细胞中伤寒沙门菌DNA  

Detection of Salmonella typhi in peripheral blood of typhoid fever by single tube nested polymerase chain reaction

作　　者：刘宏[1,2] 马志如[1,2] 陈宏础[1,2] 黄天禄[1,2] 邓兵 朱静[1,2] 张玉洪 

机构地区：[1]重庆医科大学附属第一医院检验科 [2]重庆医科大学儿童医院血液遗传二室

出　　处：《中华医学检验杂志》1997年第3期172-174,共3页

摘　　要：目的建立适用于临床应用的血中伤寒沙门菌（Ｓ．ｔｙｐｈｉ）ＤＮＡ扩增的方法。方法分离患者粒细胞，采用裂解法制备模板ＤＮＡ，单管套式聚合酶链反应扩增Ｓ．ｔｙｐｈｉ鞭毛基因。结果６例血培养阴性而临床拟诊伤寒及２３例由血培养确诊的伤寒患者粒细胞均获得了３４３ｂｐ的扩增产物，其他３４例发热待查患者粒细胞无扩增产物出现。连续１１０稀释Ｓ．ｔｙｐｈｉＤＮＡ，最低可检测到４０ｆｇＤＮＡ（约１０个菌）。扩增产物的ＤＮＡ序列测定结果也证实了３４３ｂｐ的扩增产物是Ｓ．ｔｙｐｈｉ鞭毛基因。结论该方法是一项简便、省时、灵敏和特异的实验室诊断伤寒的新技术，尤其对培养阴性的伤寒患者可提供早期、快速的实验室诊断，套式扩增反应在同一反应管中完成，能有效避免标本间的污染。Objective To establish a new amplication method for Salmonella typhi (S.typhi) DNA in peripheral blood. Methods A single tube nested polymerase chain reaction (PCR) was designed to amplify flagellin gene of S.typhi DNA prepared by boiling method. Results The granulocytes from 23 patients with typhoid fever confirmed by blood culture were positive for 343 by fragment of the flagellin gene of S. typhi, whereas 34 blood specimens of patients with other febrile disease were negative. S.typhi DNAs were detected from blood specimens of 6 patients with suspected typhoid fever on the basis of clinical features but with negative cultures. The single tube nested PCR could detect 40 fg of target DNA (corresponding to 10 organisms). The result of sequencing DNA confirmed that the amplified segment was flagellin gene. Conclusions The single tube nested PCR technique was a simple, time saving, sensitive, and specific method for detection of typhoid fever, particularly in culture negative cases. This method eliminated crosscontamination between samples because the amplifications were performed in a single tube.

关 键 词：伤寒 聚合酶链反应 DNA 

分 类 号：R516.304[医药卫生—内科学]