PDF《·实验研究·》

现代生物医学进展 www.

shengwuyixue.com Progress in Modern Biomedicine Vol.11 NO.21 NOV.2011 ・ 实验研究 ・ 复合探针荧光定量 PCR 法检测布鲁氏菌的实验研究 * 吴忠华 吕沁风 郑伟李禾(浙江国际旅行卫生保健中心 浙江 杭州

310003 ) 摘要 目的: 建立用复合探针荧光定量 PCR 快速检测布鲁氏菌的方法. 方法: 研究根据 BSCP31 基因编码 31KDa 的布鲁氏杆菌表 面蛋白的核苷酸序列设计特异引物, 通过 PCR 法的特异性、 灵敏度和重复性研究, 建立了复合探针荧光定量 PCR 检测布鲁氏菌 的方法, 用于布鲁氏菌病的筛选和诊断.结果: 结果表明该检测方法的特异性为 100%, 最低可检出

10 个拷贝的质粒 DNA 分子, 可对 1*101 -1*106 拷贝范围内的模板进行定量, 最低可检测至 1*102 CFU/ml 细菌.该方法的精密度好, 阳性质控品和阴性质控 品不同时间测定三次及同一时间五次重复实验结果 CV 值均小于 5%.结论: 本研究建立的复合探针实时荧光定量 PCR 检测布 鲁氏杆菌的方法, 可对布鲁氏病原菌进行快速检测, 对布病的筛选和确诊具有重要意义. 关键词: 复合探针;

荧光定量 PCR;

布鲁氏杆菌 中图分类号: R446, R378.5 文献标识码: A 文章编号: 1673-6273 (2011 ) 21-4054-04 Experimental Research on Brucella Bacteria Detection by Fluorescent Quantitative PCR with Composite Probe* WU Zhong-hua, LV Qin-feng, ZHENG Wei, LI He (International Travel Healthcare Center, Zhejiang, Hangzhou

310003 China) ABSTRACT Objective: To establish a composite probe fluorescent quantitative PCR assay for Brucella detection. Methods: BSCP31 gene nucleotide sequences specific for 31KDa surface protein of Brucella was used for the PCR primers design. According to the specificity, sensitivity and reproducibility of primers, a composite probe fluorescent quantitative PCR assay for Brucella detection was established for screening and diagnosis of Brucella disease. Results: The specificity of composite probe fluorescent quantitative PCR as- say was 100%. The assay can detect the minimum

10 copies of plasmid DNA molecules. Accurate quantization can be achieved with template samples detected within

1 *

101 -1 *

106 copies. The minimum bacteria concentration can be detected by the assay reaching

1 *

102 CFU / ml. The assay has good precision. The positive controls and negative control materials measured three times at different times or at the same time repeat the experiment five times, from which the CV values were calculated less than 5%. Conclusion: This study established the composite prober fluorescent quantitative PCR assay for Brucella detection, which can rapidly detect Brucella bac- teria and has important meaning on screening and diagnosis of brucellosis. Key words: Composite probe;

Fluorescent quantitative PCR;

Brucella bacteria Chinese Library Classification(CLC): R446 Document code: A Article ID: 1673-6273 (2011 ) 21-4054-04 * 基金项目: 国家质量监督检验检疫总局科研基金 (2007IK197 ) 作者简介: 吴忠华,男,硕士,副主任技师,主要研究方向:分子生物学

电话:13757195558, Email:[email protected] (收稿日期: 2011-06-27 接受日期: 2011-07-22 ) 前言 布鲁氏菌病 (布病) 由布鲁氏菌引起的人畜共患自然疫源 性传染病, 它危害严重, 流行广.布鲁氏菌可感染人类、 多种家 畜和野生动物, 引起发热、 流产与不育、 慢性关节炎及神经损 害, 是一种重要的生物战剂和生物恐怖剂.多年来对布病诊断 采用细菌学和血清学技术, 前者费事费力, 而且检出率低;