小鼠棒状杆菌-中国试验动物学报.docVIP

鼠棒状杆菌PCR检测方法的建立及其应用 　　　　　　唐连飞1，周智君2，蔡婧怡1，孟芳1，魏颖1，俞远京2，苏志杰2 　 1.湖南出入境检验检疫局，长沙 410004；２.中南大学湘雅医学院实验动物学部，长沙　410008 摘要：目的 建立鼠棒状杆菌PCR检测方法并应用于临床样本检测。 方法 用脑心浸出液培养基复苏、培养鼠棒状杆菌（corynebacterium kutscheri, C. kutscheri）并提取基因组DNA作模板； 根据GenBank中C. kutscheri的16S基因序列设计合成引物，建立鼠棒状杆菌PCR检测方法并进行敏感性和特异性的评价；人工感染昆明鼠，建立小鼠棒状杆菌感染模型，采集肝脏和肾脏，提取DNA进行检测。结果 成功建立了鼠棒状杆菌PCR检测方法，该方法可检测到100个阳性质粒；对小鼠沙门氏菌、肺炎链球菌和巴氏杆菌无交叉反应；全部8个人工感染样本全部检测为阳性。结论 建立的鼠棒状杆菌PCR检测方法灵敏度高、特异性好，可作为鼠棒状杆菌感染的快速检测方法。 关键词：鼠棒状杆菌；PCR检测方法；16S 核糖体RNA 中图分类号：S858.91 Development and Application of a PCR for the Detection of Corynebacterium Kutscheri in Mice TANG Lian-fei1, ZHOU Zhi-jun2, CAI Jing-yi1, MENG Fang1, WEI Ying1，YU Yuan-jing2, SU Zhi-jie2 1. Hunan Entry-Exit Inspection and Quarantine Bureau, Changsha 410004, China; 2. Department of Laboratory Animal of Xiangya Medicine School, Centre South University, Changsha 410008, China Abstract: Objective To develop a PCR for the detection of corynebacterium kutscheri （C. kutscheri） and apply it to clinical samples. Methods Genomic DNA was extracted as template for PCR from C. kutscheri (ATCC 11306) recoveried and cultivated in brain heart infusion medium. According to the C. kutscheri 16S rRNA gene sequence available in GenBank a pair of primes were designed and synthesized in order to develop a PCR for detection of C. kutscheri. After evaluated for sensitivity and specificity the PCR method was applied to detect the C. kutscheri in clinical livers and kidneys of mice artificially infected with C. kutscheri. Results The PCR for the detection of C. kutscheri was developed successfully and were specific enough to distinguish C. kutscheri from salmonella, streptococcus pneumoniae and Pasteurella. A minimum of 100 positive plasmids could be detected, indicating a good sensitivity of the assay. Conclusion This method repoted here is specific, sensitive and provides a fast detection of C. kutscheri and could be used for C. kutscheri clinical diagnosis. Key words: Corynebacteri