PCR法快速鉴定眼源性蜡样芽胞杆菌  

PCR Assay for rapid detection of Bacillus cereus from eye infection

作　　者：袁珊珊[1] 楼永良[1] 钟毓红[1] 毛丽萍[2] 吴娅娅[2] 陈静思[1] 沙栋杰[1] 郑美琴[2] 

机构地区：[1]温州医学院检验医学院,浙江温州325027 [2]温州医学院附属眼视光医院,浙江温州325027

出　　处：《中国微生态学杂志》2012年第3期273-277,282,共6页Chinese Journal of Microecology

基　　金：浙江省大学生新苗人才计划项目(2011R413022)

摘　　要：目的建立一种快速、灵敏、特异的眼源性蜡样芽胞杆菌PCR检测方法,为蜡样芽胞杆菌性眼内炎患者的快速诊断提供依据。方法选择编码蜡样芽胞杆菌细胞毒素的cytK为靶基因设计引物,建立检测眼源性蜡样芽胞杆菌PCR;PCR产物用琼脂糖凝胶电泳鉴定,基因序列与GenBank比对验证扩增产物;将计数过的5株蜡样芽胞杆菌菌悬液,梯度稀释后分别提取DNA进行PCR扩增,确定检测方法的灵敏度;分别用眼部常见感染菌金黄色葡萄球菌、表皮葡萄球菌、甲型溶血性链球菌、化脓性链球菌、藤黄微球菌、铜绿假单胞菌、大肠埃希菌、普通变形杆菌和白假丝酵母菌以及枯草芽胞杆菌DNA进行特异性试验;进一步将该方法应用到人工污染致病蜡样芽胞杆菌的房水标本中,并分析其灵敏度。结果 5株分离自眼内炎患者标本中的蜡样芽胞杆菌均扩增出360 bp左右的DNA片段,测序结果与GenBank比对一致;该法检测在5 h内完成,方法灵敏度达7.5～15.0 CFU/mL;其他菌株检测未出现非特异性扩增;对模拟感染房水标本的PCR鉴定结果与分离培养对比,二者符合率为100%,模拟标本的检测灵敏度与纯菌结果一致。结论 cytK基因为靶基因的PCR用于眼源性蜡样芽胞杆菌的快速检测,具有简便、快速、敏感、特异等特点,为眼内炎患者的快速诊断提供依据,在实际检验工作中有良好的应用前景。Objective To establish a rapid, sensitive and specific PCR assay for the detection of Bacillus cereus from eye infection, and provide basis for effective and rapid diagnosis of endophthalmitis caused by the pathogen. Method cytK gene was selected as the target gene to design primers for establishing a PCR to detect Bacillus cereus. The PCR product was examined by agarose gel electronphoresis, sequenced and compared to the GenBank. The sensitivity of the assay was deter- mined by amplifying DNA from 5 Bacillus cereus strains at different dilutions. Other bacteria including Staphylococcus aureus, Staphylococcus epidermidis, a-hemolytic streptococcus, Streptococcus pyogenes, Micrococcus luteus, Pseudomonas aeruginosa, Escherichia coli, Proteus vulgaris： Monilia albican and Bacillus subtils were used simultaneously to determine the specificity of this PCR assay. Futhermore, aqueous humor specimens artificially contaminated with pathogenic Bacillus cereus were used to e- valuate the sensitivity of the assay. Result The 360 bp fragments of DNA were detected in 5 Bacillus cereus isolated from the specimens of endophthalmitis patients. The sequence analysis revealed that the amplicons were the same as the sequences in the GenBank. The assay was completed in 5 hours with a sensitivity of 7.5-15 CFU/mL. There was no cross reaction with other bacteria. The detection results of simulation aqueous humor specimens were consistent with those of culture strains. Conclu- sion The PCR assay, with cytK gene as the target gene, is an easy, rapid, sensitive and highly specific in diagnostic detection of endophthalmitis. It has a fine application prospect in actual operations.

关 键 词：蜡样芽胞杆菌 cytK基因 聚合酶链式反应 眼内炎 快速检测 

分 类 号：R446[医药卫生—诊断学]