《细菌学》教学课件：06 细菌感染的检测与预防

1、第5章 细菌感染的检测方法与预防原则,The microbe must be present in every case of the disease and not present in healthy animals It must be isolated and grown in a pure culture The same disease must result when the isolated microbe is inoculated into a healthy host The same microbe must be isolated again from the dis

2、eased host,Kochs Postulates,一、细菌感染实验室诊断,查细菌 传统方法病原菌分离鉴定 快速诊断 1、形态观察 2、抗原检测 3、核酸检测 查抗体 用已知细菌抗原测病人血清中相应抗体,临床标本,增 菌,接种培养基，分离单个菌落,各种鉴定试验,涂片染色,快速诊 断试验,生化试验,血清学实验,涂片染色,药敏试验,动物试验,初步诊 断报告,细菌学检验一般程序,标本材料,因感染的不同而定，通常为病人或可疑者的血、尿、粪、痰、脑脊液、脓汁等。,举例：如腹泻、脑膜炎、伤寒等病人,细菌学诊断,标本的采集和送检六原则,避免杂菌污染,不同期不同标本,用抗生素以前,采集病变明显部位,注意记

3、录、运送和保存,标本必须新鲜,取材部位特殊，形态染色典型,淋病患者尿道分泌物直接涂片作革兰染色,脑脊液涂片作革兰染色,结核病人痰涂片作抗酸染色,深部创口取材涂片染色,S.S培养基上伤寒沙门菌形成的黑色菌落,罗氏培养基上结核分枝杆菌形成的菜花心样菌落,亚碲酸钾培养基上白喉杆菌形成的黑色菌落,巧克力色培养基上脑膜炎萘瑟菌形成的露滴状菌落,1,2,4,1,2,4,1,2,4,1,2,4,ONPG,GLU,LDC,ARA,CIT,ODC,H2S,URE,IND,TDA,OX,NO2, , , , ,7504：肺炎克雷伯氏菌肺炎亚种,7,0,5,4,原理,血清学诊断,双份血清标本，恢复期血清中抗体效价比

4、急性期血清中抗体效价升高4倍者方有意义,种类,(已知抗原/抗体相应的抗体/抗原),标本,凝集试验：,沉淀试验：,补体结合试验：,中和试验：,肥达试验、外斐试验、显微镜凝集试验等,梅毒VDRL、RPR试验等,抗“O”试验等,Q热柯克斯体抗体检测等,微生物学诊断新技朮,分子生物学技术 核酸杂交 聚合酶链反应(PCR) Western Blot 厌氧菌快速诊断 气液相色谱法 流行病学调查 噬菌体分型 细菌素分型,核酸分子杂交 （Molecular hybridization ）,核酸分子杂交方法比较：,PCR(polymerase chain reaction),目的：扩增目的基因片段 特点：特异，

5、敏感，快速 引物：约20个碱基，与扩增片段两端互补,变性（94）dsDNA ssDNA 复性（50-65）引物与ssDNA配对 延伸（72）ssDNA为模板合成新链,30个周期,凝胶电泳 检测,RT-PCR and RT-LAMP,确定某种新发感染性疾病的病原体： Kochs postulate 确定常见感染性疾病的病原体： 细菌培养、涂片、血清学、分子诊断技术等 确定病原体的毒力基因： Molecular Kochs postulate,The phenotype or property under investigation should be associated with pathog

6、enic members of a genus or pathogenic strains of a species. Additionally, the gene in question should be found in all pathogenic strains of the genus or species but be absent from nonpathogenic strains. Specific inactivation of the gene(s) associated with the suspected virulence trait should lead to

7、 a measurable loss in pathogenicity or virulence. Virulence of the microorganism with the inactivated gene must be less than that of the unaltered microorganism in an appropriate animal model.,Molecular Kochs postulates,originally described by Dr. Falkow, 1988,Reversion or allelic replacement of the

8、 mutated gene should lead to restoration of pathogenicity. In other words, reintroduction of the gene into the microbe should restore virulence in the animal model. The gene, which causes virulence, must be expressed during infection. Immunity must be protective.,Molecular Kochs postulates (continued),二、细菌感染的防控原则,控制传染源 切断传播途径 提高人群免疫力,疫苗,菌体疫苗： 死疫苗 减毒活疫苗 类毒素（Toxoid）： 外毒素经甲醛处理，失去毒性，仍保留免疫原性，人工自动免疫后可刺激产生抗毒素抗体。 亚单位疫苗 蛋白或抗原成分,多糖疫苗： 理化提取得细菌表面多糖成分，如脑膜炎球菌荚膜多糖疫苗。 联合疫苗 如DPT三联疫苗 核酸疫苗： 即DNA疫