2020/1/6,1,第一篇 微生物学基础,第八章 病原学诊断与防治 Laboratory Diagnosis and Prevention Principles 第一节 细菌学诊断 第二节 病毒学诊断 第三节 真菌学诊断 第四节 特异性预防与治疗,2020/1/6,2,Outline,Laboratory diagnosis for bacterial infections Morphological - microscopic Molecular Serological Laboratory diagnosis for viral infections Morphological - electron microscopic Isolation Molecular: nucleic acids and proteins Serological Laboratory diagnosis for fungal infections Morphological - microscopic Isolation,2020/1/6,3,2020/1/6,4,Assays,Morphological assays Light or electron microscopies Isolation and differentiation Serological assays Antigen-antibody assays Molecular assays Microorganisms gene (DNA & RNA),2020/1/6,5,Principles for Specimen Collection,Sterile manipulation, avoid contamination Obtain a specimen from the infected site according to the disease phase before administrating antibiotics Double sera for antibody detections, acute and recovery phase each Positive if the titer of the later specimen is above 4X to the first specimen Transport, and store correctly as soon as possible Store at -4oC for most bacterial specimens, but keep warm for Neisseria meningitidis and Neisseria gonorrhoeae Store at -4oC -70oC for viral specimens,2020/1/6,6,Laboratory Diagnosis for Bacterial Infections,Morphological Protein and Nucleic acids Antibody,2020/1/6,7,标本的采集和送检六原则,无菌取材,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,运送注意保存,标本必须新鲜,2020/1/6,8,病原菌的检查程序,药物敏感试验,标本,直接涂片镜检,分离培养,生化试验,血清学试验,动物试验,明确诊断,初步诊断,分子生物学技术,其他检测技术,2020/1/6,9,Bacteriological Diagnosis,Identifying the organism Morphological Isolation & culture Biochemical reaction Serological assays,Pathogenesis & antibiotics susceptibility Animal experiment Virulence test antibiotics susceptibility,2020/1/6,10,Morphological,Non-stained microscopic observation Dark-field microscopy Observing the movement of live bacteria Stained microscopic observations Gram stain Acid-fast stain Fluorescence stain,2020/1/6,11,2020/1/6,12,2020/1/6,13,2020/1/6,14,2020/1/6,15,2020/1/6,16,2020/1/6,17,Isolation & Culture: Colony,Size Shape Color Surface features Smooth - Rough Transparency Hemolysis,2020/1/6,18,2020/1/6,19,2020/1/6,20,分离培养 isolation and cultivation,分离培养是微生物学诊断的金标准，是最可靠的传统诊断技术。 （图为普通培养箱）,2020/1/6,21,厌氧罐 厌氧袋,2020/1/6,22,Biochemical Reactions,Sugar Fermentation H2S Test Citrate utilization,2020/1/6,23,Serological Assays,Detection antibody in the patients serum A current infection should be IgM positive A 4-fold or greater rise on antibody titer between the acute serum sample and the convalescent serum sample Major drawbacks A single IgG antibody titer is difficult to interpret because it is unclear whether it represents a current or a previous infection the convalescent sample is usually taken 10-14 days after the acute sample. By this time, the patient has often recovered and the diagnosis becomes a retrospective one Some exceptions In certain diseases, a single titer of sufficient magnitude can be used as presumptive evidence of a current infection,2020/1/6,24,基本原则 用已知抗体检测未知抗原； 用已知抗原检测未知抗体。 最简易的方法有玻片凝集等试验。 常用的方法有：凝集试验、沉淀试验、补体结合试验、ELISA、免疫荧光等。,免疫学试验,2020/1/6,25,Animal experiment,Animals Mouse Guinea Pig Rabbit Dog Monkey Inoculation routes Intradermal Subcutaneous Intraperitoneal Intravenous Intracranial / intracerebral Intraspinal Intranasal Lavage,2020/1/6,26,Virulence Test,Median lethal dose, LD50 Median infective dose, ID50 Elek Plate Diphtherotoxin Corynebacterium diphtheriae Enterotoxin enterotoxigenic E. coli,2020/1/6,27,Antibiotic Susceptibility Test,Method,2020/1/6,28,MIC & MBC,Minimum Inhibitory Concentration，MIC Minimum Bactericidal Concentration，MBC Bactericidal drugs usually have an MBC equal or very similar to the MIC Bacteriostatic drugs usually have an MBC significantly higher than the MIC,2020/1/6,29,2020/1/6,30,Bacterial Proteins, DNA & RNA,Antigens (Proteins) Known antibodies Agglutination, coagulation, precipitation, ELISA, Immunofluorescence, radioimmunoassay DNA & RNA PCR Nucleic acid hybridization,2020/1/6,31,Detection of Bacterial Antigens,Precipitation test Coagglutination test Immunoflorecence, IF McAb technique,2020/1/6,32,2020/1/6,33,2020/1/6,34,Bacterial DNA & RNA,PCR DNA probe & hybridization DNA hybridization (southern blot) Plasmid fingerprint analysis Restrict multimorphologic analysis,2020/1/6,35,2020/1/6,36,2020/1/6,37,PCR,2020/1/6,38,分子生物学技术 molecular biological techniques,核酸杂交 nucleotide hybridization 原位杂交 in situ hybridization 斑点杂交 dot blot Southern blot Northern blot PCR 技术：多聚酶链反应 (polymerase chain reaction，PCR ),2020/1/6,39,PCR 是一种无细胞的分子克隆 技术，能在体外将一个基因或 一段DNA 序列经过多次循 环的DNA复制，在数小时内扩 增上百万倍。既便于基因诊断，又利于 研究。,2020/1/6,40,2020/1/6,41,Gene Chip / microarray,2020/1/6,42,Diagnosis Strategy for Bacterial Infection,2020/1/6,43,第25章 病毒感染的诊断 P238 Laboratory Diagnosis for Viral Infection,Morphological Isolation & Culture Protein & Nucleic acids Serological,2020/1/6,44,病毒感染的检测,一、标本的采集与运送,急性期标本；