1、 第5章 細菌感染的檢測方法與預防原則The microbe must be present in every case of the disease and not present in healthy animalsIt must be isolated and grown in a pure cultureThe same disease must result when the isolated microbe is inoculated into a healthy host The same microbe must be isolated again from the disea

2、sed hostKochs Postulates一、細菌感染實驗室診斷 查細菌 傳統方法病原菌分離鑒定 快速診斷 1、形態觀察 2、抗原檢測 3、核酸檢測 查抗體 用已知細菌抗原測病人血清中相應抗體臨床標本增 菌接種培養基，分離單個菌落各種鑒定試驗涂片染色快速診斷試驗生化試驗血清學實驗涂片染色藥敏試驗動物試驗初步診斷報告最終診斷報告細菌學檢驗一般程序預處理標本材料因感染的不同而定，通常為病人或可疑者的血、尿、糞、痰、腦脊液、膿汁等。舉例：如腹瀉、腦膜炎、傷寒等病人細菌學診斷標本的采集和送檢六原則避免雜菌污染不同期不同標本用抗生素以前采集病變明顯部位注意記錄、運送和保存標本必須新鮮取材部位特殊，

3、形態染色典型淋病患者尿道分泌物直接涂片作革蘭染色腦脊液涂片作革蘭染色結核病人痰涂片作抗酸染色深部創口取材涂片染色S.S培養基上傷寒沙門菌形成的黑色菌落羅氏培養基上結核分枝桿菌形成的菜花心樣菌落亞碲酸鉀培養基上白喉桿菌形成的黑色菌落巧克力色培養基上腦膜炎萘瑟菌形成的露滴狀菌落124124124124ONPGGLULDCARACITODCH2SUREINDTDAOXNO2 7504：肺炎克雷伯氏菌肺炎亞種7054原理血清學診斷 雙份血清標本，恢復期血清中抗體效價比急性期血清中抗體效價升高4倍者方有意義種類(已知抗原/抗體相應的抗體/抗原)標本凝集試驗：沉淀試驗：補體結合試驗：中和試驗： 肥達試驗、

4、外斐試驗、顯微鏡凝集試驗等梅毒VDRL、RPR試驗等抗“O”試驗等Q熱柯克斯體抗體檢測等微生物學診斷新技朮分子生物學技術核酸雜交聚合酶鏈反應(PCR)Western Blot 厭氧菌快速診斷 氣液相色譜法流行病學調查噬菌體分型細菌素分型 核酸分子雜交（Molecular hybridization ） 核酸分子雜交方法比較：Southern /Northern blotDot blotIn situ blot標本DNA/RNA血清組織切片瓊脂糖電泳硝酸纖維素膜變性+探針+顯色反應+用途診斷分子量測定診斷診斷胞內定位 PCR(polymerase chain reaction)目的：擴增目的基因

5、片段特點：特異，敏感，快速引物：約20個堿基，與擴增片段兩端互補 變性（94）dsDNA ssDNA復性（50-65）引物與ssDNA配對延伸（72）ssDNA為模板合成新鏈30個周期凝膠電泳 檢測RT-PCR and RT-LAMP確定某種新發感染性疾病的病原體：Kochs postulate確定常見感染性疾病的病原體：細菌培養、涂片、血清學、分子診斷技術等確定病原體的毒力基因：Molecular Kochs postulateThe phenotype or property under investigation should be associated with pathogenic

6、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 a me

7、asurable 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 postulatesoriginally described by Dr. Falkow, 1988 Reversion or allelic replacement of the mutat

8、ed 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、細菌感染的防控原則 控制傳染源 切斷傳播途徑 提高人群免疫力疫苗 菌體疫苗： 死疫苗 減毒活疫苗類毒素（Toxoid）： 外毒素經甲醛處理，失去毒性，仍保留免疫原性，人工自動免疫后可刺激產生抗毒素抗體。亞單位疫苗蛋白或抗原成分多糖疫苗： 理化提取得細菌表面多糖成分，如腦膜炎球菌莢膜多糖疫苗。聯合疫苗 如DPT三聯疫苗核酸疫苗： 即DNA疫苗，目前只在動物上試用。基因工程疫苗區別死疫苗活疫苗制劑特點死，強毒株活減毒株接種劑量與特點量較大，23次量較小，1次保存及有效期易保存，約1年不易保存免疫效果較低，維