本文關鍵詞： 分枝桿菌 聚合酶鏈反應 單鏈構象多態(tài)性 菌種鑒定 反向斑點雜交　出處：《北京市結核病胸部腫瘤研究所》2005年博士論文　論文類型：學位論文

【摘要】：目的:建立一種簡便、快速、靈敏、特異的分枝桿菌菌種鑒定方法,研究膜反向斑點雜交方法在快速鑒定分枝桿菌菌種方面的應用價值。 方法:通過16S rRNA聚合酶鏈反應(polymerase chain reaction,簡稱PCR)—單鏈構象多態(tài)性(single stranded conformation polymorphism,簡稱SSCP)分析253株分枝桿菌臨床分離株;設計與合成用于鑒定分枝桿菌菌種的16S rRNA寡核苷酸探針,點于硝酸纖維素膜上。與待測的28種分枝桿菌標準菌株、9種非分枝桿菌和253株分枝桿菌臨床分離株生物素標記的16S rRNA基因PCR產物進行反向斑點雜交。 結果:應用PCR—膜反向斑點雜交方法分析28種分枝桿菌標準菌株和9種非分枝桿菌菌株,結果顯示寡核苷酸探針是特異的。253株分枝桿菌臨床分離株中,經16S rRNA PCR-SSCP初步鑒定,198株為結核分枝桿菌復合群,經膜雜交,顯示a1雜交陽性,兩種鑒定方法結果一致:36株非結核分枝桿菌經膜雜交,11株鑒定為堪薩斯、瘰疬、胃、猿猴分枝桿菌,6株鑒定為胞內分枝桿菌,4株鑒定為恥垢分枝桿菌,3株鑒定為母牛分枝桿菌,3株鑒定為龜分枝桿菌,2株鑒定為戈登分枝桿菌,2株鑒定為偶發(fā)分枝桿菌,2株鑒定為結核分枝桿菌和胞內分枝桿菌復合感染株,1株鑒定為鳥胞內分枝桿菌復合感染株,1株鑒定為土分枝桿菌,1株鑒定為鳥分枝桿菌,另19株未出現分析探針陽性雜交。PCR-膜反向斑點雜交技術鑒定分枝桿菌菌種的靈敏度為92.49%,特異度為100%。 選用孔徑為0.22μm的硝酸纖維素膜,選擇各菌種特異性探針,探針終濃度為1.5pmol/μl,采用雜交溫度42℃、雜交時間60分鐘、45℃洗膜5分鐘雜交結果較理想。 結論:影響雜交的主要因素是探針序列的組成、探針濃度、雜交和洗膜溫度、洗膜時間等。PCR-膜反向斑點雜交技術可簡便、快速、特異地鑒定分枝桿菌菌種,僅需2天時間。
[Abstract]:Objective : To establish a simple , rapid , sensitive and specific method for the identification of Mycobacterium tuberculosis strains . Methods : The 16S rRNA oligonucleotide probes were analyzed by 16S rRNA polymerase chain reaction ( PCR ) - single strand conformation polymorphism ( SSCP ) . Results : The PCR - membrane reverse dot blot was used to analyze 28 strains of Mycobacterium tuberculosis and 9 strains of non - Mycobacterium tuberculosis . The results showed that the oligonucleotide probe was a specific strain of Mycobacterium tuberculosis . A cellulose nitrate membrane with pore size of 0.22 渭m was selected , the specific probes were selected , the final concentration of the probe was 1.5 pmol / 渭l , the hybridization temperature was 42 鈩，

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