【摘要】：目的：克隆結(jié)核分枝桿菌rpoB、katG常見基因突變位點(diǎn),為核酸薄膜導(dǎo)流雜交技術(shù)快速檢測(cè)臨床標(biāo)本結(jié)核分枝桿菌rpoB、katG基因突變提供陽性對(duì)照,以助判斷臨床標(biāo)本結(jié)核分枝桿菌有無耐藥。 方法：前期研究發(fā)現(xiàn)本地區(qū)結(jié)核分枝桿菌rpoB、katG基因常見的4個(gè)突變位點(diǎn)和6個(gè)突變形式,選擇6株含有上述突變特征的臨床分離株,PCR擴(kuò)增其rpoB、katG基因片段進(jìn)行T-A克隆,采用HindⅢ和EcorⅠ雙酶切、PCR擴(kuò)增及重組質(zhì)粒測(cè)序鑒定重組質(zhì)粒。核酸薄膜導(dǎo)流雜交技術(shù)檢測(cè)此6個(gè)臨床分離株的rpoB、katG基因片段。 結(jié)果：構(gòu)建了6個(gè)含有rpoB、katG基因不同突變類型的重組質(zhì)粒,三種方法鑒定明確重組質(zhì)粒含有正確序列。核酸薄膜導(dǎo)流雜交技術(shù)檢測(cè)6個(gè)臨床分離株的rppB、katG基因片段結(jié)果顯示在預(yù)期的位置顯示陽性斑點(diǎn)。 結(jié)論：重組質(zhì)�？梢宰鳛楹怂岜∧�(dǎo)流雜交技術(shù)檢測(cè)臨床標(biāo)本時(shí)提示結(jié)核分枝桿菌有無耐藥的穩(wěn)定的陽性對(duì)照。
[Abstract]:Objective: to clone the common mutation sites of Mycobacterium tuberculosis rpoBhkatG gene, and to provide a positive control for rapid detection of rpoB katG gene mutation in clinical specimens by membrane diversion hybridization, in order to help determine the resistance of Mycobacterium tuberculosis in clinical specimens. Methods: four common mutation sites and six mutation forms of Mycobacterium tuberculosis rpoBnkatG gene were found in previous studies. Six clinical isolates with the above mutational characteristics were selected for T-A cloning by PCR amplification of the rpoBmkatG gene fragment of Mycobacterium tuberculosis. The recombinant plasmids were identified by Hind 鈪，

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