本文選題：洋蔥伯克氏菌群 + 鑒定��； 參考：《浙江大學(xué)》2006年博士論文

【摘要】：洋蔥伯克氏菌(Burkholderia cepacia)是一種廣泛存在于農(nóng)業(yè)環(huán)境和醫(yī)院的革蘭氏陰性細(xì)菌,現(xiàn)被國(guó)際上劃分為9個(gè)基因型,合稱洋蔥伯克氏菌群(B.cepacia complex,簡(jiǎn)稱Bcc)。Bcc雖然引起洋蔥酸皮病,但國(guó)內(nèi)主要作為生物農(nóng)藥在應(yīng)用。目前國(guó)內(nèi)外報(bào)道該菌引起洋蔥伯克氏綜合癥而致多人死亡�，F(xiàn)有研究顯示Bcc菌中生物農(nóng)藥菌、植物致病菌和人體條件致病菌難以區(qū)分。我國(guó)無人涉足Bcc菌基因型及其與醫(yī)院致病菌的比較研究。我國(guó)農(nóng)田中存在哪些Bcc基因型、它們是否與醫(yī)院同類致病菌一樣危險(xiǎn)等問題急待解決。 本研究從廣西、浙江、海南、河北4省共采集農(nóng)田灌溉水和土壤樣本43份,用選擇培養(yǎng)基Bcc富集液分離Bcc菌株,經(jīng)表型特征鑒定后確定所分離的細(xì)菌菌株中有26個(gè)為Bcc菌株。從遼寧、浙江2省的醫(yī)院獲取并用表型特征鑒定Bcc菌株41個(gè)。研究表明Biolog和脂肪酸分析(FAMEs)均只能將Bcc鑒定到種的水平,而無法鑒定到基因型。 由于各基因型Bcc菌株間同源性極高,常用的細(xì)菌分子鑒定手段16S rRNA基因序列比較無法區(qū)分各基因型。本研究采用recA基因區(qū)分Bcc各基因型菌株,同時(shí)比較了recA特異引物PCR擴(kuò)增和recA全序列分析的方法與表型鑒定方法的差異,經(jīng)表型鑒定方法定為Bcc的67個(gè)菌株中只有59個(gè)被確證為Bcc菌株,其中農(nóng)田菌株25個(gè),醫(yī)院菌株34個(gè),Biolog和FAMEs對(duì)Bcc菌的誤鑒定率為11.94%。農(nóng)田菌株中檢測(cè)到基因型Ⅰ、Ⅱ、Ⅳ和Ⅴ,其中基因型Ⅰ為優(yōu)勢(shì)類型,其次是基因型Ⅴ;醫(yī)院菌株中檢測(cè)到基因型Ⅰ、Ⅱ、ⅢA、ⅢD和Ⅴ,其中基因型ⅢA為優(yōu)勢(shì)類型,它是引起洋蔥伯克氏綜合癥的主要基因型,但同時(shí)也發(fā)現(xiàn)醫(yī)院存在較高比率的基因型Ⅰ菌。recA特異引物PCR擴(kuò)增法是針對(duì)醫(yī)院Bcc菌株的基因型鑒定設(shè)計(jì)的,該法操作簡(jiǎn)便、準(zhǔn)確率較高可以作為初步鑒定Bcc基因型的主要手段,但是由于一些基因型recA特異引物的缺少(如基因型Ⅸ)和對(duì)農(nóng)田菌株靈敏度不夠高(如基因型Ⅱ),因此recA全序列分析是鑒定和確證Bcc基因型不可或缺的手段。研究還表明一些菌株的基因型與其在TSA平板上的菌落特征有一定的對(duì)應(yīng)關(guān)系,通�；蛐廷窬昃渚鶠辄S綠色、基因型Ⅳ菌落接近無色、基因型Ⅲ的菌落多為乳白色。
[Abstract]:Burkholderia cepacia is a Gram-negative bacterium widely found in agricultural environment and hospital. It is divided into 9 genotypes in the world. It is called B.cepacia complex (BCC). BCC causes onion skin disease. However, it is mainly used as biological pesticide in China. At present, it is reported at home and abroad that the bacteria causes onion Burke's syndrome and causes many deaths. Current studies show that biological pesticide bacteria, plant pathogens and human conditioned pathogens in BCC bacteria are difficult to distinguish. A comparative study of BCC genotype and hospital pathogenic bacteria in China. The problems of which BCC genotypes exist in farmland in China and whether they are as dangerous as the similar pathogenic bacteria in hospitals need to be solved urgently. In this study, 43 farmland irrigation water and soil samples were collected from Guangxi, Zhejiang, Hainan and Hebei provinces. BCC strains were isolated by BCC enrichment medium. 26 of them were identified as BCC strains. 41 strains of BCC were obtained from hospitals in Liaoning and Zhejiang provinces and identified with phenotypic characteristics. Biolog analysis and fatty acid analysis (FAMEs) showed that BCC could only be identified at the species level, but not genotypes. Because of the high homology among the BCC strains, the 16s rRNA gene sequence of the commonly used bacterial molecular identification method can not distinguish the genotypes. In this study, recA gene was used to distinguish BCC genotypes, and the differences between recA specific primer PCR amplification and recA full sequence analysis were compared with phenotypic identification methods. Only 59 of the 67 strains identified as BCC by phenotypic identification method were identified as BCC strains, including 25 strains from farmland and 34 strains from hospitals. The false identification rate of BCC bacteria was 11.94% by Biolog and FAmes. Genotypes 鈪，

本文編號(hào)：2061473