本文關(guān)鍵詞： 鼠疫桿菌 大腸桿菌 16s rRNA 熒光PCR 毒力基因　出處：《大理學(xué)院》2013年碩士論文　論文類型：學(xué)位論文

【摘要】：目的 對從云南省西雙版納州景洪縣種豬場自斃黃胸鼠臟器分離到的1株曾疑似為鼠疫耶爾森氏菌株(以下簡稱X1菌株)進(jìn)行鼠疫菌的排除實(shí)驗(yàn)，并進(jìn)一步對該菌株進(jìn)行系統(tǒng)地表型及分子生物學(xué)鑒定，以確認(rèn)其為何種菌株。 方法 1、鼠疫菌的排除實(shí)驗(yàn) ①用鼠疫細(xì)菌學(xué)常規(guī)方法確定其生物學(xué)表型；②應(yīng)用API20E腸桿菌科生化試紙條和VITEK-2全自動測定生化鑒定儀分別鑒定X1菌株的生化特性；③應(yīng)用檢測鼠疫金標(biāo)試紙條檢測F1抗原；④應(yīng)用PCR檢測鼠疫特異基因pla、caf1；⑤動物實(shí)驗(yàn)：經(jīng)腹腔注射、灌胃、靜脈注射等三種途徑，分不同的劑量組和不同批次感染小白鼠，觀察是否有病變，同時(shí)取肝、脾、肺、心、腺進(jìn)行細(xì)菌培養(yǎng)鑒定。 2、采用16srRNA對X1菌株進(jìn)行鑒定 將X1菌株經(jīng)16srRNA全基因序列進(jìn)行PCR擴(kuò)增，產(chǎn)物純化、測序；使用DNAstar軟件包中的SeqMan對序列片段進(jìn)行拼接、編輯、校正；序列經(jīng)BLAST與GenBank中的已知序列進(jìn)行同源性比較，并構(gòu)建系統(tǒng)進(jìn)化樹，判定其屬種。 3、熒光PCR檢測 先用常見的致瀉性大腸桿菌毒素基因：STX1/STX2/EAE，LT/ST，ARRG/IPAH對X1、X11、X12三個(gè)細(xì)菌樣品進(jìn)行檢測，再用大腸桿菌O104檢測試劑對3個(gè)細(xì)菌樣品進(jìn)行檢測，最后用大腸桿菌H4檢測試劑盒檢測。 4、腸出血性大腸桿菌（EHEC）O104:H4的毒力基因檢測 用PCR進(jìn)行毒力基因檢測，檢測的毒力基因包括針對EHEC的4個(gè)基因:stx1、stx2、eaeA和ehxA；針對EAEC毒力質(zhì)粒上的3個(gè)基因：aatA、aggR、aap；以及O104特異基因wzy和H4特異基因fliC。若wzy和fliC基因檢測陽性，還需血清凝集試驗(yàn)確認(rèn)血清型是O104、鞭毛(H)抗原是04。 結(jié)果 1、①該菌株為革蘭陰性短小桿菌，兩極濃染；在37℃時(shí)能被假結(jié)核噬菌體裂解；②梅里埃API20E和生化鑒定儀的試驗(yàn)結(jié)果均未鑒定出X1菌株的屬種；③金標(biāo)試紙條檢測F1抗原為陰性；④鼠疫特異性基因pla、caf1檢測結(jié)果陰性。⑤動物實(shí)驗(yàn)：將配制的濃度為8×1010/ml的X1菌株菌液經(jīng)三種途徑感染小鼠，未出現(xiàn)死亡小鼠，將菌液濃度增加到8×1011/ml，，經(jīng)腹腔注射感染的小鼠全部死亡；將分離到的X11菌株分別配制到4×1011/ml和8×1011/ml經(jīng)腹腔注射感染的小鼠也全部死亡，分離到菌株X12，解剖觀察臟器發(fā)生病變，且X11菌株和X12菌株在37℃能被假結(jié)核噬菌體裂解。 2、①經(jīng)16srRNA鑒定，X1菌株與大腸桿菌埃希氏菌屬的一些菌株相似度達(dá)99%，初步判斷為大腸埃希氏菌屬，且與2011年新發(fā)病原菌O104:H4菌株的相似度為99%。②進(jìn)化樹顯示X1菌株與O104:H4菌株的同源性更高。③血清型鑒定試驗(yàn)菌株出現(xiàn)自凝現(xiàn)象，無法進(jìn)行血清型鑒定。 3、熒光PCR檢測結(jié)果：大腸桿菌O104基因和ipaH基因檢測結(jié)果為陽性；Stx1/Stx2/eae、LT/ST、Aggr、志賀菌/沙門菌、H4檢測結(jié)果均為陰性。 4、EHECO104:H4毒力基因檢測結(jié)果：9個(gè)毒力因子檢測結(jié)果均為陰性。結(jié)論 X1菌株非鼠疫菌株，結(jié)合形態(tài)特征、生化鑒定結(jié)果、動物實(shí)驗(yàn)、16srRNA基因鑒定結(jié)果判定其為一株大腸桿菌；對常見致病性大腸桿菌及腸出血性大腸桿菌O104:H4的13種毒力因子的檢測結(jié)果表明，X1菌株為大腸桿菌弱毒株O104菌。
[Abstract]:objective
From Yunnan Prefecture of Xishuangbanna province Jinghong County pig organs is Rattus flavipectus isolate 1 strains of plague suspected Jerson S (hereinafter referred to as X1 strain) were excluded from the study of Yersinia pestis, and further the system of surface type and molecular identification of the strain, to confirm its why strains.
Method
1, the elimination experiment of Yersinia pestis
The biological phenotype is determined by conventional bacteriology method; biochemical characteristics of API20E Enterobacteriaceae biochemical test strip and VITEK-2 automatic biochemical identification instrument X1 strains were identified; the application of colloidal gold strip for detection of plague F1 antigen detection; the application of PCR for detection of plague specific gene PLA, Caf1; the animal experiment: after intraperitoneal injection, gavage, three intravenous injection, the mice in different dose groups and different batches of infection, to observe whether lesions in the liver, spleen, lung, heart, gland bacteria were cultured and identified.
2, identification of X1 strains by 16srRNA
The X1 strain was the whole sequence of 16srRNA was amplified by PCR, purified and sequenced; using the DNAstar software package of SeqMan sequence fragment splicing, editing, correction; sequence by BLAST and GenBank in the known sequence of homology comparison and phylogenetic tree, determine the species.
3, fluorescence PCR detection
First, we used the common diarrhoea Escherichia coli toxin gene: STX1/STX2/EAE, LT/ST and ARRG/IPAH to detect three bacterial samples of X1, X11 and X12, then detected 3 bacterial samples with Escherichia coli O104 test reagents, and finally detected with E.coli H4 detection kit.
4, detection of virulence gene of entero hemorrhagic Escherichia coli (EHEC) O104:H4
The virulence gene was detected by PCR, virulence gene detection including 4 genes for EHEC: stx1, Stx2, eaeA and ehxA; the 3 genes on the virulence plasmid EAEC: aatA, aggR, AAP; and O104 and WZY genes specific to H4 gene specific fliC. if detection of WZY and fliC gene positive, serum need serum agglutination test confirmed the type is O104 (H) 4 flagella antigen
Result
1, the strains were gram negative bacillus pumilus, bipolar stain; at 37 DEG C can be pseudotuberculosis bacteriophage lysis; test the biochemical identification system of bioMerieux API20E and the results were not identified X1 strains of species; the colloidal gold strip for detection of F1 antigen was negative; the plague specific gene PLA Caf1, the test results were negative. The animal experiment: with concentration of 8 * 1010/ml X1 bacterial liquid by three ways of infected mice, there was no death in mice, the bacteria concentration increased to 8 * 1011/ml, all died of infection by intraperitoneal injection in mice; strain X11 isolated were prepared to 4 * 1011/ml and 8 * 1011/ml by intraperitoneal injection of infected mice also died, isolated strain X12, anatomical observation of organ lesions, and X11 strain and X12 strain at 37 DEG C can be pseudotuberculosis bacteriophage lysis.
2, the 16srRNA identified X1 strains of Escherichia coli strains belonging to some of the similarity of 99%, a preliminary judge for Escherichia coli, and the similarity of new O104:H4 strains of pathogenic bacteria in 2011 for the 99%. phylogenetic tree showed that X1 strain and O104:H4 strain homology higher. The serotype identification test strains since the emergence of coagulation phenomenon, not serotyped.
3, fluorescent PCR detection results: Escherichia coli O104 gene and ipaH gene test results were positive; Stx1/Stx2/eae, LT/ST, Aggr, Shigella / Salmonella, H4 detection results were negative.
4, EHECO104:H4 virulence gene detection results: the results of 9 virulence factors were all negative.
Non X1 strains of Yersinia pestis, combined with morphological characteristics, biochemical identification, animal experiment, 16srRNA gene identification results to determine their Escherichia coli; the common pathogenic Escherichia coli and intestinal test results of 13 virulence factors of Escherichia coli O104:H4 showed that the X1 strain of Escherichia coli O104 attenuated strain of bacteria.

【學(xué)位授予單位】：大理學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R378.21

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