一株大肠杆菌O104的表型及基因型鉴定

发布时间：2018-02-11 15:13

本文关键词： 鼠疫杆菌大肠杆菌 16s rRNA 荧光PCR 毒力基因　出处：《大理学院》2013年硕士论文　论文类型：学位论文

【摘要】：目的对从云南省西双版纳州景洪县种猪场自毙黄胸鼠脏器分离到的1株曾疑似为鼠疫耶尔森氏菌株(以下简称X1菌株)进行鼠疫菌的排除实验，并进一步对该菌株进行系统地表型及分子生物学鉴定，以确认其为何种菌株。方法 1、鼠疫菌的排除实验 ①用鼠疫细菌学常规方法确定其生物学表型；②应用API20E肠杆菌科生化试纸条和VITEK-2全自动测定生化鉴定仪分别鉴定X1菌株的生化特性；③应用检测鼠疫金标试纸条检测F1抗原；④应用PCR检测鼠疫特异基因pla、caf1；⑤动物实验：经腹腔注射、灌胃、静脉注射等三种途径，分不同的剂量组和不同批次感染小白鼠，观察是否有病变，同时取肝、脾、肺、心、腺进行细菌培养鉴定。 2、采用16srRNA对X1菌株进行鉴定将X1菌株经16srRNA全基因序列进行PCR扩增，产物纯化、测序；使用DNAstar软件包中的SeqMan对序列片段进行拼接、编辑、校正；序列经BLAST与GenBank中的已知序列进行同源性比较，并构建系统进化树，判定其属种。 3、荧光PCR检测先用常见的致泻性大肠杆菌毒素基因：STX1/STX2/EAE，LT/ST，ARRG/IPAH对X1、X11、X12三个细菌样品进行检测，再用大肠杆菌O104检测试剂对3个细菌样品进行检测，最后用大肠杆菌H4检测试剂盒检测。 4、肠出血性大肠杆菌（EHEC）O104:H4的毒力基因检测用PCR进行毒力基因检测，检测的毒力基因包括针对EHEC的4个基因:stx1、stx2、eaeA和ehxA；针对EAEC毒力质粒上的3个基因：aatA、aggR、aap；以及O104特异基因wzy和H4特异基因fliC。若wzy和fliC基因检测阳性，还需血清凝集试验确认血清型是O104、鞭毛(H)抗原是04。结果 1、①该菌株为革兰阴性短小杆菌，两极浓染；在37℃时能被假结核噬菌体裂解；②梅里埃API20E和生化鉴定仪的试验结果均未鉴定出X1菌株的属种；③金标试纸条检测F1抗原为阴性；④鼠疫特异性基因pla、caf1检测结果阴性。⑤动物实验：将配制的浓度为8×1010/ml的X1菌株菌液经三种途径感染小鼠，未出现死亡小鼠，将菌液浓度增加到8×1011/ml，，经腹腔注射感染的小鼠全部死亡；将分离到的X11菌株分别配制到4×1011/ml和8×1011/ml经腹腔注射感染的小鼠也全部死亡，分离到菌株X12，解剖观察脏器发生病变，且X11菌株和X12菌株在37℃能被假结核噬菌体裂解。 2、①经16srRNA鉴定，X1菌株与大肠杆菌埃希氏菌属的一些菌株相似度达99%，初步判断为大肠埃希氏菌属，且与2011年新发病原菌O104:H4菌株的相似度为99%。②进化树显示X1菌株与O104:H4菌株的同源性更高。③血清型鉴定试验菌株出现自凝现象，无法进行血清型鉴定。 3、荧光PCR检测结果：大肠杆菌O104基因和ipaH基因检测结果为阳性；Stx1/Stx2/eae、LT/ST、Aggr、志贺菌/沙门菌、H4检测结果均为阴性。 4、EHECO104:H4毒力基因检测结果：9个毒力因子检测结果均为阴性。结论 X1菌株非鼠疫菌株，结合形态特征、生化鉴定结果、动物实验、16srRNA基因鉴定结果判定其为一株大肠杆菌；对常见致病性大肠杆菌及肠出血性大肠杆菌O104:H4的13种毒力因子的检测结果表明，X1菌株为大肠杆菌弱毒株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.

【学位授予单位】：大理学院
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R378.21

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