广西鸡源致病性沙门氏菌流行优势菌株血清型及耐药性的研究

发布时间：2018-02-09 00:51

本文关键词： 鸡致病性沙门氏菌血清型药物敏感性耐药基因多重PCR TaqMan-MGB荧光定量PCR bla_(NDM-1)基因　出处：《广西大学》2015年硕士论文　论文类型：学位论文

【摘要】：沙门氏菌可以引起鸡白痢、鸡伤寒和禽副伤寒,具有垂直传播和水平传播的特点,是严重危害养鸡业的重要病原菌。广西是养鸡大省,年饲养量超过10亿羽,掌握当前广西鸡源致病性沙门氏菌的流行优势菌株及其耐药特点,对鸡沙门氏菌病的防治具有重要的指导意义。1.鸡源致病性沙门氏菌多重PCR检测方法的建立及应用根据GenBank中发表的沙门氏菌属特异性毒力基因invA.质粒毒力基因spvR.鸡宿主特异性毒力基因fliC的序列,利用Primer Express 3.0软件设计合成了3对特异性引物。经过反应条件的优化,建立了检测鸡源致病性沙门氏菌的多重PCR方法。该方法可以特异性地扩增携带毒力质粒的致病性鸡沙门氏菌,而与大肠杆菌、多杀性巴氏杆菌、志贺氏痢疾杆菌及普通变形杆菌均无交叉反应；对沙门氏菌菌液的检出下限为4.5×10~2 CFU/mL,对重组质粒标准品的检出下限为1.67×10~3拷贝/μL。应用所建立的方法,对采集的310份临床病料进行检测,结果检出invA基因阳性34份,占10.97%,其中invA+spvR基因阳性20份,占6.45%; invA+fliC基因阳性2份,占O.65%; invA+spvR+fliC基因阳性12份,占3.87%。2.鸡源致病性沙门氏菌的分离鉴定及血清型和药敏性分析采集疑似病鸡的组织病料,对沙门氏菌进行增菌和分离培养,对分离菌株运用VITEK System ATB Expression法ID 32E肠杆菌鉴定试剂条进行生化试验鉴定,应用玻片凝集试验进行血清型鉴定,采用ATB VET药敏试剂条对28种肠杆菌抗菌药物进行药敏性试验。结果,从310份疑似鸡病料中分离鉴定出34株致病性沙门氏茵；这些分离菌株中有A群1株、C2群1株、B群14株和D群14株,另有3株未定型,其中以B群鼠伤寒沙门氏菌和D群鸡白痢沙门氏菌为优势菌株；34株分离株对大观霉素和安普霉素全部敏感,对氯霉素、卡那霉素、庆大霉素的耐药率小于10%,对阿莫西林、链霉素、氟甲喹、恶喹酸、磺胺甲恶唑、四环素和呋喃妥因耐药率介于50%和90%之间,而对青霉素、苯唑西林、夫西地酸、利福平和甲硝唑的耐药率达到100%,并且存在多重耐药现象。3.鸡源沙门氏菌耐药性与耐药基因和血清群的相关性分析为研究沙门氏菌分离株耐药表型与耐药基因和血清群之间的关系,对血清型鉴定为B群和D群的29株鸡源致病性沙门氏菌,采用ATB VET药敏试剂条法对20种抗菌药物进行敏感性测定,应用PCR技术对这些抗菌药物19种相关耐药基因进行检测。结果,29株沙门氏菌分离株中,青霉素、苯唑西林的耐药率最高(100%),耐药率超过50%的还有磺胺甲恶唑(82.76%)、链霉素(75.86%)、恶喹酸(75.86%)、氟甲喹(65.52%)、恩诺沙星(55.17%)、四环素(55.17%)、阿莫西林(51.72%),而对大观霉素、庆大霉素、安普霉素、氯霉素、多粘菌素E没有出现耐药性。所有29个分离株至少对6种抗菌药物具有耐药性,以6-7重耐药为主(占51.73%),最高可达14重耐药(占3.45%)。所检测的19种耐药基因中,共检测到14种耐药基因,其中blaTEM检出率最高(100%),检出率超过40%的还有aadAl(62.07 %) Sul2 (62.07%)、Sul3(51.72%)、tetA (48.28%)、qnrA (44.83%),而blaPSE、aadA2、tetG、tetM、tetX未能检出。所有29个分离株至少携带2种耐药基因,以携带4～-6种耐药基因为主(占79.31%),最多者携带10种耐药基因(占3.45%)。B血清群和D血清群的耐药表型和耐药基因携带率没有明显的差异。表明,广西鸡源致病性沙门氏菌耐药性及多重耐药性非常普遍,耐药表型与相关耐药基因检出率呈正相关,而与血清群之间无明显的相关性。4.超级细菌blaNDM-1基因TaqMan-MGB荧光定量PCR检测方法的建立及应用为建立快速检测超级细菌编码新德里金属p-内酰胺酶1(NDM-1)的blaNDM-1基因的方法,针对blaNDM-1及其变异体的基因序列设计一对特异性引物和一条MGB探针,以构建的含有blaNDM-1基因片段的重组质粒作为阳性标准品,经过反应条件的优化,成功建立了检测blaNDM-1基因的TaqMan-MGB荧光定量PCR方法。该方法可以特异性扩增blaNDM-1基因重组质粒标准品,而对鸡白痢沙门氏菌、大肠杆菌、多杀性巴氏杆菌和鼠伤寒沙门氏菌的标准菌株为阴性；检出下限为2.59拷贝/μL,比常规PCR敏感性高100倍；重复性试验的变异系数小于1.5%。应用所建立的方法对34株鸡源致病性沙门氏菌分离株进行检测,结果均未检测到目的条带,所有分离株均没有携带blaNDM-1基因。表明,本研究建立的TaqMan-MGB荧光定量PCR方法特异性强、敏感性高、重复性好,可用于监测携带blaNDM-1基因及其变异体的超级细菌。综上所述,本研究基于沙门氏菌属特异性毒力基因invA.沙门氏菌鸡宿主特异性基因fliC以及沙门氏菌质粒毒力基因spvR,成功建立了鉴别检测鸡源致病性沙门氏菌的多重PCR方法；对广西当前鸡源致病性沙门氏菌进行分离鉴定和耐药性分析,发现以B群鼠伤寒沙门氏菌和D群鸡白痢沙门氏菌为流行优势菌株,并且耐药性及多重耐药性非常普遍；针对当前流行优势菌株进行耐药表型与耐药基因、血清群的相关性分析,发现耐药表型与耐药基因呈正相关,而耐药表型与血清群之间无明显的相关性；针对超级细菌编码新德里金属p-内酰胺酶1(NDM-1)的blaNDM-1基因及其变异体,成功建立了检测bla_(NDM-1)基因的TaqMan-MGB荧光定量PCR方法。
[Abstract]:Salmonella can cause pullorum disease, fowl typhoid and paratyphoid bird, has the characteristics of vertical and horizontal communication, is an important pathogen of serious harms to the poultry industry. Guangxi province is chicken, raising the amount of more than 1 billion feather, master current dominant strains and drug resistance characteristics of Guangxi Chicken Pathogenic Salmonella the significance of.1. Chicken Pathogenic Salmonella from multiple PCR detection method for important prevention of salmonellosis establishment and application according to the sequence specific spvR. virulence gene invA. plasmid virulence gene of chicken host specific virulence gene fliC of Salmonella species published in GenBank, 3 pairs of specific primers. Synthesis using Primer Express 3 software design. After optimization of the reaction conditions, a multiplex PCR method for detection of avian pathogenic Salmonella. This method can specifically amplify the virulence plasmid Disease of chicken Salmonella, and Escherichia coli, Pasteurella multocida, Shigella dysenteriae and Bacillus proteus. There was no cross reaction; detection of Salmonella bacteria liquid limit of 4.5 * 10~2 CFU/mL, the detection of recombinant plasmid standard limit of 1.67 * 10~3 / L. application copy method the establishment, on the acquisition of 310 clinical samples were detected, the detection of invA gene was detected in 34 samples, accounting for 10.97%, in which the invA+spvR gene was detected in 20 samples, accounting for 6.45%; the invA+fliC gene was detected in 2 samples, accounting for O.65%; the invA+spvR+fliC gene was detected in 12 samples, accounting for 3.87%.2. of Chicken Pathogenic Salmonella isolation and serum type and drug sensitivity analysis of collected tissue suspected diseased chickens, the Salmonella bacteria and cultured on isolated strains using VITEK Expression method System ATB ID 32E Enterobacteriaceae identification reagent strip biochemical tests, application of slide coagulation The test set for serotype identification, drug sensitivity by ATB VET reagent of 28 Escherichia coli antimicrobial susceptibility test results, identified 34 strains of Pathogenic Salmonella isolated from 310 samples of chicken disease materials; these isolates in the 1 strains of group A, 1 strains of group C2, B group 14 strains and 14 strains of group D, while 3 were not stereotypes, of which B group D group of Salmonella typhimurium and Salmonella pullorum were the dominant strains; 34 strains to spectinomycin and Apramycin are sensitive to chloramphenicol, kanamycin, gentamicin resistant rate of less than 10% for, amoxicillin, streptomycin, flumequine, oxolinic acid, sulfamethoxazole, tetracycline and nitrofurantoin resistant rate between 50% and 90%, and to penicillin, oxacillin, fusidic acid, metronidazole and rifampin resistance rate reached 100%, and there is the phenomenon of multi drug resistant.3. chicken Salmonella resistance and resistance gene The analysis and correlation of serum of Salmonella isolates the relationship between the resistance phenotype and drug resistance gene and serum group, 29 strains of avian pathogenic Salmonella serotypes of B group and D group, on the 20 kinds of antimicrobial susceptibility was determined by ATB VET susceptibility test strip method, application PCR technology to detect these 19 kinds of antimicrobial resistance genes. The results showed that 29 strains of Salmonella isolates, resistance to penicillin, oxacillin was the highest (100%), the resistance rate of more than 50% and sulfamethoxazole (82.76%), streptomycin (75.86%), oxolinic acid and flumequine ((75.86%) 65.52%), enrofloxacin (55.17%), tetracycline (55.17%), amoxicillin (51.72%), and to spectinomycin, gentamicin, Apramycin, chloramphenicol, polymyxin E showed no resistance. All 29 isolates of at least 6 kinds of antimicrobial agents with resistance to 6-7 heavy resistance. (51.73%), up to 14 antibiotics (3.45%). 19 kinds of resistance genes detected in 14 resistant genes were detected, with the highest detection rate of blaTEM (100%), the detection rate of more than 40% and aadAl (62.07%) Sul2 (62.07%), Sul3 (51.72%), tetA (48.28%), qnrA (44.83%), blaPSE, aadA2, tetG, tetM, tetX was not detected. All 29 isolates carrying at least 2 kinds of resistance genes, to carry the 4 ~ -6 resistant genes mainly (79.31%), most carrying 10 resistant genes (3.45%) drug resistance phenotype and drug resistance gene.B in serum group and D blood group with no significant differences in rates. Show that Guangxi Chicken Pathogenic Salmonella resistance and multidrug resistance is very common, and the related drug resistance phenotype gene detection rate were positively correlated, while no correlation between.4. blaNDM-1 gene in super bacteria TaqMan-MGB fluorescence quantitative PCR detection method with significant serum groups The establishment and application of law for the establishment of rapid detection of super bacteria encoding New Delhi metal p- lactam enzyme 1 (NDM-1) method of blaNDM-1 gene, a pair of specific primers and a MGB probe designed according to the sequence of blaNDM-1 gene and its variants, to construct a recombinant plasmid containing the blaNDM-1 gene fragment was used as positive standard, after optimization the reaction conditions, we established the TaqMan-MGB fluorescence quantitative PCR method for detection of blaNDM-1 gene. This method can blaNDM-1 gene recombinant plasmid standard specific amplification of Escherichia coli and Salmonella pullorum, and standard strains of Pasteurella multocida and Salmonella typhimurium were negative; detection limit of 2.59 copies per mu L, 100 times higher than the conventional PCR method sensitivity; variation coefficient of repeatability test is less than 1.5%. by the 34 strains of avian pathogenic Salmonella isolates were detected Test results were not detected in the target band, all the isolates were not carrying blaNDM-1 gene. This study shows that the establishment of PCR TaqMan-MGB fluorescence quantitative method with strong specificity, high sensitivity, good reproducibility, and can be used for monitoring the super bacteria carrying blaNDM-1 gene and its variants. In summary, this study is based on the specific virulence gene invA. Salmonella in chicken host specific gene fliC and Salmonella plasmid virulence gene spvR of Salmonella, successfully established a multiplex PCR method for detection and identification of avian pathogenic Salmonella in Guangxi; current source of Chicken Pathogenic Salmonella isolation and drug resistance analysis, found in Salmonella typhimurium B group D group of bacteria and Salmonella pullorum were prevalent strains, and drug resistance and multi drug resistance is very common; resistance phenotype and drug resistance gene for the dominant strain of blood. Correlation analysis of the Qing group, found a positive resistance phenotype and resistance related genes, but there is no obvious correlation between resistance phenotype and serum group; for the super bacteria encoding New Delhi metal p- lactam enzyme 1 (NDM-1) blaNDM-1 gene and its variants, is successfully established for the detection of bla_ (NDM-1) TaqMan-MGB fluorescence quantitative PCR gene.

【学位授予单位】：广西大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.31

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