本文關(guān)鍵詞： 雞 致病性沙門氏菌 血清型 藥物敏感性 耐藥基因 多重PCR TaqMan-MGB熒光定量PCR bla_(NDM-1)基因　出處：《廣西大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：沙門氏菌可以引起雞白痢、雞傷寒和禽副傷寒,具有垂直傳播和水平傳播的特點(diǎn),是嚴(yán)重危害養(yǎng)雞業(yè)的重要病原菌。廣西是養(yǎng)雞大省,年飼養(yǎng)量超過10億羽,掌握當(dāng)前廣西雞源致病性沙門氏菌的流行優(yōu)勢菌株及其耐藥特點(diǎn),對雞沙門氏菌病的防治具有重要的指導(dǎo)意義。1.雞源致病性沙門氏菌多重PCR檢測方法的建立及應(yīng)用根據(jù)GenBank中發(fā)表的沙門氏菌屬特異性毒力基因invA.質(zhì)粒毒力基因spvR.雞宿主特異性毒力基因fliC的序列,利用Primer Express 3.0軟件設(shè)計(jì)合成了3對特異性引物。經(jīng)過反應(yīng)條件的優(yōu)化,建立了檢測雞源致病性沙門氏菌的多重PCR方法。該方法可以特異性地?cái)U(kuò)增攜帶毒力質(zhì)粒的致病性雞沙門氏菌,而與大腸桿菌、多殺性巴氏桿菌、志賀氏痢疾桿菌及普通變形桿菌均無交叉反應(yīng)；對沙門氏菌菌液的檢出下限為4.5×10~2 CFU/mL,對重組質(zhì)粒標(biāo)準(zhǔn)品的檢出下限為1.67×10~3拷貝/μL。應(yīng)用所建立的方法,對采集的310份臨床病料進(jìn)行檢測,結(jié)果檢出invA基因陽性34份,占10.97%,其中invA+spvR基因陽性20份,占6.45%; invA+fliC基因陽性2份,占O.65%; invA+spvR+fliC基因陽性12份,占3.87%。2.雞源致病性沙門氏菌的分離鑒定及血清型和藥敏性分析采集疑似病雞的組織病料,對沙門氏菌進(jìn)行增菌和分離培養(yǎng),對分離菌株運(yùn)用VITEK System ATB Expression法ID 32E腸桿菌鑒定試劑條進(jìn)行生化試驗(yàn)鑒定,應(yīng)用玻片凝集試驗(yàn)進(jìn)行血清型鑒定,采用ATB VET藥敏試劑條對28種腸桿菌抗菌藥物進(jìn)行藥敏性試驗(yàn)。結(jié)果,從310份疑似雞病料中分離鑒定出34株致病性沙門氏茵；這些分離菌株中有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群鼠傷寒沙門氏菌和D群雞白痢沙門氏菌為優(yōu)勢菌株；34株分離株對大觀霉素和安普霉素全部敏感,對氯霉素、卡那霉素、慶大霉素的耐藥率小于10%,對阿莫西林、鏈霉素、氟甲喹、惡喹酸、磺胺甲惡唑、四環(huán)素和呋喃妥因耐藥率介于50%和90%之間,而對青霉素、苯唑西林、夫西地酸、利福平和甲硝唑的耐藥率達(dá)到100%,并且存在多重耐藥現(xiàn)象。3.雞源沙門氏菌耐藥性與耐藥基因和血清群的相關(guān)性分析為研究沙門氏菌分離株耐藥表型與耐藥基因和血清群之間的關(guān)系,對血清型鑒定為B群和D群的29株雞源致病性沙門氏菌,采用ATB VET藥敏試劑條法對20種抗菌藥物進(jìn)行敏感性測定,應(yīng)用PCR技術(shù)對這些抗菌藥物19種相關(guān)耐藥基因進(jìn)行檢測。結(jié)果,29株沙門氏菌分離株中,青霉素、苯唑西林的耐藥率最高(100%),耐藥率超過50%的還有磺胺甲惡唑(82.76%)、鏈霉素(75.86%)、惡喹酸(75.86%)、氟甲喹(65.52%)、恩諾沙星(55.17%)、四環(huán)素(55.17%)、阿莫西林(51.72%),而對大觀霉素、慶大霉素、安普霉素、氯霉素、多粘菌素E沒有出現(xiàn)耐藥性。所有29個(gè)分離株至少對6種抗菌藥物具有耐藥性,以6-7重耐藥為主(占51.73%),最高可達(dá)14重耐藥(占3.45%)。所檢測的19種耐藥基因中,共檢測到14種耐藥基因,其中blaTEM檢出率最高(100%),檢出率超過40%的還有aadAl(62.07 %) Sul2 (62.07%)、Sul3(51.72%)、tetA (48.28%)、qnrA (44.83%),而blaPSE、aadA2、tetG、tetM、tetX未能檢出。所有29個(gè)分離株至少攜帶2種耐藥基因,以攜帶4～-6種耐藥基因?yàn)橹?占79.31%),最多者攜帶10種耐藥基因(占3.45%)。B血清群和D血清群的耐藥表型和耐藥基因攜帶率沒有明顯的差異。表明,廣西雞源致病性沙門氏菌耐藥性及多重耐藥性非常普遍,耐藥表型與相關(guān)耐藥基因檢出率呈正相關(guān),而與血清群之間無明顯的相關(guān)性。4.超級細(xì)菌blaNDM-1基因TaqMan-MGB熒光定量PCR檢測方法的建立及應(yīng)用為建立快速檢測超級細(xì)菌編碼新德里金屬p-內(nèi)酰胺酶1(NDM-1)的blaNDM-1基因的方法,針對blaNDM-1及其變異體的基因序列設(shè)計(jì)一對特異性引物和一條MGB探針,以構(gòu)建的含有blaNDM-1基因片段的重組質(zhì)粒作為陽性標(biāo)準(zhǔn)品,經(jīng)過反應(yīng)條件的優(yōu)化,成功建立了檢測blaNDM-1基因的TaqMan-MGB熒光定量PCR方法。該方法可以特異性擴(kuò)增blaNDM-1基因重組質(zhì)粒標(biāo)準(zhǔn)品,而對雞白痢沙門氏菌、大腸桿菌、多殺性巴氏桿菌和鼠傷寒沙門氏菌的標(biāo)準(zhǔn)菌株為陰性；檢出下限為2.59拷貝/μL,比常規(guī)PCR敏感性高100倍；重復(fù)性試驗(yàn)的變異系數(shù)小于1.5%。應(yīng)用所建立的方法對34株雞源致病性沙門氏菌分離株進(jìn)行檢測,結(jié)果均未檢測到目的條帶,所有分離株均沒有攜帶blaNDM-1基因。表明,本研究建立的TaqMan-MGB熒光定量PCR方法特異性強(qiáng)、敏感性高、重復(fù)性好,可用于監(jiān)測攜帶blaNDM-1基因及其變異體的超級細(xì)菌。綜上所述,本研究基于沙門氏菌屬特異性毒力基因invA.沙門氏菌雞宿主特異性基因fliC以及沙門氏菌質(zhì)粒毒力基因spvR,成功建立了鑒別檢測雞源致病性沙門氏菌的多重PCR方法；對廣西當(dāng)前雞源致病性沙門氏菌進(jìn)行分離鑒定和耐藥性分析,發(fā)現(xiàn)以B群鼠傷寒沙門氏菌和D群雞白痢沙門氏菌為流行優(yōu)勢菌株,并且耐藥性及多重耐藥性非常普遍；針對當(dāng)前流行優(yōu)勢菌株進(jìn)行耐藥表型與耐藥基因、血清群的相關(guān)性分析,發(fā)現(xiàn)耐藥表型與耐藥基因呈正相關(guān),而耐藥表型與血清群之間無明顯的相關(guān)性；針對超級細(xì)菌編碼新德里金屬p-內(nèi)酰胺酶1(NDM-1)的blaNDM-1基因及其變異體,成功建立了檢測bla_(NDM-1)基因的TaqMan-MGB熒光定量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.

【學(xué)位授予單位】：廣西大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S858.31

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