本文選題：大腸埃希菌　切入點：目標捕獲　出處：《寧波大學》2015年碩士論文　論文類型：學位論文

【摘要】：目的建立目標捕獲和高通量測序相結(jié)合的方法,應(yīng)用于臨床耐藥細菌的檢測,達到同時獲得菌株鑒定結(jié)果、耐藥基因型及用于MLST分型的管家基因序列的目的,節(jié)約臨床診斷的時間,綜合分析本地區(qū)耐藥基因的分布情況,為臨床合理用藥和進行流行病學監(jiān)測提供科學依據(jù)。方法參考相關(guān)文獻,選取大腸埃希菌常見的52種耐藥基因,Genbank上下載耐藥基因序列、大腸埃希菌16S r RNA基因序列及8個管家基因序列,序列交由美國Agilent公司設(shè)計合成適配于高通量測序儀的Sureselect目標捕獲試劑盒。對臨床收集的4株產(chǎn)ESBLs大腸埃希菌進行檢測,建立目標捕獲-高通量測序方法,并驗證該方法的準確性和可重復(fù)性。應(yīng)用目標捕獲-高通量測序方法檢測臨床收集的22株產(chǎn)ESBLs大腸埃希菌,檢測結(jié)果經(jīng)BLAST比對,獲得檢測菌株的耐藥基因型,結(jié)合藥敏試驗結(jié)果分析本地區(qū)大腸埃希菌的耐藥特點;獲得管家基因序列,使用Pasture軟件進行MLST分型,分析菌株之間的流行病學關(guān)系。結(jié)果(1)運用我們建立的目標捕獲-高通量測序方法檢測臨床收集的4株產(chǎn)ESBLs大腸埃希菌,得到的16S r RNA基因序列經(jīng)BLAST比對后證實為大腸埃希菌,與VITEK-2檢測系統(tǒng)鑒定結(jié)果相一致。選取部分耐藥基因采用PCRSanger測序,兩者測序結(jié)果完全一致。運用該方法重復(fù)檢測這4株菌株,兩次得到的結(jié)果一致。(2)臨床收集產(chǎn)ESBLs大腸埃希菌22株,運用所建立的目標捕獲-高通量測序方法進行檢測,得到的16S r RNA基因序列經(jīng)BLAST比對后證實所有菌株為大腸埃希菌,與VITEK-2檢測系統(tǒng)鑒定結(jié)果相一致。(3)得到的耐藥基因序列經(jīng)BLAST比對,共得到24種耐藥基因型。有20株檢出8種ESBLs基因,其中CTX-M-14、CTX-M-55、CTX-M-15、CTX-M-27、CTX-M-65、SHV-12、OXA-1、OXA-10型的檢出率分別為68.18%、45.45%、13.64%、4.55%、9.09%、18.18%、4.55%和4.55%。有3株檢出Amp C類耐藥基因,均為DHA-1型。有20株檢出6種氨基糖苷類藥物耐藥基因,其中aac(3)-II、aac(6’)-Ib、ant(3’’)-I、aph(3’)-I、aad A、rmt B型的檢出率分別為63.63%、18.18%、13.64%、9.09%、31.82%和4.55%。有17株檢出6種喹諾酮類藥物耐藥基因,其中g(shù)yr A、gyr B、qnr B、qnr S、aac(6’)-Ib-cr、qep A型的檢出率分別為45.45%、4.55%、4.55%、9.09%、36.36%和22.73%。有18株檢出2種四環(huán)素類耐藥基因,其中tet(A)和tet(B)型的檢出率分別為68.18%、45.45%。TEM-1型基因檢出率為90.91%,其他耐藥基因均未檢出。所有菌株均檢出三種及以上不同種類藥物耐藥基因。藥敏結(jié)果顯示所有菌株對第三代頭孢菌素類藥物耐藥嚴重,多重耐藥株在50%以上。(4)得到的管家基因序列使用Pasture軟件進行MLST分型,共得到16種不同的ST型,檢出率最高的是ST38型,共檢出3株,其他還有ST131型、ST405型等。結(jié)論(1)成功構(gòu)建了可同時完成菌株鑒定、耐藥基因檢測及流行病學分型的目標捕獲-高通量測序方法,證明了該方法有很好的準確性和可重復(fù)性,初步證實該方法可應(yīng)用于臨床耐藥菌的檢測,給臨床快速診斷和用藥提供一定的科學依據(jù)。(2)產(chǎn)ESBLs大腸埃希菌耐藥情況嚴峻,耐藥基因檢出率高。與β-內(nèi)酰胺類相關(guān)的耐藥基因主要為TEM-1型和CTX-M-14型,與氨基糖苷類藥物耐藥相關(guān)的主要為aac(3)-II型和aad A型,與喹諾酮類藥物耐藥相關(guān)的主要為gyr A型與aac(6’)-Ib-cr型,與四環(huán)素類藥物耐藥相關(guān)的主要為tet(A)型和tet(B)型。(3)本研究中產(chǎn)ESBLs大腸埃希菌遺傳背景具有多樣性,ST38型為主要流行ST型,ICU內(nèi)有交叉感染的可能,需加強監(jiān)測。
[Abstract]:Objective to establish a method of target acquisition and high-throughput sequencing combined detection, for the clinical application of drug resistant bacteria, at the same time the identification of strains, resistance gene and housekeeping gene MLST type for the purpose of saving the time of clinical diagnosis, the distribution of the area of resistance gene of comprehensive analysis, to provide a scientific basis for clinical the rational use of drugs and epidemiological monitoring methods. Reference to the relevant literature, selected 52 kinds of common resistance genes of Escherichia coli, Download resistance gene sequence Genbank and sequence R gene of Escherichia coli 16S RNA and 8 housekeeping gene sequence, sequence by the American Agilent company to design and synthesize suitable for high-throughput sequencing of Sureselect target acquisition kit. The clinical collection of 4 ESBLs producing strains of Escherichia coli were detected, establish the target capture method of high-throughput sequencing, and verify the accuracy of the method And repeatability. Application of target capture method of high-throughput sequencing to detect clinically collected 22 strains of ESBLs producing Escherichia coli, the detection results by BLAST comparison, resistance gene type detection of strains, combined with the characteristics of local resistance of Escherichia coli in analysis of drug susceptibility tests; obtain sequences of the housekeeping genes, MLST type Pasture software, analysis of epidemiological relationship between strains. Results (1) we use established target acquisition method of high-throughput sequencing detection of clinically collected 4 strains of ESBLs producing Escherichia coli 16S R RNA gene sequences obtained by BLAST comparison confirmed for Escherichia coli, and the detection results of VITEK-2 system identification selection consistent. Some resistant gene by PCRSanger sequencing, the sequencing results are completely consistent. Using this method, repeated detection of these 4 strains, the results obtained two times. (2) clinical ESBLs producing Escherichia coli collected 甯岃弻22鏍，

本文編號：1585541