基于目标捕获和高通量测序检测耐药细菌的研究

发布时间：2018-03-08 21:07

  本文选题：大肠埃希菌　切入点：目标捕获　出处：《宁波大学》2015年硕士论文　论文类型：学位论文

【摘要】：目的建立目标捕获和高通量测序相结合的方法,应用于临床耐药细菌的检测,达到同时获得菌株鉴定结果、耐药基因型及用于MLST分型的管家基因序列的目的,节约临床诊断的时间,综合分析本地区耐药基因的分布情况,为临床合理用药和进行流行病学监测提供科学依据。方法参考相关文献,选取大肠埃希菌常见的52种耐药基因,Genbank上下载耐药基因序列、大肠埃希菌16S r RNA基因序列及8个管家基因序列,序列交由美国Agilent公司设计合成适配于高通量测序仪的Sureselect目标捕获试剂盒。对临床收集的4株产ESBLs大肠埃希菌进行检测,建立目标捕获-高通量测序方法,并验证该方法的准确性和可重复性。应用目标捕获-高通量测序方法检测临床收集的22株产ESBLs大肠埃希菌,检测结果经BLAST比对,获得检测菌株的耐药基因型,结合药敏试验结果分析本地区大肠埃希菌的耐药特点;获得管家基因序列,使用Pasture软件进行MLST分型,分析菌株之间的流行病学关系。结果(1)运用我们建立的目标捕获-高通量测序方法检测临床收集的4株产ESBLs大肠埃希菌,得到的16S r RNA基因序列经BLAST比对后证实为大肠埃希菌,与VITEK-2检测系统鉴定结果相一致。选取部分耐药基因采用PCRSanger测序,两者测序结果完全一致。运用该方法重复检测这4株菌株,两次得到的结果一致。(2)临床收集产ESBLs大肠埃希菌22株,运用所建立的目标捕获-高通量测序方法进行检测,得到的16S r RNA基因序列经BLAST比对后证实所有菌株为大肠埃希菌,与VITEK-2检测系统鉴定结果相一致。(3)得到的耐药基因序列经BLAST比对,共得到24种耐药基因型。有20株检出8种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型的检出率分别为68.18%、45.45%、13.64%、4.55%、9.09%、18.18%、4.55%和4.55%。有3株检出Amp C类耐药基因,均为DHA-1型。有20株检出6种氨基糖苷类药物耐药基因,其中aac(3)-II、aac(6’)-Ib、ant(3’’)-I、aph(3’)-I、aad A、rmt B型的检出率分别为63.63%、18.18%、13.64%、9.09%、31.82%和4.55%。有17株检出6种喹诺酮类药物耐药基因,其中gyr A、gyr B、qnr B、qnr S、aac(6’)-Ib-cr、qep A型的检出率分别为45.45%、4.55%、4.55%、9.09%、36.36%和22.73%。有18株检出2种四环素类耐药基因,其中tet(A)和tet(B)型的检出率分别为68.18%、45.45%。TEM-1型基因检出率为90.91%,其他耐药基因均未检出。所有菌株均检出三种及以上不同种类药物耐药基因。药敏结果显示所有菌株对第三代头孢菌素类药物耐药严重,多重耐药株在50%以上。(4)得到的管家基因序列使用Pasture软件进行MLST分型,共得到16种不同的ST型,检出率最高的是ST38型,共检出3株,其他还有ST131型、ST405型等。结论(1)成功构建了可同时完成菌株鉴定、耐药基因检测及流行病学分型的目标捕获-高通量测序方法,证明了该方法有很好的准确性和可重复性,初步证实该方法可应用于临床耐药菌的检测,给临床快速诊断和用药提供一定的科学依据。(2)产ESBLs大肠埃希菌耐药情况严峻,耐药基因检出率高。与β-内酰胺类相关的耐药基因主要为TEM-1型和CTX-M-14型,与氨基糖苷类药物耐药相关的主要为aac(3)-II型和aad A型,与喹诺酮类药物耐药相关的主要为gyr A型与aac(6’)-Ib-cr型,与四环素类药物耐药相关的主要为tet(A)型和tet(B)型。(3)本研究中产ESBLs大肠埃希菌遗传背景具有多样性,ST38型为主要流行ST型,ICU内有交叉感染的可能,需加强监测。
[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，

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