耐药基因及病原体高通量检测芯片的研制及初步应用

发布时间：2018-08-07 14:19

【摘要】：病原体引起的感染性疾病严重威胁人类健康。随着生物技术的进步，抗生素大量不合理使用，导致多重耐药菌的产生，近年更出现了超级细菌，导致临床治疗难度大，，也构成了严重的公共卫生问题。且随着环境污染日趋严重，一些从前未出现过的疾病又给人类带来了新一轮的威胁。传统病原体检测方法存在耗时长、受培养条件限制、只对少量样品检测等缺点，越来越不能满足临床的需要。因此，发展快速、准确、检测病原体的方法一直是人们追求的目标。基因芯片技术（Genechip）是90年代中期以来快速发展起来的分子生物学高新技术，因其高通量、大规模、并行化处理的特点，为病原体的分子诊断提供了有力的技术条件。它可以实现对多种目标基因的平行高通量鉴定，能够很好地对多种感染性疾病的病原体，耐药性及毒素因子进行检测，提高病原体的检出水平，这正是有效预防感染性疾病发生和控制其迅速传播的前提和关键。本文的研究目的是建立针对多种耐药基因及病原体检测的高通量基因芯片，为快速确定细菌耐药性、指导临床合理选用抗菌药及快速筛查病原菌提供依据。根据NCBI数据库中提供的各类已发表基因序列，按照探针设计原则，筛选和设计合成有代表性的17大类耐药性基因探针115条（包括超广谱β内酰胺酶、头孢菌素酶、碳青霉烯酶、整合酶类基因、四环素家族耐药基因、氨基糖甙类药物耐药基因、耐消毒剂基因、红霉素耐药相关基因、大环内酯类外排基因、耐万古霉素耐药基因、多药耐药外排泵基因、耐莫匹罗星基因、磺胺类耐药基因、泰洛星耐药基因、氟喹诺酮类药物耐药基因，金葡菌金标准mecA基因和常用基因工程耐药基因），8类病原体种类特异基因探针32条（包括鼻疽伯克霍尔德氏菌和类鼻疽伯克霍尔德氏菌、布鲁氏菌、沙门氏菌、鼠疫耶尔森菌、炭疽芽孢杆菌、土拉弗朗西斯菌、志贺菌属和侵袭性大肠埃希菌、霍乱弧菌）及7类毒素基因探针25条（包括白喉毒素、志贺毒素、肉毒毒素、篦麻毒素、破伤风毒素、葡萄球菌肠毒素和霍乱毒素）共172条探针，分别建立了耐药基因芯片和耐药基因及病原体检测芯片。探讨核酸提取方法，优化全基因组DNA的扩增标记体系，并探讨了探针浓度、点样温度及湿度、杂交液成分及浓度、杂交温度及时间对基因芯片杂交结果的影响，筛选出了基因芯片杂交的最适条件。在此基础上，评价其灵敏度、特异性和重复性等性能指标，并将其运用于临床病原菌及耐药细菌的初步检测中。本实验以抗卡那霉素重组耐药菌株（E.coliDH5α/pET28）为例，确定了合适的基因组DNA提取方法为CTAB/NaCl法。最适点样条件为温度25℃，湿度55%；最适杂交条件为：杂交温度为42℃，杂交时间为4h，杂交液成分为50%去离子甲酰胺，5×SSC，0.1%SDS，0.5μg/μL鲑鱼精DNA。基因芯片的检测灵敏度为20ng/μL的DNA。建立的耐药芯片检测常用的基因工程受体菌、基因工程耐药菌、7株已测序参考菌株均得到正确结果，显示其良好的特异性及准确性。耐药基因及病原体检测芯片初步检测霍乱弧菌，O157：H7标准株，沙门氏菌、志贺氏菌、葡萄球菌及基因工程耐药菌均得到正确检测结果。对42株临床疑似耐药菌进行检测，其中6株为混合耐药菌，与常规药敏试验法相比一致。本研究建立的耐药基因及病原体高通量检测芯片特异性及灵敏性良好，可快速准确检出细菌的耐药性及病原体种类，大大减轻工作量。该芯片检测方法不仅可用于临床耐药菌株的耐药谱检测，指导抗生素的合理使用，而且可用于新发和突发传染病等公共卫生事件的病原体筛查，对在预防和控制感染性疾病的传播及流行中具有重要意义。
[Abstract]:Infectious diseases caused by pathogens are serious threat to human health. With the progress of biotechnology, antibiotics are unreasonably used, resulting in the emergence of multidrug resistant bacteria. In recent years, the emergence of superbacteria has resulted in the difficulty of clinical treatment and serious public health problems. With the environmental pollution becoming more and more serious, some have not been produced before. The existing disease has brought a new round of threat to human beings. The traditional detection methods of traditional pathogens are time-consuming, limited by culture conditions, only a small number of samples are detected and other shortcomings, more and more can not meet the needs of the clinical. Therefore, the development of rapid, accurate, detection of pathogens has been the goal of the people's pursuit.
Gene chip technology (Genechip) is a high and new technology developed rapidly since the mid 90s. Because of its high flux, large scale and parallel processing characteristics, it provides powerful technical conditions for molecular diagnosis of pathogens. It can realize parallel high flux identification for various target bases, and can be very good to a variety of sense. Detection of pathogens, drug resistance and toxin factors of dyed diseases to improve the detection level of pathogens is the prerequisite and key to the effective prevention of infectious diseases and the control of its rapid transmission. The aim of this study is to establish a high throughput gene chip for multiple resistance genes and pathogenic tests for rapid identification of bacterial resistance. It provides guidance for clinical rational selection of antibiotics and rapid screening of pathogens.
According to the various published gene sequences provided in the NCBI database, according to the principle of probe design, 115 kinds of representative 17 major resistance gene probes (including hyper broad-spectrum beta lactamase, cephalosporinase, carbapenem, integrase gene, four cyclin family resistance gene, aminoglycoside drug resistance gene) are screened and designed. Anti disinfectant gene, erythromycin resistance related genes, macrolide resistant gene, vancomycin resistant gene, multidrug resistant efflux pump gene, mopiopicin gene, sulfonamide resistance gene, Tylox resistance gene, fluoroquinolone resistance gene, Jin Pu gold standard mecA gene and common gene engineering resistance gene, and the 8 types of gene engineering resistance genes. 32 pathogenic specific gene probes (including Burke Holder and Burke Holder, Brucella, Salmonella, Jerson, Bacillus plague, Bacillus anthracis, Turafrancisrand, Shigella and invasive Escherichia coli, Vibrio cholerae) and 7 toxin gene probes (including diphtheria toxin, Chronicles) A total of 172 probes were used to establish a microarray of drug-resistant gene chips, drug resistance genes and pathogen detection chips, and to explore the method of nucleic acid extraction to optimize the amplification and labeling system of the whole genome DNA, and to explore the concentration of the probe, the temperature and humidity of the point, and the hybridization. The effects of liquid composition and concentration, hybridization temperature and time on the results of gene chip hybridization were screened out the optimum conditions for gene chip hybridization. On this basis, the sensitivity, specificity and repeatability of the hybridization were evaluated and used in the preliminary detection of clinical pathogens and drug resistant bacteria.
In this experiment, a recombinant resistant strain of kanamycin resistant strain (E.coliDH5 alpha /pET28) was used as an example to determine a suitable genomic DNA extraction method for CTAB/NaCl. The optimum point sample was 25 C and 55% humidity, and the optimum hybridization conditions were: hybridization temperature was 42, hybridization time was 4h, the composition of hybrids was 50% deionalamide, 5 x SSC, 0.1%SDS, 0.5 u g/. The detection sensitivity of the DNA. gene chip of the micron L salmon sperm was 20ng/ micron L for the detection of the common gene engineering receptor bacteria, the gene engineering resistant bacteria and the 7 sequence reference strains all obtained the correct results, showing its good specificity and accuracy. The drug resistance gene and the detection chip of the disease Mycoplasma were used for the preliminary detection of Vibrio cholerae, O157: H7 standard strain, Salmonella, Shigella, Staphylococcus and gene engineering resistant bacteria were detected correctly. 42 clinically suspected drug-resistant bacteria were detected, 6 of them were mixed resistant bacteria, which were in accordance with the conventional drug sensitivity test.
The resistance gene and high throughput detection chip established in this study have good specificity and sensitivity, which can quickly and accurately detect the bacterial resistance and pathogens, and greatly reduce the amount of work. This chip detection method can be used not only for the detection of drug resistance spectrum of clinical drug resistant strains, but also for the rational use of antibiotics, and can be used in new hair and new hair. Screening of pathogens in public health events such as emergent infectious diseases is of great significance in preventing and controlling the spread and epidemic of infectious diseases.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R346

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