【摘要】：病原體引起的感染性疾病嚴(yán)重威脅人類健康。隨著生物技術(shù)的進(jìn)步，抗生素大量不合理使用，導(dǎo)致多重耐藥菌的產(chǎn)生，近年更出現(xiàn)了超級(jí)細(xì)菌，導(dǎo)致臨床治療難度大，，也構(gòu)成了嚴(yán)重的公共衛(wèi)生問(wèn)題。且隨著環(huán)境污染日趨嚴(yán)重，一些從前未出現(xiàn)過(guò)的疾病又給人類帶來(lái)了新一輪的威脅。傳統(tǒng)病原體檢測(cè)方法存在耗時(shí)長(zhǎng)、受培養(yǎng)條件限制、只對(duì)少量樣品檢測(cè)等缺點(diǎn)，越來(lái)越不能滿足臨床的需要。因此，發(fā)展快速、準(zhǔn)確、檢測(cè)病原體的方法一直是人們追求的目標(biāo)。 基因芯片技術(shù)（Genechip）是90年代中期以來(lái)快速發(fā)展起來(lái)的分子生物學(xué)高新技術(shù)，因其高通量、大規(guī)模、并行化處理的特點(diǎn)，為病原體的分子診斷提供了有力的技術(shù)條件。它可以實(shí)現(xiàn)對(duì)多種目標(biāo)基因的平行高通量鑒定，能夠很好地對(duì)多種感染性疾病的病原體，耐藥性及毒素因子進(jìn)行檢測(cè)，提高病原體的檢出水平，這正是有效預(yù)防感染性疾病發(fā)生和控制其迅速傳播的前提和關(guān)鍵。本文的研究目的是建立針對(duì)多種耐藥基因及病原體檢測(cè)的高通量基因芯片，為快速確定細(xì)菌耐藥性、指導(dǎo)臨床合理選用抗菌藥及快速篩查病原菌提供依據(jù)。 根據(jù)NCBI數(shù)據(jù)庫(kù)中提供的各類已發(fā)表基因序列，按照探針設(shè)計(jì)原則，篩選和設(shè)計(jì)合成有代表性的17大類耐藥性基因探針115條（包括超廣譜β內(nèi)酰胺酶、頭孢菌素酶、碳青霉烯酶、整合酶類基因、四環(huán)素家族耐藥基因、氨基糖甙類藥物耐藥基因、耐消毒劑基因、紅霉素耐藥相關(guān)基因、大環(huán)內(nèi)酯類外排基因、耐萬(wàn)古霉素耐藥基因、多藥耐藥外排泵基因、耐莫匹羅星基因、磺胺類耐藥基因、泰洛星耐藥基因、氟喹諾酮類藥物耐藥基因，金葡菌金標(biāo)準(zhǔn)mecA基因和常用基因工程耐藥基因），8類病原體種類特異基因探針32條（包括鼻疽伯克霍爾德氏菌和類鼻疽伯克霍爾德氏菌、布魯氏菌、沙門氏菌、鼠疫耶爾森菌、炭疽芽孢桿菌、土拉弗朗西斯菌、志賀菌屬和侵襲性大腸埃希菌、霍亂弧菌）及7類毒素基因探針25條（包括白喉毒素、志賀毒素、肉毒毒素、篦麻毒素、破傷風(fēng)毒素、葡萄球菌腸毒素和霍亂毒素）共172條探針，分別建立了耐藥基因芯片和耐藥基因及病原體檢測(cè)芯片。探討核酸提取方法，優(yōu)化全基因組DNA的擴(kuò)增標(biāo)記體系，并探討了探針濃度、點(diǎn)樣溫度及濕度、雜交液成分及濃度、雜交溫度及時(shí)間對(duì)基因芯片雜交結(jié)果的影響，篩選出了基因芯片雜交的最適條件。在此基礎(chǔ)上，評(píng)價(jià)其靈敏度、特異性和重復(fù)性等性能指標(biāo)，并將其運(yùn)用于臨床病原菌及耐藥細(xì)菌的初步檢測(cè)中。 本實(shí)驗(yàn)以抗卡那霉素重組耐藥菌株（E.coliDH5α/pET28）為例，確定了合適的基因組DNA提取方法為CTAB/NaCl法。最適點(diǎn)樣條件為溫度25℃，濕度55%；最適雜交條件為：雜交溫度為42℃，雜交時(shí)間為4h，雜交液成分為50%去離子甲酰胺，5×SSC，0.1%SDS，0.5μg/μL鮭魚精DNA�；蛐酒臋z測(cè)靈敏度為20ng/μL的DNA。建立的耐藥芯片檢測(cè)常用的基因工程受體菌、基因工程耐藥菌、7株已測(cè)序參考菌株均得到正確結(jié)果，顯示其良好的特異性及準(zhǔn)確性。耐藥基因及病原體檢測(cè)芯片初步檢測(cè)霍亂弧菌，O157：H7標(biāo)準(zhǔn)株，沙門氏菌、志賀氏菌、葡萄球菌及基因工程耐藥菌均得到正確檢測(cè)結(jié)果。對(duì)42株臨床疑似耐藥菌進(jìn)行檢測(cè)，其中6株為混合耐藥菌，與常規(guī)藥敏試驗(yàn)法相比一致。 本研究建立的耐藥基因及病原體高通量檢測(cè)芯片特異性及靈敏性良好，可快速準(zhǔn)確檢出細(xì)菌的耐藥性及病原體種類，大大減輕工作量。該芯片檢測(cè)方法不僅可用于臨床耐藥菌株的耐藥譜檢測(cè)，指導(dǎo)抗生素的合理使用，而且可用于新發(fā)和突發(fā)傳染病等公共衛(wèi)生事件的病原體篩查，對(duì)在預(yù)防和控制感染性疾病的傳播及流行中具有重要意義。
[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.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R346

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 吳玲;聶根新;鄭新添;;基因芯片技術(shù)在現(xiàn)代畜牧業(yè)中的應(yīng)用[J];現(xiàn)代農(nóng)業(yè)科技;2007年21期

2 王洪敏;何冬梅;周輝;柯碧霞;鄧小玲;朱海明;陳經(jīng)雕;黎薇;楊杏芬;柯昌文;;華南地區(qū)常見的8種食源性致病菌檢測(cè)基因芯片研制[J];南方醫(yī)科大學(xué)學(xué)報(bào);2010年11期

3 孫菲;萬(wàn)向陽(yáng);季芳;;革蘭氏陰性菌耐藥基因檢測(cè)芯片的研制及應(yīng)用[J];國(guó)際檢驗(yàn)醫(yī)學(xué)雜志;2010年11期

4 陳昱;潘迎捷;趙勇;金維榮;秦紅友;徐曉晶;唐明未;;基因芯片技術(shù)檢測(cè)3種食源性致病微生物方法的建立[J];微生物學(xué)通報(bào);2009年02期

5 張海燕;馬文麗;黃吉城;商濤;廖之君;郭波旋;鄭文嶺;;60mer寡核苷酸芯片制備及雜交系統(tǒng)的優(yōu)化[J];現(xiàn)代預(yù)防醫(yī)學(xué);2008年18期

6 代敏,王紅寧,吳琦;PCR和核酸探針檢測(cè)豬源沙門氏菌四環(huán)素耐藥基因tetC的研究[J];畜牧獸醫(yī)學(xué)報(bào);2005年05期

7 金慧英,陶開華,李越希,李法卿,陳華標(biāo),張錦海;檢測(cè)霍亂弧菌的基因芯片的制備[J];中國(guó)公共衛(wèi)生;2004年04期

8 靳連群,李君文,王升啟,晁福寰,王新為;基因芯片技術(shù)檢測(cè)環(huán)境中常見致病菌的初步研究[J];中華微生物學(xué)和免疫學(xué)雜志;2003年01期

9 洪幫興,江麗芳,胡玉山,方丹云,郭輝玉;23S rRNA基因序列分析及其在細(xì)菌鑒別診斷中的應(yīng)用[J];中華微生物學(xué)和免疫學(xué)雜志;2004年03期

10 李君文 ,晁福寰 ,靳連群 ,王新為 ,鄭金來(lái) ,宋農(nóng) ,王升啟;基因芯片技術(shù)快速檢測(cè)水中常見致病菌[J];中華預(yù)防醫(yī)學(xué)雜志;2002年04期

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