【作者】 鲁卫平；

【导师】 涂植光；

【作者基本信息】 重庆医科大学 ， 临床检验诊断学， 2009， 博士

【摘要】 随着分子生物学和生物技术的发展,DNA分子识别技术不断提高。相比传统的以微生物表型特征为基础的检测方法,基于DNA识别技术的分子生物学检测方法,具有快速、敏感性和特异性高的优点,非常适用于病原微生物的临床检测和鉴定。同时随着越来越多的动植物、微生物基因组序列得到测定,这些巨量的信息为我们开启了进行大量生物学分析和检测的大门。DNA生物传感器是集分子生物学、现代物理、现代化学、微电子技术于一体的新型基因分析技术,具有快速、敏感、高效、高通量等优点,在生物工程、临床医学、环境监测、食品分析等领域具有重要的意义。目前已有包括光学传感器、石英晶体传感器、电化学传感器等众多类型的DNA传感器,根据是否选用标记物,这些传感器可分为标记型和非标记型两大类。标记型DNA传感器对DNA的识别,是通过检测DNA上标记的信号分子所引起的光学、电化学等信号的改变。非标记型DNA传感器则直接检测DNA杂交形成复合体过程中引起的光学、质量以及电化学的改变。本研究主要分两部分,第一部分我们将真菌核糖体分型技术、DNA生物传感器技术和纳米金信号放大技术相结合,以纳米金为信号分子,建立了一种适合临床实验室应用的病原性真菌检测DNA传感器检测系统。第二部分我们将表面等离子体共振(surface plasmonresonance,SPR)传感器技术与肽核酸探针技术相结合,对无信号分子的DNA的直接、简便、快速检测方法进行了初步探索。实验的主要方法及结果如下:第一部分基于纳米金的标记型生物传感器同时检测多个病原性真菌1.通过查阅文献报道并进行临床调查,确定以20余种临床常见的病原性真菌为检测目标,包括念珠菌、曲霉菌、毛霉菌、皮肤癣菌等。2.通过临床常见病原性真菌,包括酵母菌、浅部真菌和深部真菌共14个属30种真菌的核糖体RNA(ribosomal RNA,rRNA)基因序列的比对,以真菌5.8S rRNA基因和28S rRNA基因的保守序列,设计真菌通用引物,可扩增真菌5.8S rDNA部分序列、ITS 2(内转录间隔区2).全序列、28S rDNA的部分序列。利用该通用引物建立了真菌rRNA基因通用PCR扩增方法并进行了优化。该通用PCR方法对20种靶真菌均可扩增出目的DNA带,大小在259～531 bp之间。所有非真菌样品扩增结果均为阴性。3.为简化PCR方法和减少标本用量,利用基因释放剂直接制备病原性真菌DNA模板。该方法操作非常快速、简单,整个模板制备过程仅需30min左右,而传统方法通常都需要2h以上。4.在相同条件下利用AlleleID 6.0软件设计了针对各靶真菌ITS2序列的种特异性寡核苷酸探针,利用生物信息学方法对所有探针进行分析筛选,并利用反向膜杂交技术进行验证。结果表明所选用探针特异性强、可靠性好,且探针间Tm值相差仅1℃,具有相同的杂交条件。5.建立并优化了纳米金显色目视DNA芯片检测系统,包括:最佳点样探针浓度;最适杂交温度、杂交时间;最佳银染显色方法;自身质量控制系统。该技术具有很好的特异性,对热带念珠菌扩增产物和白色念珠菌的检测敏感性分别达到50fmol/L和10cfu/ml,且检测结果可以用肉眼直接观察或普通光学仪器记录。6.为验证该检测技术用于临床样品检测的可靠性和实用性。用该检测系统对临床样品直接进行检测,分别检测了真菌感染标本的阳性培养物、加入标准菌株制备的模拟临床标本以及含菌的临床标本。本法的检测结果与常规经典检测鉴定方法结果一致,证明本检测方法准确可靠,可用于临床样品的检测。以上结果表明:我们建立的通用PCR结合纳米金DNA传感器技术,具有敏感、特异、操作简单快速的优点。整个检测过程可在6h内完成,而传统方法需要2～4天。与传统的基因芯片技术相比,其技术和设备要求、检测成本大大降低。能部分满足临床检测的通量要求,适合在临床开展,有望成为临床微生物实验室传统真菌检测技术理想的替代方法。第二部分基于SPR和bis-PNA的非标记型生物传感器1.采用一种全新的二维SPR折射率成像方法—“并行扫描光谱表面等离子体共振成像方法”。这项技术具有SPR传感器一贯的高折射率灵敏度的特点,且能提供定量的被测平面的二维折射率分布图,其中每点的亮度值与折射率直接线性对应;本方法采用线形光束照明探测,一次探测一维区域,扫描探测二维平面区域,探测速度快,具有很高的信息通量。2.针对流感病毒M基因,设计合成了bis-PNA探针。其中Hoogsteen链的C碱基用J(pseudoisocytosine)碱基检测替换,消除了C碱基在杂交过程中需要质子化的影响,使探针在中性杂交环境中具有较好的稳定性,既保证其高结合率又有较好的特异性。3.将巯基修饰的bis-PNA探针固定在金膜上,制成微阵列,直接与甲型流感病毒M基因扩增产物杂交,然后进行SPR扫描分析。该技术检测敏感性为1pmol/L的双链DNA分子。结合并行扫描光谱表面等离子体共振传感器系统和bis-PNA技术,我们构建了一种无需任何标记的生物传感器技术,并进行了初步验证。结果表明该方法可直接检测双链DNA,无需对其进行变性处理。具有操作简单快速、检测通量高的优点。但目前离实际应用还有一段距离,在探针的设计、杂交条件、检测系统的稳定性等方面还需进一步优化提高。更多还原

【Abstract】 The DNA recognition technology is continuously improved with the development of molecular biology and biotechnology.Compared to traditional detection methods based on phenotypic characteristics of the pathogenic microorganisms,the molecular diagnostic techniques based on DNA recognition technology are the preferable choice for the detection and identification of the pathogenic microorganism since they can offer the high speed,sensitivity and specificity.The information obtained from genome sequencing projects has opened the door to tremendous biological analysis and detection.DNA sensor technology integrates molecular biology with physics,chemistry and micro-electronic technology,has advantages of high speed,sensitivity and specificity,and high throughput. It can be used in the fields of biological engineering,clinical medicine, environmental monitoring,foodstuff analysis,and so on.According to label or not,the DNA biosensors can be divided into two categories,one is label-based DNA biosensor and another is label-free biosensor.The label-based DNA biosensor is based on changes of optical and electrochemical signal caused by signaling molecule.The label-free biosensor is based on changes of optical,mass or electrochemical signal caused by hybridization of nucleic acid.The dissertation includes two parts.In partⅠ,employing the ribosomal gene-typing technology,DNA biosensor technology and nano-amplification technique,a label-based DNA biosensor technique for simultaneously detection and identification clinical common pathogenic fungi was developed.Due to the high efficiency,low cost,good specificity and sensitivity,this technique offers a reliable alternative to conventional methods for the detection and identification of clinical-important fungal pathogens.In partⅡ,we explore a label-free biosensor based on surface plasmon resonance(SPR) and dimeric peptide nucleic acid(bis-PNA) as a platform for detection of indicator free DNA.The main results were as follows:PartⅠ:A DNA biosensor based on nanogold for simultaneously detection and identification clinical common pathogenic fungi.1.In accordance with the literature report and the clinical investigation,20 kinds of clinical important pathogenic fungi were selected as the target pathogen in this study,including Candida, Aspergillus,mucoraceae,dermatophytes,and so on.2.Based on the multi-alignment of the ribosome RNA(rRNA) gene sequences among 30 species of pathogenic fungi belonging to 14 genera,a pair of universal primers were designed for the amplification of the ITS2 regions of rRNA.The forward primer is complementary to the conserved sequence of 5.8S rRNA,and the reverse primer is complementary to the conserved sequence of 28S rDNA.A universal PCR amplification method was established and optimized to common pathogenic fungal strains. Using this PCR amplification method,PCR products of 20 target fungi showed target band of 259-531 bp respectively,and all non-fungal samples were no PCR products.3.By using GeneReleaser,the procedure of the preparation of DNA templates is much more simple and faster(0.5h) than that of conventional methods(＞2h).4.The species-specific oligonucleotide probes targeting ITS2 sequences were designed using AlleleID 6.0,analyzed and screened by bioinformatical technology,then verified by a universal PCR combined reverse blot hybridization.It turned out that the selected probes were all of high specificity and reliability,and had the same hybridization conditions.5.A DNA biosensor technique integrating nano-gold labeling,silver stain enhancement and the microarray technique was developed and optimized,including the optimal spotting concentrations of probes,the optimal hybridization temperature and time,the optimal procedure of silver stain enhancement and perfect self quality control system.The results showed that the method had ideal specificity and its detection sensitivity reached 50fmol/L for amplicon of Candida tropicalis and 10 cells/ml for Candida albicans.The hybridization signal can be observed visually or recorded by optic scanner.6.The method was further verified by fugus cultures from clinical specimens,mimic clinical samples spiked with standard fungal strains and clinical infectious samples.It was showed that the results of the DNA biosensor were completely consistent with that of traditional techniques, and our method was feasible for detection of clinical samples obtained from patients suffering from fungal pathogens.Therefore,the experiments mentioned above have proved that established nanogold-amplification technique-based DNA biosensor combining with universal PCR provides a simple,rapid,sensitive and specific method for the identification of fungal pathogens.The analysis is simple to perform and can be completed in 6 hours,compared with 2～4 days for conventional methods.Compared with the classical DNA biosensor,our method do not requires the expensive labeling material and complex detection equipment,so the efficacy/cost is greatly increased.It can meet the high throughput demand of clinical detection,and promise to be a alternative to conventional methods for the detection and identification of fungal pathogens in the clinical laboratories.PartⅡA label-based DNA biosensor based on SPR and bis-PNA 1.In this study,a parallel scan SPR imaging technique was adopted into SPR biosensor.This technique has the advamage of high sensitivity as the other SPR technique.The technique can offer two-dimensional quantificational distribution map of reflective index(RI) on the sensing plane.The pixels on the map have brightness linearly correspond to their RI.Another advantage of this technique is that the parallel scan method makes it a fast imaging,high-throughput analyzing technique.2.A specific bis-PNA probe was designed for M gene of influenza virus.The cytosine bases in N-terminal half of bis-PNA are replaced by pseudoisocytosine bases(J base),which do not require protonation to form triplexes,so it has high affinity to dsDNA and high specificity at neutral pH.3.The thiol-modified bis-PNA probe was immobilized to the surface of gold membrane by Au-SH bond.The dsDNA could hybridize with bis-PNA without denaturation.The detection sensitivity of the method reached 1pmol/L.Combining with SPR senseor based on parallel scan SPR imaging technique and bis-PNA probe,a label-free DNA biosensor was built and the preliminary verification tests were conducted.According to the result, the target dsDNA can hybridize with bis-PNA and be directly detected without any label and denaturation.The biosensor technique provides a simple,rapid,and high throughput means for the detection of nucleic acid. However,for application in clinical diagnosis,some conditions of this technique should be further optimized and improved,such as the probe design,hybridization conditions,the stability of detection systems,etc.更多还原