采用分子生物学技术诊断隐球菌脑膜脑炎的研究

目的采用分子检测技术对疑似隐球菌感染的脑膜炎病例进行诊断。方法收集患者的脑脊液样本,提取DNA,设计引物进行PCR扩增,采用DNA芯片技术对扩增产物进行分子检测。结果显示样本新生隐球菌阳性。结论通过ITS保守序列设计引物进行PCR扩增和DNA芯片技术对常规真菌学检查不能确定的疑似隐球菌脑膜炎患者脑脊液样本进行非培养检测,具有实验室诊断参考价值。     

Detection of Hepatitis B Virus (HBV) DNA at femtomolar concentrations using a silica nanoparticle-enhanced microcantilever sensor

We report Hepatitis B Virus (HBV) DNA detection using a silica nanoparticle-enhanced dynamic microcantilever biosensor. A 243-mer nucleotide of HBV DNA precore/core region was used as the target DNA. For this assay, the capture probe on the microcantilever surface and the detection probe conjugated with silica nanoparticles were designed specifically for the target DNA. For efficient detection of the HBV target DNA using silica nanoparticle-enhanced DNA assay, the size of silica nanoparticles and the dimension of microcantilever were optimized by directly binding the silica nanoparticles through DNA hybridization. In addition, the correlation between the applied nanoparticle concentrations and the resonant frequency shifts of the microcantilever was discussed clearly to validate the quantitative relationship between mass loading and resonant frequency shift.HBV target DNAs of 23.1 fM to 2.31 nM which were obtained from the PCR product were detected using a silica nanoparticle-enhanced microcantilever. The HBV target DNA of 243-mer was detected up to the picomolar (pM) level without nanoparticle enhancement and up to the femtomolar (fM) level using a nanoparticle-based signal amplification process. In the above two cases, the resonant frequency shifts were found to be linearly correlated with the concentrations of HBV target DNAs. We believe that this linearity originated mainly from an increase in mass that resulted from binding between the probe DNA and HBV PCR product, and between HBV PCR product and silica nanoparticles for the signal enhancement, even though there is another potential factor such as the spring constant change that may have influenced on the resonant frequency of the microcantilever.     

DNA hybridization detection with organic thin film transistors: toward fast and disposable DNA microarray chips

We demonstrate a novel DNA hybridization detection method with organic thin film transistors. DNA molecules are immobilized directly on the surface of organic semiconductors, producing an unambiguous doping-induced threshold voltage shift upon hybridization. With these shifts, single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) are differentiated successfully. This method is expected to result in higher sensitivity than the main competitive technology, ISFET-based sensors because of the direct exposure of DNA molecules to sensitive layers. Factors that influence sensor sensitivity have been analyzed and optimum conditions have been determined using statistically designed experiments. Under the optimum conditions, the maximum difference between saturation current ratios caused by ssDNA and dsDNA reaches as high as 70%. In order to make DNA detection fast, we also demonstrate rapid on-chip electrically enhanced hybridization using the TFTs. These technologies together will enable the realization of disposable, rapid-turnaround tools for field-deployable genomic diagnosis.     

PLGA纳米/微球作为核酸载体的研究进展

生物可降解材料[poly(lactide-co-glycolide acid), PLGA]颗粒在持续释放和定位递送各种药剂包括核酸有很大的研究和应用价值。本文综述了PLGA作为核酸载体的制备及其用于基因载体和疫苗佐剂的研究。     

Optical detection of DNA hybridization based on fluorescence quenching of tagged oligonucleotide probes by gold nanoparticles

A novel system for the detection of DNA hybridization in a homogeneous format is developed. This method is based on fluorescence quenching by gold nanoparticles used as both nanoscaffolds for the immobilization of capture sequences and nanoquenchers of fluorophores attached to detection sequences. The oligonucleotide-functionalized gold nanoparticles are synthesized by derivatizing the colloidal gold solution with 5'-thiolated 12-base oligonucleotides. Introduction of sequence-specific target DNAs (24 bases) into the mixture containing dye-tagged detection sequences and oligonucleotide-functionalized gold nanoparticles results in the quenching of carboxytetramethylrhodamine-labeled DNA fluorescence because DNA hybridization occurs and brings fluorophores into close proximity with oligonucleotide-functionalized gold nanoparticles. The quenching efficiency of fluorescence increases with the target DNA concentration and provides a quantitative measurement of sequence-specific DNA in sample. A linearity is obtained within the range from 1.4 to 92 nM. The target sequence is detected down to 2 nM. This new system not only overcomes many of the drawbacks inherent in radioisotopic measurement or enzyme-linked assay but also avoids the requirement for the stem-loop structure compared with conventional molecular beacons. Furthermore, the background signal that is defined as fluorescence quenching arising from electrostatic attraction between positively charged fluorophores and negatively charged gold nanoparticles is comparatively low due to electrostatic repulsion between negatively charged oligonucleotides. In addition, this is a homogeneous assay that can offer the potential to be monitored in real time, be amenable to automation, eliminate washing steps, and reduce the risk of contamination.     

纳米粒子标记DNA 探针在电化学DNA 生物传感器中的应用

介绍了纳米电化学DNA生物传感器的基本概念和分类,并介绍了用于DNA标记的纳米粒子的六种类型及其三大检测方法,在此基础上对纳米电化学DNA生物传感器在基因检测、疾病诊断、DNA检测等方面的最新进展进行了综述与讨论。     

用DNA内在分子电荷来探测DNA

随着生命科学的发展,DNA的标记和检测已经成为关键的瓶颈技术。利用DNA内在的分子电荷,并结合半导体传感器技术,可以对DNA分子电荷状态进行快速检测,为基因诊断开辟全新的技术途径。     

Microwave-accelerated method for ultra-rapid extraction of Neisseria gonorrhoeae DNA for downstream detection

Nucleic acid-based detection of gonorrhea infections typically require a two-step process involving isolation of the nucleic acid, followed by detection of the genomic target often involving polymerase chain reaction (PCR)-based approaches. In an effort to improve on current detection approaches, we have developed a unique two-step microwave-accelerated approach for rapid extraction and detection of Neisseria gonorrhoeae (gonorrhea, GC) DNA. Our approach is based on the use of highly focused microwave radiation to rapidly lyse bacterial cells, release, and subsequently fragment microbial DNA. The DNA target is then detected by a process known as microwave-accelerated metal-enhanced fluorescence (MAMEF), an ultra-sensitive direct DNA detection analytical technique. In the current study, we show that highly focused microwaves at 2.45 GHz, using 12.3-mm gold film equilateral triangles, are able to rapidly lyse both bacteria cells and fragment DNA in a time- and microwave power-dependent manner. Detection of the extracted DNA can be performed by MAMEF, without the need for DNA amplification, in less than 10 min total time or by other PCR-based approaches. Collectively, the use of a microwave-accelerated method for the release and detection of DNA represents a significant step forward toward the development of a point-of-care (POC) platform for detection of gonorrhea infections.     

基于表面等离子体共振和电化学联用的DNA传感器研究进展

表面等离子共振(surface plasmon resonance,SPR)技术旨在检测物体表面附近折射率的变化,其特点是无标记、实时、灵敏和快速,该技术多用于研究分子的相互作用,包括动力学、效率常数和大分子构象变化等。电化学(electrochemical,EC)技术是一项用于定性定量研究电子转移、物质氧化还原、界面吸附等过程的成熟技术,具有简单、低成本和设备小型化的优点。现有的DNA杂交技术,例如光学、电化学或压电转导技术,主要关注于提高DNA杂交检测系统的选择性和灵敏度。传统的SPR在DNA分析方面,由于无法测量折射率的极小变化而在超灵敏检测中的应用受到限制。因此,随着纳米材料的研发和联用技术的飞速发展,SPR与EC联用的生物传感器研究越来越成为人们关注的热点。近年来,关于SPR和EC联用在DNA检测方面的综述鲜有报道。对SPR和EC检测DNA的技术原理、联用方法、应用进展等方面作出了简要的介绍,以期为表面等离子共振和电化学联用的DNA传感器相关研究提供参考。     

Gold nanoparticle-based detection of genomic DNA targets on microarrays using a novel optical detection system

The development of a nanoparticle-based detection methodology for sensitive and specific DNA-based diagnostic applications is described. The technology utilizes gold nanoparticles derivatized with thiol modified oligonucleotides that are designed to bind complementary DNA targets. A glass surface with arrays of immobilized oligonucleotide capture sequences is used to capture DNA targets, which are then detected via hybridization to the gold nanoparticle probes. Amplification with silver allows for detection and quantitation by measuring evanescent wave induced light scatter with low-cost optical detection systems. Compared to Cy3-based fluorescence, silver amplified gold nanoparticle probes provide for a approximately 1000-fold increase in sensitivity. Furthermore, direct detection of non-amplified genomic DNA from infectious agents is afforded through increased specificity and even identification of single nucleotide polymorphisms (SNP) in human genomic DNA appears feasible.     

Gold Nanoparticle Based FRET for DNA Detection

The nanoscience revolution that sprouted throughout the 1990s is having great impact in current and future DNA detection technology around the world. In this review, we report our recent progress on gold nanoparticle based fluorescence resonance energy transfer assay to monitor DNA hybridization as well as the cleavage of DNA by nucleases. We tried to discuss a reasonable account of the science and the important fundamental work carried out in this area. We also report the development of a compact, highly specific, inexpensive and user-friendly optical fiber laser-induced fluorescence sensor based on fluorescence quenching by nanoparticles to detect single-strand DNA hybridization at femtomolar level.     

Prospects of nanoparticle–DNA binding and its implications in medical biotechnology

Bio-nanotechnology is a new interdisciplinary R&D area that integrates engineering and physical science with biology through the development of multifunctional devices and systems, focusing biology inspired processes or their applications, in particular in medical biotechnology. DNA based nanotechnology, in many ways, has been one of the most intensively studied fields in recent years that involves the use and the creation of bio-inspired materials and their technologies for highly selective biosensing, nanoarchitecture engineering and nanoelectronics. Increasing researches have been offered to a fundamental understanding how the interactions between the nanoparticles and DNA molecules could alter DNA molecular structure and its biochemical activities. This minor review describes the mechanisms of the nanoparticle–DNA binding and molecular interactions. We present recent discoveries and research progresses how the nanoparticle–DNA binding could vary DNA molecular structure, DNA detection, and gene therapy. We report a few case studies associated with the application of the nanoparticle–DNA binding devices in medical detection and biotechnology. The potential impacts of the nanoparticles via DNA binding on toxicity of the microorganisms are briefly discussed. The nanoparticle–DNA interactions and their impact on molecular and microbial functionalities have only drown attention in recent a few years. The information presented in this review can provide useful references for further studies on biomedical science and technology.     

High performance DNA nano-carriers of carbonate apatite: multiple factors in regulation of particle synthesis and transfection efficiency

Increasing attention is being paid on synthetic DNA delivery systems considering some potential life-threatening effects of viral particles, for development of gene-based nanomedicine in the 21st century. In the current nonviral approaches, most of the efforts have been engaged with organic macromolecules like lipids, polymers, and peptides, but comparatively fewer attempts were made to evaluate the potential of inorganic materials for gene delivery. We recently reported that biodegradable nanoparticles of carbonate apatite are highly efficient in transfecting a wide variety of mammalian cells. Here we show that a number of parameters actively regulate synthesis of the nanoparticles and their subsequent transfection efficacy. Development of "supersaturation", which is the prerequisite for generation of such particles, could be easily modulated by reactant concentrations, pH of the buffered solution, and incubation temperatures, enabling us to establish a flexible particle generation process for highly productive trans-gene delivery. Carbonate incorporation into the particles have been proposed for generating nano-size particles resulting in cellular uptake of huge amount of plasmid DNA as well as endosome destabilization facilitating significant release of DNA from the endosomes.     

DNA point mutation detection based on DNA ligase reaction and nano-Au amplification: a piezoelectric approach

A novel piezoelectric method for DNA point mutation detection based on DNA ligase reaction and nano-Au-amplified DNA probes is proposed. A capture probe was designed with the potential point mutation site located at the 3' end and a thiol group at the 5' end to be immobilized on the gold electrode surface of quartz crystal microbalance (QCM). Successive hybridization with the target DNA and detection probe of nano-Au-labeled DNA forms a double-strand DNA (dsDNA). After the DNA ligase reaction and denaturing at an elevated temperature, the QCM frequency would revert to the original value for the target with single-base mismatch, whereas a reduced frequency response would be obtained for the case of the perfect match target. In this way, the purpose of point mutation discrimination could be achieved. The current approach is demonstrated with the identification of a single-base mutation in artificial codon CD17 of the beta-thalassemia gene, and the wild type and mutant type were discriminated successfully. The scanning electron microscope (SEM) image showing that plenty of gold nanoparticles remained on the electrode surface demonstrated that the nano-Au label served as an efficient signal amplification agent in QCM assay. A detection limit of 2.6 x 10(-9)mol/L of oligonucleotides was achieved. Owing to its ease of operation and low detection limit, it is expected that the proposed procedure may hold great promise in both research-based and clinical genomic assays.     

血浆游离DNA全基因组甲基化测序的实用稳定性评估

随着液体活检技术的发展,血浆游离DNA成为当前的研究热点之一。血浆游离DNA的全基因组甲基化测序被认为在癌症检测等医学应用拥有巨大潜力,但目前尚缺乏针对该实验流程的实用稳定性评估。文中利用两名志愿者在不同时间采样的血浆游离DNA,在不同实验平台分别进行DNA甲基化的重亚硫酸盐转化前建库(Pre-BS)、转化后建库(Post-BS)和常规DNA建库,获取多因素影响下的测序数据样本。在此基础上,建立了一套血浆游离DNA测序数据分析的质量控制参考流程,综合评估了血液采集提取、游离DNA建库测序过程的实用稳定性,为血浆游离DNA全基因组甲基化测序应用于临床液体活检提供实用性的基础参考。     

A comparison of methods used to extract bacterial DNA from raw milk and raw milk cheese

Aims: In this study, we compare seven different methods which have been designed or modified to extract total DNA from raw milk and raw milk cheese with a view to its subsequent use for the PCR of bacterial DNA. Materials and Results: Seven extraction methods were employed to extract total DNA from these foods, and their relative success with respect to the yield and purity of the DNA isolated, and its quality as a template for downstream PCR, was compared. Although all of the methods were successful with respect to the extraction of DNA naturally present in cheese, they varied in their relative ability to extract DNA from milk. However, when milk was spiked with a representative Gram‐positive (Listeria monocytogenes EGDe) or Gram‐negative (Salmonella enterica serovar Typhimurium LT2) bacterium, it was established that all methods successfully extracted DNA which was suitable for subsequent detection by PCR. Conclusions: Of the seven approaches, the PowerFood? Microbial DNA Isolation kit (MoBio Laboratories Inc.) was found to most consistently extract highly concentrated and pure DNA with a view to its subsequent use for PCR‐based amplification and also facilitated accurate detection by real‐time quantitative PCR. Significance and Impact of the Study: Accurately assessing the bacterial composition of milk and cheese is of great importance to the dairy industry. Increasingly, DNA‐based technologies are being employed to provide an accurate assessment of this microbiota. However, these approaches are dependent on our ability to extract DNA of sufficient yield and purity. This study compares a number of different options and highlights the relative success of these approaches. We also highlight the success of one method to extract DNA from different microbial populations as well as DNA which is suitable for real‐time PCR of microbes of interest, a challenge often encountered by the food industry.     

A universal platform for sensitive and selective colorimetric DNA detection based on Exo III assisted signal amplification

Rapid growth of available sequence data has made the detection of nucleic acids critical to the development of modern life sciences. Many amplification methods based on gold nanoparticles and endonuclease for sensitive DNA detection have been developed. However, these approaches require specific target sequence for endonuclease recognition, which cannot be fulfilled in all systems. Replacing the restriction enzyme with a nuclease that does not require any specific recognition sequence may offer a universally adaptable system. Here we have developed a novel homogeneous, colorimetric DNA detection method, which consists of Exo III, a linker DNA, and two DNA-modified gold nanoparticles. This system is simple, low-cost, sensitive and selective. By coupling cyclic enzymatic cleavage and gold nanoparticle for signal amplification, our system provides a colorimetric detection limit of 15 pM, which is 3 orders of magnitude more sensitive than that of a general three-component sandwich assay format. Due to the intrinsic property of Exo III, our method shows excellent detection selectivity for single-base discrimination. More importantly, superior to other methods based on nicking and FokI endonuclease, our target sequence-independent platform is generally applicable for DNA sensing. This new approach could be widely applied to sensitive nucleic acids detection.     

非标记表面增强拉曼光谱在DNA检测中的应用

表面增强拉曼光谱(SERS)是一种超灵敏的生化分析技术,已经被广泛运用于细胞、核酸、蛋白质等生物分子的检测,在生物医学领域表现出了巨大的应用潜力。近年来,将表面增强拉曼光谱技术应用于遗传物质DNA的精准检测,引起了人们广泛的关注。本文简要叙述了表面增强拉曼光谱技术的基本原理及其在DNA检测中的优势,主要介绍了非标记的DNA-SERS检测应用进展,其中包括本项目组的相关工作。研究表明,非标记DNA-SERS技术有望成为一种快速、准确的临床诊断方式。     

DNA sequencing research group: 2006 general survey of DNA sequencing facilities.

Over the past few years, technological advances in automated DNA sequencing have had a profound effect on the nature of DNA sequencing laboratories. To characterize the changes occurring within DNA sequencing facilities, the DNA Sequencing Research Group conducted three previous studies, in 1998, 2000, and 2003. A new general survey has been designed and conducted by the DSRG to capture the current status of DNA sequencing facilities in all sectors. Included were questions regarding facility administration, pricing, instrumentation, technology, protocols, and operation. The results of the survey are presented here, accompanied by comparisons to the previous surveys. These comparisons formed a basis for the discussion of trends within the facilities in response to the dynamics of a changing technology.