基因芯片技术在检测肠道致病菌方面的应用

基因芯片技术具有高通量、自动化、快速检测等特点,因此被广泛地应用于各种研究领域,如细菌分子流行病学、细菌基因鉴定、致病分子机理、基因突变及多态性分析、表达谱分析、DNA测序和药物筛选等。现介绍基因芯片检测肠道致病菌方面的国外研究进展,基因芯片应用于检测肠道致病菌的3个方面:结合多重PCR对致病菌的毒力因子或者特异性基因进行鉴定;直接检测细菌的DNA或者RNA;以致病细菌核糖体RNA作为检测的靶基因同时检测多种肠道致病菌。由于其检测的高效率,该技术要优于其他分子生物学检测方法。基因芯片技术在肠道致病菌检测中有着巨大的应用价值,具有广阔的应用前景。     

Separation procedures capable of revealing DNA adducts

Detection and quantification of DNA adducts are very important in relation to diseases such as cancer. Both high sensitivity and high selectivity are required for the detection of DNA adducts because the content of adducts in DNA is very small compared with those of normal bases and only small amounts of DNA samples are available for analysis in general cases. In this paper are described separation procedures such as liquid chromatography, gas chromatography and capillary electrophoresis combined with a detection and identification method such as 32P-postlabeling, mass spectrometry, electrochemical detection, fluorescence detection and immunoassay. The merits and demerits of the procedures are also discussed.     

Identification of procaryotic repetitive DNA suitable for use as fingerprinting probes.

We have developed a method which enables the cloning and identification of procaryotic repetitive DNA suitable for use as DNA fingerprinting probes. The method involves shotgun cloning of restricted genomic DNA with subsequent selection of clones containing repetitive DNA by reverse-probed genomic hybridizations, in which the plasmid DNA clones are probed with labelled genomic DNA. Confirmation that the clones contained repeated sequences was by Southern hybridization, gene copy equivalence, and DNA sequencing. The sequences were used for highly specific and sensitive detection of bacteria and as target sequences for the mediation of chromosomal integration of reporter gene constructs.     

PCR-Free Detection of Genetically Modified Organisms Using Magnetic Capture Technology and Fluorescence Cross-Correlation Spectroscopy

The safety of genetically modified organisms (GMOs) has attracted much attention recently. Polymerase chain reaction (PCR) amplification is a common method used in the identification of GMOs. However, a major disadvantage of PCR is the potential amplification of non-target DNA, causing false-positive identification. Thus, there remains a need for a simple, reliable and ultrasensitive method to identify and quantify GMO in crops. This report is to introduce a magnetic bead-based PCR-free method for rapid detection of GMOs using dual-color fluorescence cross-correlation spectroscopy (FCCS). The cauliflower mosaic virus 35S (CaMV35S) promoter commonly used in transgenic products was targeted. CaMV35S target was captured by a biotin-labeled nucleic acid probe and then purified using streptavidin-coated magnetic beads through biotin-streptavidin linkage. The purified target DNA fragment was hybridized with two nucleic acid probes labeled respectively by Rhodamine Green and Cy5 dyes. Finally, FCCS was used to detect and quantify the target DNA fragment through simultaneously detecting the fluorescence emissions from the two dyes. In our study, GMOs in genetically engineered soybeans and tomatoes were detected, using the magnetic bead-based PCR-free FCCS method. A detection limit of 50 pM GMOs target was achieved and PCR-free detection of GMOs from 5 µg genomic DNA with magnetic capture technology was accomplished. Also, the accuracy of GMO determination by the FCCS method is verified by spectrophotometry at 260 nm using PCR amplified target DNA fragment from GM tomato. The new method is rapid and effective as demonstrated in our experiments and can be easily extended to high-throughput and automatic screening format. We believe that the new magnetic bead-assisted FCCS detection technique will be a useful tool for PCR-free GMOs identification and other specific nucleic acids.     

Rapid, one-step DNA extraction for insect pest identification by using DNA barcodes

Early detection of economically important insects is critical to preventing their establishment as serious pests. To accomplish this, tools for rapid and accurate species identification are needed. DNA barcoding, using short DNA sequences as species "genetic identification tags," has already shown large potential as a tool for rapid and accurate detection of economically important insects. DNA extraction is the critical first step in generating DNA barcodes and can be a rate-limiting step in very large barcoding studies. Consequently, a DNA extraction method that is rapid, easy to use, cost-effective, robust enough to cope with range of qualities and quantities of tissue, and can be adapted to robotic systems will provide the best method for high-throughput production of DNA barcodes. We tested the performance of a new commercial kit (prepGEM), which uses a novel, streamlined approach to DNA extraction, and we compared it with two other commercial kits (ChargeSwitch and Aquapure), which differ in their method of DNA extraction. We compared performance of these kits by measuring percentage of polymerase chain reaction (PCR) success and mean PCR product yield across a variety of arthropod taxa, whichincluded freshly collected, ethanol-preserved, and dried specimens of different ages. ChargeSwitch and prepGEM performed equally well, but they outperformed Aquapure. prepGEM was much faster, easier to use, and cheaper than ChargeSwitch, but ChargeSwitch performed slightly better for older (> 5-yr-old) dried insect specimens. Overall, prepGEM may provide a highly streamlined method of DNA extraction for fresh, ethanol-preserved, and young, dried specimens, especially when adapted for high-throughput, robotic systems.     

Microarray detection of food-borne pathogens using specific probes prepared by comparative genomics

In order to design a method for the accurate detection and identification of food-borne pathogens, we used comparative genomics to select 70-mer oligonucleotide probes specific for 11 major food-borne pathogens (10 overlapping probes per pathogen) for use in microarray analysis. We analyzed the hybridization pattern of this constructed microarray with the Cy3-labeled genomic DNA of various food-borne pathogens and other bacteria. Our microarray showed a highly specific hybridization pattern with the genomic DNA of each food-borne pathogen; little unexpected cross-hybridization was observed. Microarray data were analyzed and clustered using the GenePix Pro 6.0 and GeneSpring GX 7.3.1 programs. The analyzed dendrogram revealed the discriminating power of constructed microarray. Each food-borne pathogen clustered according to its hybridization specificity and non-pathogenic species were discriminated from pathogenic species. Our method can be applied to the rapid and accurate detection and identification of food-borne pathogens in the food industry. In addition, this study demonstrates that genome sequence comparison and DNA microarray analysis have a powerful application in epidemiologic and taxonomic studies, as well as in the food safety and biodefense fields.     

Rapid DNA chemical ligation for amplification of RNA and DNA signal

Enzymatic ligation methods are useful in the diagnostic detection of DNA sequences. Here, we describe the investigation of nonenzymatic phosphorothioate--iodoacetyl DNA chemical ligation as a method for the detection and identification of RNA and DNA. The specificity of ligation on the DNA target is shown to allow the discrimination of a single point mutation with a drop in the ligation yield of up to 16.1-fold. Although enzymatic ligation has very low activity for RNA targets, this reaction is very efficient for RNA targets. The speed of the chemical ligation with an RNA target achieves a 70% yield in 5 s, which is equal to or better than that of ligase-enzyme-mediated ligation with a DNA target. The reaction also exhibits a significant level of signal amplification under thermal cycling in periods as short as 100-120 min, with the RNA or DNA target acting in a catalytic way to ligate multiple pairs of probes.     

Nonenzymatic autoligation in direct three-color detection of RNA and DNA point mutations

Enzymatic ligation methods are useful in diagnostic detection of DNA sequences. Here we describe the investigation of nonenzymatic phosphorothioate-iodide DNA autoligation chemistry as a method for detection and identification of both RNA and DNA sequences. Combining ligation specificity with the hybridization specificity of the ligated product is shown to yield discrimination of a point mutation as high as >10(4)-fold. Unlike enzymatic ligations, this reaction is found to be equally efficient on RNA or DNA templates. The reaction is also shown to exhibit a significant level of self-amplification, with the template acting in catalytic fashion to ligate multiple pairs of probes. A strategy for fluorescence labeling of three autoligating energy transfer (ALET) probes and directly competing them for autoligation on a target sequence is described. The method is tested in several formats, including solution phase, gel, and blot assays. The ALET probe design offers direct RNA detection, combining high sequence specificity with an easily detectable color change by fluorescence resonance energy transfer (FRET).     

基于特异性PCR和荧光检测技术快速鉴定艾纳香

为建立快速准确的艾纳香分子鉴定方法。采取筛选艾纳香及其混伪品基因组DNA的提取方法,针对艾纳香特异性位点设计引物,优化PCR扩增条件,荧光检测扩增产物。结果表明碱裂解法更适于艾纳香基因组DNA的提取;叶绿体基因（tDNA）特异引物能特异性扩增艾纳香DNA,其扩增产物荧光检测呈绿色,混伪品无反应发生。试验结果显示该法简化了分子鉴定过程,省时节力,且结果准确可靠,可作为艾纳香植物和药材的鉴定方法。     

Detection and identification of minor nucleotides in intact deoxyribonucleic acids by mass spectrometry.

A mass spectral method is described for the detection and identification of unusual nucleotide residues present in DNAs. Analysis by this method of intact, underivatized DNA from salmon sperm, calf thymus, mouse L-cells, wheat germ, M. lysodeikticus, E. Coli, and the bacteriophages 0X-174, fd, and lamda, yields diagnostic ions for the four common components of DNA as well as characteristic ions for 5-methyldeoxycytidine residues. The spectrum from T2 DNA contains ions indicative of 5-hydroxymethyldeoxycytidine and 5-methyldoxycytidine components but no ions corresponding to deoxycytidine residues. The DNAs of phages fd and 0X-174 also display ion products indicative of N6-methyldeoxyadenosine residues. Additional series of ions in the spectra of all four bacteriophage DNAs suggest the presence of 5-substituted deoxyuridine residues. The detection method exhibits considerable sensitivity in that amounts of DNA as low as 0.01 A260nm units can be used in the analysis, and thus, the procedure should prove of some value in the detection and location of modified components in specific regions of the various genomes by analysis of the appropriate endonuclease restriction fragments.     

DNA microarray to detect and identify trichothecene- and moniliformin-producing Fusarium species

AIMS: To develop a DNA microarray for easy and fast detection of trichothecene- and moniliformin-producing Fusarium species. METHOD AND RESULTS: A DNA microarray was developed for detection and identification of 14 trichothecene- and moniliformin-producing species of the fungal genus Fusarium. The array could also differentiate between four species groups. Capture probes were designed based on recent phylogenetic analyses of translation elongation factor-1 alpha (TEF-1alpha) sequences. Particular emphasis was put on designing capture probes corresponding to groups or species with particular mycotoxigenic synthetic abilities. A consensus PCR amplification of a part of the TEF-1alpha is followed by hybridization to the Fusarium chip and the results are visualized by a colorimetric Silverquant detection method. We validated the Fusarium chip against five naturally infected cereal samples for which we also have morphological and chemical data. The limit of detection was estimated to be less than 16 copies of genomic DNA in spiked commercial wheat flour. CONCLUSIONS: The current Fusarium chip proved to be a highly sensitive and fast microarray for detection and identification of Fusarium species. We postulate that the method also has potential for (semi-)quantification. SIGNIFICANCE AND IMPACT OF THE STUDY: The Fusarium chip may prove to be a very valuable tool for screening of cereal samples in the food and feed production chain, and may facilitate detection of new or introduced Fusarium spp.     

混合DNA样品池扩增法及其应用

将个体 DNA提取出来后 ,按一定方式进行混合 ,构成混合 DNA样品池。这种混合 DNA样品可用于病因未明的遗传性及遗传易感性疾病的研究。在研究常染色体隐性遗传性耳聋致病基因时 ,发现与染色体 9q的 D9S92 2和 D9S30 1位点有相关性。此方法比通常的连锁分析法省时省力。在肿瘤相关基因或责任基因的研究、法医学的个体认定、基因突变的检测等方面均显示出实用性 ,值得推广     

微囊藻毒素合成酶基因的PCR检测方法

针对微囊藻毒素合成酶基因簇的核酸序列,筛选特异性引物,探索一种适用于自然水样中微囊藻产毒潜能检测的全细胞PCR方法。经灵敏度测试表明,这种PCR方法的检测下限相当于100cells。该方法不需要提取基因组DNA,检测所需水样量少,具有操作简便、快速、成本低、灵敏度高等优点,能应用于水库等饮用水源水体中具有产毒潜能的微囊藻的检测。     

基因芯片技术检测3种食源性致病微生物方法的建立

建立一种运用多重PCR和基因芯片技术检测和鉴定志贺氏菌、沙门氏菌、大肠杆菌O157的方法, 为3种食源性致病菌的快速检测和鉴定提供了准确、快速、灵敏的方法。分别选取编码志贺氏菌侵袭性质粒抗原H基因(ipaH)、沙门氏菌肠毒素(stn)基因和致泻性大肠杆菌O157志贺样毒素(slt)基因设计引物和探针, 进行三重PCR扩增, 产物与含特异性探针的芯片杂交。对7种细菌共26株菌进行芯片检测, 仅3种菌得到阳性扩增结果, 证明此方法具有很高的特异性。3种致病菌基因组DNA和细菌纯培养物的检测灵敏度约为8 pg。对模拟食品样品进行直接检测, 结果与常规细菌学培养结果一致, 检测限为50 CFU/mL。结果表明：所建立的基因芯片检测方法特异性好, 灵敏度高, 为食源性致病菌的检测提供了理想手段, 有良好的应用前景。     

Quaternionic periodicity transform: an algebraic solution to the tandem repeat detection problem

MOTIVATION: One of the main tasks of DNA sequence analysis is identification of repetitive patterns. DNA symbol repetitions play a key role in a number of applications, including prediction of gene and exon locations, identification of diseases, reconstruction of human evolutionary history and DNA forensics. RESULTS: A new approach towards identification of tandem repeats in DNA sequences is proposed. The approach is a refinement of previously considered method, based on the complex periodicity transform. The refinement is obtained, among others, by mapping of DNA symbols to pure quaternions. This mapping results in an enhanced, symbol-balanced sensitivity of the transform to DNA patterns, and an unambiguous threshold selection criterion. Computational efficiency of the transform is further improved, and coupling of the computation with the period value is removed, thereby facilitating parallel implementation of the algorithm. Additionally, a post-processing stage is inserted into the algorithm, enabling unambiguous display of results in a convenient graphical format. Comparison of the quaternionic periodicity transform with two well-known pattern detection techniques shows that the new approach is competitive with these two techniques in detection of exact and approximate repeats.     

Biotechnology offers revolution to fish health management

Biotechnology has many applications in fish health management. The application of monoclonal antibodies (mAbs) provides a rapid means of pathogen identification; antibodies to immunoglobulins from different fish species can be used to monitor the host response following vaccination; and mAbs also have the potential for screening broodstock for previous exposure to pathogens. Luminex technology exemplifies a novel antibody-based method that can be applied to both pathogen detection and vaccine development. Molecular technologies, such as the polymerase chain reaction (PCR), real time PCR and nucleic acid sequence-based amplification (NASBA), have enabled detection, identification and quantification of extremely low levels of aquatic pathogens, and microarray technologies offer a new dimension to multiplex screening for pathogens and host response. Recombinant DNA technology permits large-scale, low-cost vaccine production, moreover DNA vaccination, proteomics, adjuvant design and oral vaccine delivery will undoubtedly foster the development of effective fish vaccines in the future.     

QCM detection of DNA targets with single-base mutation based on DNA ligase reaction and biocatalyzed deposition amplification

A novel biosensing technique for highly specific identification of gene with single-base mutation is proposed based on the implementation of the DNA ligase reaction and the biocatalyzed deposition of an insoluble product. The target gene mediated deposition of an insoluble precipitate is then transduced by quartz crystal microbalance (QCM) measurements. In this method, the DNA target hybridizes with a capture DNA probe tethered onto the gold electrode and then with a biotinylated allele-specific detection DNA. A ligase reaction is performed to generate the ligation between the capture and the detection probes, provided there is perfect match between the DNA target and the detection probe. Otherwise even when there is an allele mismatch between them, no ligation would take place. After thermal treatment at an elevated temperature, the formed duplex melts apart that merely allows the detection probe perfectly matched with the target to remain on the electrode surface. The presence of the biotinylated allele-matched probe is then detected by the QCM via the binding to streptavidin-peroxide horseradish (SA-HRP), which catalyzes the oxidative precipitation of 3,3-diaminobenzidine (DAB) by H2O2 on the electrode and provides an amplified frequency response. The proposed approach has been successfully implemented for the identification of single-base mutation in -28 site of the beta-thalassemia gene with a detection limit of 0.1 nM, demonstrating that this method provides a highly specific and cost-efficient approach for point mutation detection.     

A rapid PCR-based method for identification of four important Candida species

The rapid detection and identification of Candida species in clinical laboratories are extremely important for the management of patients with hematogenous candidosis. Currently available culture and biochemical methods for detection and identification of Candida species are time-consuming. This study describes the use of a simple and rapid PCR method using species-specific oligonucleotides for the detection of clinical isolates of Candida species. These species-specific oligonucleotides are complementary to unique sequences within the intergenic transcribed spacer 2, located in between the 5.8S and 28S ribosomal DNA, and generated DNA fragments by both the conventional and hemi-nested PCR reactions. Conventional PCR produced a single DNA fragment of variable size in all isolates, while the hemi-nested PCR produced two discrete DNA fragments, both with the expected sizes of 111bp/57bp (C. albicans), 84bp/42bp (C. glabrata), 94bp/45bp (C. krusei) and 95bp/49bp (C. parapsilosis). In conclusion, the PCR-based method described in this study is fast and specific for the identification of clinically important Candida species.     

A simple and rapid technique for identification of large numbers of individual mosquitoes using DNA hybridization

A general method for obtaining species-specific repetitive DNA sequences is described. The method is based on the detection of recombinant DNA clones containing repetitive sequences using labeled total genomic DNA. These repetitive DNA sequences can be used to identify individual mosquito adults, pupae, and larvae squashed on filter membranes (squash blots). This technique was used to distinguish individuals of the four sibling species of the Anopheles quadrimaculatus complex. Repetitive DNA sequences and squash blots can be of use for rapid identification of other insect species in field collections.