基因芯片及其在性传播疾病病原体研究中的应用

基因芯片是近年发展起来的一种高通量的核酸分析技术,已成为“后基因组时代”的重要分析工具之一。本简述了基因芯片的概念、分类及特点,并对基因芯片技术在性传播疾病病原体淋球菌、沙眼衣原体、解脲脲原体和人乳头瘤病毒研究中的应用作了综述。     

单核苷酸多态性检测方法的研究进展

单核苷酸多态性（single nucleotide polymorphism, SNP）的研究已成为人类后基因组时代的主要内容之一。因此建立高度自动化和高通量的SNP检测分析技术十分重要。文章系统地介绍了最新发展的几种SNP检测技术的原理和检测平台,详细阐述了等位基因特异性杂交、内切酶酶切技术、引物延伸法、寡核苷酸连接反应等SNP检测原理,以及平板读数仪、基因芯片、微球阵列技术和质谱仪等检测平台,并对SNP高通量检测技术的发展进行了展望。     

单核苷酸多态性检测方法的研究进展

单核苷酸多态性(single nucleotide polymorphism,SNP)作为第三代遗传标记已经广泛应用于基因作图、疾病相关性分析、群体遗传学及药物研究等领域.因此建立高度自动化和高通量的SNP检测分析技术十分重要.简要介绍了国内外几种主要SNP检测技术的原理和检测分析手段,并对SNP高通量检测技术的发展进行了展望.     

利用基因芯片技术检测P53基因突变

基因芯片技术是后基因组时代基因功能分析的最重要技术之一。利用基因芯片技术检测P53基因突变,具有快速、准确、高通量和自动化的特点。本文阐述了基因芯片技术的基本原理及其检测P53基因突变的方法。     

利用基因芯片技术检测P53基因突变

基因芯片技术是后基因组时代基因功能分析的最重要技术之一。利用基因芯片技术检测Ｐ５３基因突变,具有快速、准确、高通量和自动化的特点。本文阐述了基因芯片技术的基本原理及其检测Ｐ５３基因突变的方法。     

病毒的基因芯片分型技术及其应用

生物芯片技术是上世纪90年代发展起来的新型生物技术,它具有高通量、高度集成化、微型化、高敏感、高度平行性等特点。近年来,基因芯片技术已逐渐被应用于病毒的基因分型中。病毒的基因分型芯片是直接把病毒分型探针固定在基片上从而制成的基因芯片,本文主要对病毒的基因分型芯片技术及其应用进展进行综述。     

乳酸菌基因芯片应用研究进展

基因芯片技术是上世纪90年代兴起的一种对成百上千甚至上万个基因同时进行检测的新技术,具有高通量、并行化的特点,广泛应用于基因表达谱测定、基因功能预测、基因突变检测和多态性分析等方面。多种乳酸菌基因组全序列以及其大量EST、16S rDNA、16S-23S基因间区和功能基因序列测定的完成,有力地推动了基因芯片技术在乳酸菌研究中的应用。介绍了基因芯片的基本原理及乳酸菌基因芯片在基因表达、种属鉴定等研究中的应用进展,以期更好地利用和开发乳酸菌基因芯片。     

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。     

基因芯片与高通量测序技术的原理与应用的比较

随着后基因组时代的到来,基因芯片和高通量测序已成为生物化学和分子生物学研究中的两大重要技术。从检测效率、准确性以及自动化程度证实,这两大技术都较传统的遗传学方法有了新的突破。基因芯片技术是一种具有高通量、高效率以及高自动化特点的方法,发展至今无论在核心技术还是工业应用方面都得到广泛的推广。高通量DNA测序技术建立较晚,但是其发展速度快,特别是在技术方面的更新换代极快,不断地改进使得测序的高通量、高准确率在生命科学中的应用也是占据不可逾越的优势。二者在原理上存在着显著的差异,却在应用方面上常常交融。基于此背景,本文以基因芯片技术与高通量测序技术二者在原理和基因拷贝数变异、肠道微生物、农业等应用方面作简要论述和对比。     

使用基因芯片时应注意的几个问题

基因芯片技术从兴起到投入商业应用只有短短十年左右时间 ,在临床疾病诊断、新药开发、军事、环保、进出口检疫、司法、农业、林业等领域具有广泛的应用前景。由于基因芯片的高通量和平行检测的特点 ,在一块基因芯片上能同时检测成千上万个基因的表达情况 ,与传统检测基因表达方法比较 ,具有省时、高效、费用相对较低等优点 ,因而获得了医学、药学、环保、农业和林业等科研工作者的青睐。但是 ,基因芯片作为一项新兴技术 ,要求应用者具有一定的分子生物学知识。在实际使用中 ,有的使用者对基因芯片的知识不太了解 ,出现了一些不该出现的问题…     

Identification of upper respiratory tract pathogens using electrochemical detection on an oligonucleotide microarray

Bacterial and viral upper respiratory infections (URI) produce highly variable clinical symptoms that cannot be used to identify the etiologic agent. Proper treatment, however, depends on correct identification of the pathogen involved as antibiotics provide little or no benefit with viral infections. Here we describe a rapid and sensitive genotyping assay and microarray for URI identification using standard amplification and hybridization techniques, with electrochemical detection (ECD) on a semiconductor-based oligonucleotide microarray. The assay was developed to detect four bacterial pathogens (Bordetella pertussis, Streptococcus pyogenes, Chlamydia pneumoniae and Mycoplasma pneumoniae) and 9 viral pathogens (adenovirus 4, coronavirus OC43, 229E and HK, influenza A and B, parainfluenza types 1, 2, and 3 and respiratory syncytial virus. This new platform forms the basis for a fully automated diagnostics system that is very flexible and can be customized to suit different or additional pathogens. Multiple probes on a flexible platform allow one to test probes empirically and then select highly reactive probes for further iterative evaluation. Because ECD uses an enzymatic reaction to create electrical signals that can be read directly from the array, there is no need for image analysis or for expensive and delicate optical scanning equipment. We show assay sensitivity and specificity that are excellent for a multiplexed format.     

Simultaneous detection of major enteric viruses using a combimatrix microarray

Various enteric viruses including norovirus, rotavirus, adenovirus, and astrovirus are the major etiological agents of food-borne and water-borne disease outbreaks and frequently cause non-bacterial gastroenteritis worldwide. Sensitive and high-throughput detection methods for these viral pathogens are compulsory for diagnosing viral pathogens and subsequently improving public health. Hence, we developed a sensitive, specific, and high-throughput analytical assay to detect most major enteric viral pathogens using “Combimatrix” platform oligonucleotide probes. In order to detect four different enteric viral pathogens in a sensitive and simultaneous manner, we first developed a multiplex RT-PCR assay targeting partial gene sequences of these viruses with fluorescent labeling for the subsequent microarray. Then, five olignonucleotides specific to each of the four major enteric viruses were selected for the microarray from the oligonulceotide pools targeting the specific genes obtained by multiplex PCR of these viruses. The oligonucleotide microarray was evaluated against stool specimens containing single or mixed viral species. As a result, we demonstrated that the multiplex RT-PCR assay specifically amplified partial sequences of four enteric viruses and the subsequent microarray assay was capable of sensitive and simultaneous detection of those viruses. The developed method could be useful for diagnosing enteric viruses in both clinical and environmental specimens.     

Multipathogen oligonucleotide microarray for environmental and biodefense applications

Food-borne pathogens are a major health problem. The large and diverse number of microbial pathogens and their virulence factors has fueled interest in technologies capable of detecting multiple pathogens and multiple virulence factors simultaneously. Some of these pathogens and their toxins have potential use as bioweapons. DNA microarray technology allows the simultaneous analysis of thousands of sequences of DNA in a relatively short time, making it appropriate for biodefense and for public health uses. This paper describes methods for using DNA microarrays to detect and analyze microbial pathogens. The FDA-1 microarray was developed for the simultaneous detection of several food-borne pathogens and their virulence factors including Listeria spp., Campylobacter spp., Staphylococcus aureus enterotoxin genes and Clostridium perfringens toxin genes. Three elements were incorporated to increase confidence in the microarray detection system: redundancy of genes, redundancy of oligonucleotide probes (oligoprobes) for a specific gene, and quality control oligoprobes to monitor array spotting and target DNA hybridization. These elements enhance the reliability of detection and reduce the chance of erroneous results due to the genetic variability of microbes or technical problems with the microarray. The results presented demonstrate the potential of oligonucleotide microarrays for detection of environmental and biodefense relevant microbial pathogens.     

Viral discovery and sequence recovery using DNA microarrays

Because of the constant threat posed by emerging infectious diseases and the limitations of existing approaches used to identify new pathogens, there is a great demand for new technological methods for viral discovery. We describe herein a DNA microarray-based platform for novel virus identification and characterization. Central to this approach was a DNA microarray designed to detect a wide range of known viruses as well as novel members of existing viral families; this microarray contained the most highly conserved 70mer sequences from every fully sequenced reference viral genome in GenBank. During an outbreak of severe acute respiratory syndrome (SARS) in March 2003, hybridization to this microarray revealed the presence of a previously uncharacterized coronavirus in a viral isolate cultivated from a SARS patient. To further characterize this new virus, approximately 1 kb of the unknown virus genome was cloned by physically recovering viral sequences hybridized to individual array elements. Sequencing of these fragments confirmed that the virus was indeed a new member of the coronavirus family. This combination of array hybridization followed by direct viral sequence recovery should prove to be a general strategy for the rapid identification and characterization of novel viruses and emerging infectious disease.     

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.     

The microbial detection array combined with random Phi29-amplification used as a diagnostic tool for virus detection in clinical samples

A common technique used for sensitive and specific diagnostic virus detection in clinical samples is PCR that can identify one or several viruses in one assay. However, a diagnostic microarray containing probes for all human pathogens could replace hundreds of individual PCR-reactions and remove the need for a clear clinical hypothesis regarding a suspected pathogen. We have established such a diagnostic platform for random amplification and subsequent microarray identification of viral pathogens in clinical samples. We show that Phi29 polymerase-amplification of a diverse set of clinical samples generates enough viral material for successful identification by the Microbial Detection Array, demonstrating the potential of the microarray technique for broad-spectrum pathogen detection. We conclude that this method detects both DNA and RNA virus, present in the same sample, as well as differentiates between different virus subtypes. We propose this assay for diagnostic analysis of viruses in clinical samples.     

Optimization and clinical validation of a pathogen detection microarray

DNA microarrays used as 'genomic sensors' have great potential in clinical diagnostics. Biases inherent in random PCR-amplification, cross-hybridization effects, and inadequate microarray analysis, however, limit detection sensitivity and specificity. Here, we have studied the relationships between viral amplification efficiency, hybridization signal, and target-probe annealing specificity using a customized microarray platform. Novel features of this platform include the development of a robust algorithm that accurately predicts PCR bias during DNA amplification and can be used to improve PCR primer design, as well as a powerful statistical concept for inferring pathogen identity from probe recognition signatures. Compared to real-time PCR, the microarray platform identified pathogens with 94% accuracy (76% sensitivity and 100% specificity) in a panel of 36 patient specimens. Our findings show that microarrays can be used for the robust and accurate diagnosis of pathogens, and further substantiate the use of microarray technology in clinical diagnostics.