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.     

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。     

Detection of representative enteropathogenic bacteria, Vibrio spp., pathogenic Escherichia coli, Salmonella spp., Shigella spp., and Yersinia enterocolitica, using a virulence factor gene-based oligonucleotide microarray

Rapid identification of enteropathogenic bacteria in stool samples is critical for clinical diagnosis and antimicrobial therapy. In this study, we describe the development of an approach that couples multiplex PCR with hybridization to a DNA microarray, to allow the simultaneous detection of the 10 pathogens. The microarray was synthesized with 20-mer oligonucleotide probes that were designed to be specific for virulence-factor genes of each strain. The detection limit for genomic DNA from a single strain was approximately 10 fg. In the presence of heterogeneous non-target DNA, the detection sensitivity of the array decreased to approximately 100 fg. We did not observe any non-specific hybridization. In addition, we successfully used this oligonucleotide-based DNA microarray to identify the causative agents in clinical stool samples from patients with food-borne enteritis.     

基因芯片技术检测3种肠道病原微生物方法的建立

目的:建立一种运用多重PCR和基因芯片技术检测和鉴定伤寒沙门氏菌、痢疾杆菌和单核细胞增生利斯特菌的方法。方法:分别选取伤寒沙门氏菌染色体ViaB区域中编码调控Vi抗原表达的基因(vipR)、痢疾杆菌编码侵袭质粒抗原H基因(ipaH)和单核细胞增生利斯特菌溶血素基因(hlyA)设计引物和探针,探针3'端进行氨基修饰,下游引物标记荧光素Cy3。在优化的PCR和杂交反应条件下,进行三重PCR扩增,产物与包括3种致病菌特异性探针的基因芯片杂交。在评价基因芯片的特异性和灵敏度之后,对临床样本进行检测。结果:只有3种目的致病菌的PCR产物在相应探针位置出现特异性信号,其他阴性细菌均无信号出现;3种致病菌的检测灵敏度均可达到103CFU/mL;检测30例临床样本的结果与常规细菌学培养结果一致。结论:所建立的可同时检测伤寒沙门氏菌、痢疾杆菌和单核细胞增生利斯特菌的基因芯片方法快速、准确,特异性高,重复性好,为3种肠道致病菌的快速检测和鉴定提供了新方法和新思路。     

DNA芯片技术在食品病原体检测中的应用

病原体的存在,尤其是食品中的病原体,给人类健康带来了威胁。DNA芯片技术是一种非常有效的病原体检测工具,具有众多传统检测方法所不具备的优势,受到广泛关注。我们简要论述了DNA芯片在细菌病原体、寄生虫、病毒病原体、微生物耐药性等的检测中的应用,并进一步综述了DNA芯片技术在食品检测中存在的问题、解决方法及发展方向。     

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.     

Microarray analysis of microbial virulence factors.

Hybridization with oligonucleotide microchips (microarrays) was used for discrimination among strains of Escherichia coli and other pathogenic enteric bacteria harboring various virulence factors. Oligonucleotide microchips are miniature arrays of gene-specific oligonucleotide probes immobilized on a glass surface. The combination of this technique with the amplification of genetic material by PCR is a powerful tool for the detection of and simultaneous discrimination among food-borne human pathogens. The presence of six genes (eaeA, slt-I, slt-II, fliC, rfbE, and ipaH) encoding bacterial antigenic determinants and virulence factors of bacterial strains was monitored by multiplex PCR followed by hybridization of the denatured PCR product to the gene-specific oligonucleotides on the microchip. The assay was able to detect these virulence factors in 15 Salmonella, Shigella, and E. coli strains. The results of the chip analysis were confirmed by hybridization of radiolabeled gene-specific probes to genomic DNA from bacterial colonies. In contrast, gel electrophoretic analysis of the multiplex PCR products used for the microarray analysis produced ambiguous results due to the presence of unexpected and uncharacterized bands. Our results suggest that microarray analysis of microbial virulence factors might be very useful for automated identification and characterization of bacterial pathogens.     

Detection of genes involved in biodegradation and biotransformation in microbial communities by using 50-mer oligonucleotide microarrays

To effectively monitor biodegrading populations, a comprehensive 50-mer-based oligonucleotide microarray was developed based on most of the 2,402 known genes and pathways involved in biodegradation and metal resistance. This array contained 1,662 unique and group-specific probes with <85% similarity to their nontarget sequences. Based on artificial probes, our results showed that under hybridization conditions of 50 degrees C and 50% formamide, the 50-mer microarray hybridization can differentiate sequences having <88% similarity. Specificity tests with representative pure cultures indicated that the designed probes on the arrays appeared to be specific to their corresponding target genes. The detection limit was approximately 5 to 10 ng of genomic DNA in the absence of background DNA and 50 to 100 ng of pure-culture genomic DNA in the presence of background DNA or 1.3 x 10(7) cells in the presence of background RNA. Strong linear relationships between the signal intensity and the target DNA and RNA were observed (r(2) = 0.95 to 0.99). Application of this type of microarray to analyze naphthalene-amended enrichment and soil microcosms demonstrated that microflora changed differently depending on the incubation conditions. While the naphthalene-degrading genes from Rhodococcus-type microorganisms were dominant in naphthalene-degrading enrichments, the genes involved in naphthalene (and polyaromatic hydrocarbon and nitrotoluene) degradation from gram-negative microorganisms, such as Ralstonia, Comamonas, and Burkholderia, were most abundant in the soil microcosms. In contrast to general conceptions, naphthalene-degrading genes from Pseudomonas were not detected, although Pseudomonas is widely known as a model microorganism for studying naphthalene degradation. The real-time PCR analysis with four representative genes showed that the microarray-based quantification was very consistent with real-time PCR (r(2) = 0.74). In addition, application of the arrays to both polyaromatic-hydrocarbon- and benzene-toluene-ethylbenzene-xylene-contaminated and uncontaminated soils indicated that the developed microarrays appeared to be useful for profiling differences in microbial community structures. Our results indicate that this technology has potential as a specific, sensitive, and quantitative tool in revealing a comprehensive picture of the compositions of biodegradation genes and the microbial community in contaminated environments, although more work is needed to improve detection sensitivity.     

Oligonucleotide microarray for the identification of potential mycotoxigenic fungi

Background  

Mycotoxins are secondary metabolites which are produced by numerous fungi and pose a continuous challenge to the safety and quality of food commodities in South Africa. These toxins have toxicologically relevant effects on humans and animals that eat contaminated foods. In this study, a diagnostic DNA microarray was developed for the identification of the most common food-borne fungi, as well as the genes leading to toxin production.     

Microarray probe expression measures, data normalization and statistical validation

DNA microarray technology is a high-throughput method for gaining information on gene function. Microarray technology is based on deposition/synthesis, in an ordered manner, on a solid surface, of thousands of EST sequences/genes/oligonucleotides. Due to the high number of generated datapoints, computational tools are essential in microarray data analysis and mining to grasp knowledge from experimental results. In this review, we will focus on some of the methodologies actually available to define gene expression intensity measures, microarray data normalization, and statistical validation of differential expression.     

应用DNA芯片技术对禽致病性大肠杆菌可能致病基因体外表达水平的研究

应用DNA芯片研究禽致病性大肠杆菌可能致病基因的表达.构建禽致病性大肠杆菌毒力基因、潜在毒力基因的DNA芯片,应用基因芯片技术对同属O2血清型的禽高致病性大肠杆菌E058株和低致病性大肠杆菌E526株在体外LB培养基和鸡血清培养状态下进行差异表达分析.结果:在体外LB静置培养状态下,低致病株E526与高致病株E058相比共有16个差异基因,均为下调基因.在鸡血清静置培养中,E526与E058相比共有15个差异基因,均为下调基因.应用基因芯片成功筛选了禽致病性大肠杆菌在体外不同条件下的毒力基因及可能毒力基因中差异表达基因,表明一些铁摄取系统相关基因对APEC的毒力较重要,同时也筛选出了一些新的可能致病基因aes-1,aes-2,aes-3,aes-4,aes-6,aes-8,aes-10,aes-13,aes-15,aes-31等.     

基于功能基因的微生物碳循环分子生态学研究进展

碳循环是生态系统中重要的生物地球化学元素循环之一。微生物参与碳固定、甲烷代谢、碳降解等多个重要的碳循环过程,深入了解微生物群落在碳循环过程中的功能和作用,有助于获悉微生物对全球气候变化的响应、适应和反馈机制,这也是微生物生态学研究的关键问题之一。传统的研究多集中于微生物分离培养技术,无法覆盖绝大部分未培养微生物,并且无法深入解析碳循环过程中微生物群落的结构和功能,宏基因组学技术的出现克服了这些缺陷,成为研究微生物群落结构和功能的有效手段。本文对目前宏基因组学的主要技术——定量PCR、DNA分子指纹图谱、基因芯片、克隆文库和高通量测序等技术进行了简要介绍,着重介绍了参与碳固定、甲烷生成和氧化、碳降解等主要碳循环过程的关键功能基因的研究现状,最后对碳循环过程中微生物宏基因组学研究的未来发展进行了总结与展望。     

Genomic Portraits of the Nervous System in Health and Disease

As the human genome project moves toward its goal of sequencing the entire human genome, gene expression profiling by DNA microarray technology is being employed to rapidly screen genes for biological information. In this review, we will introduce DNA microarray technology, outline the basic experimental paradigms and data analysis methods, and then show with some examples how gene expression profiling can be applied to the study of the central nervous system in health and disease.     

A virulence and antimicrobial resistance DNA microarray detects a high frequency of virulence genes in Escherichia coli isolates from Great Lakes recreational waters

Escherichia coli is generally described as a commensal species with occasional pathogenic strains. Due to technological limitations, there is currently little information concerning the prevalence of pathogenic E. coli strains in the environment. For the first time, using a DNA microarray capable of detecting all currently described virulence genes and commonly found antimicrobial resistance genes, a survey of environmental E. coli isolates from recreational waters was carried out. A high proportion (29%) of 308 isolates from a beach site in the Great Lakes carried a pathotype set of virulence-related genes, and 14% carried antimicrobial resistance genes, findings consistent with a potential risk for public health. The results also showed that another 8% of the isolates had unusual virulence gene combinations that would be missed by conventional screening. This new application of a DNA microarray to environmental waters will likely have an important impact on public health, epidemiology, and microbial ecology in the future.     

DNA microarray technology: a new tool for the epidemiological typing of bacterial pathogens?

Genomic hybridization on whole genome arrays detects the presence or absence of similar DNA regions in sufficiently related microorganisms, allowing genome-wide comparison of their genetic contents. A whole genome array is based on a sequenced bacterial isolate, and is a collection of DNA probes fixed on a solid support. In a single hybridization experiment, the absence/presence status of all genes of the sequenced microbe in the queried isolate can be examined. The objective of this minireview is to summarize the past usage of DNA microarray technology for microbial strain characterizations, and to estimate its future utilization in epidemiological studies and molecular typing of bacterial pathogens. The studies reviewed here confirm the usefulness of microarray technology for the detection of genetic polymorphisms. However, the construction or purchase of DNA microarrays and the performance of strain to strain hybridization experiments are still prohibitively expensive for routine application. Future use of arrays in epidemiology is likely to depend on the development of more cost-effective protocols, more robust and simplified formats, and the adequate evaluation of their performance (efficacy) and convenience (efficiency) compared with other genotyping methods. It seems more likely that a more focused assay, concentrating on genomic regions of variability previously detected by genome-wide microarrays, will find broad application in routine bacterial epidemiology.     

Quantitative assessment of DNA microarrays--comparison with Northern blot analyses

DNA microarray is a powerful technology that provides the expression profile of thousands of genes. However, less attention has been paid to its quantitative aspect. In this study, we constructed a small-scale DNA microarray that contains 84 genes and characterized its quantitative aspect. Analyses with this microarray showed that 17 genes were induced, whereas 8 genes were suppressed at least twofold during the differentiation of mouse embryonic stem cells. When repeated with the same combination of fluorescent dyes for probe labeling, the microarray produced consistent data (correlation coefficient = 0.991). In contrast, data were less consistent when repeated with the reverse combination of dyes (correlation coefficient = 0.945). The effect of dye combination was particularly evident in several genes. Total RNA (15 microg) and poly(A) RNA (0.5 microg) showed comparable sensitivity and produced essentially identical data (correlation coefficient = 0.983). The sensitivity of the DNA microarrays was slightly inferior to that of Northern blot analyses. In most genes, data obtained with the two methods were consistent. However, in 4 of 46 genes compared, DNA microarrays failed to detect the expression changes that were revealed by Northern blot. These data demonstrated that DNA microarrays provide quantitative data comparable to Northern blot in general, but a few issues must be considered when analyzing data.