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.     

Detection of bacterial pathogens in municipal wastewater using an oligonucleotide microarray and real-time quantitative PCR

As a first step toward building a comprehensive microarray, two low density DNA microarrays were constructed and evaluated for the accurate detection of wastewater pathogens. The first one involved the direct hybridization of wastewater microbial genomic DNA to the functional gene probes while the second involved PCR amplification of 23S ribosomal DNA. The genomic DNA microarray employed 10 functional genes as detection targets. Sensitivity of the microarray was determined to be approximately 1.0 microg of Esherichia coli genomic DNA, or 2 x 10(8) copies of the target gene, and only E. coli DNA was detected with the microarray assay using municipal raw sewage. Sensitivity of the microarray was enhanced approximately by 6 orders of magnitude when the target 23S rRNA gene sequences were PCR amplified with a novel universal primer set and allowed hybridization to 24 species-specific oligonucleotide probes. The minimum detection limit was estimated to be about 100 fg of E. coli genomic DNA or 1.4 x 10(2) copies of the 23S rRNA gene. The PCR amplified DNA microarray successfully detected multiple bacterial pathogens in wastewater. As a parallel study to verify efficiency of the DNA microarray, a real-time quantitative PCR assay was also developed based on the fluorescent TaqMan probes (Applied Biosystems).     

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.     

Waterborne pathogen detection by use of oligonucleotide-based microarrays

A small-oligonucleotide microarray prototype was designed with probes specific for the universal 16S rRNA and cpn60 genes of several pathogens that are usually encountered in wastewaters. In addition to these two targets, wecE-specific oligonucleotide probes were included in the microarray to enhance its discriminating power within the Enterobacteriaceae family. Universal PCR primers were used to amplify variable regions of 16S rRNA, cpn60, and wecE genes directly in Escherichia coli and Salmonella enterica serovar Typhimurium genomic DNA mixtures (binary); E. coli, S. enterica serovar Typhimurium, and Yersinia enterocolitica genomic DNA mixtures (ternary); or wastewater total DNA. Amplified products were fluorescently labeled and hybridized on the prototype chip. The detection sensitivity for S. enterica serovar Typhimurium was estimated to be on the order of 0.1% (10(4) S. enterica genomes) of the total DNA for the combination of PCR followed by microarray hybridization. The sensitivity of the prototype could be increased by hybridizing amplicons generated by PCR targeting genes specific for a bacterial subgroup, such as wecE genes, instead of universal taxonomic amplicons. However, there was evidence of PCR bias affecting the detection limits of a given pathogen as increasing amounts of a different pathogen were spiked into the test samples. These results demonstrate the feasibility of using DNA microarrays in the detection of waterborne pathogens within mixed populations but also raise the problem of PCR bias in such experiments.     

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。     

Pattern‐mapped multiple detection of 11 pathogenic bacteria using a 16s rDNA‐based oligonucleotide microarray

Pathogen detection is an important issue in human health due to the threats posed by severe communicable diseases. In the present study, to achieve efficient and accurate multiple detection of 11 selected pathogenic bacteria, we constructed a 16S rDNA oligonucleotide microarray containing doubly specific capture probes. Many target pathogens were specifically detected by the microarray with the aid of traditional perfect match‐based analysis using our previously proposed two‐dimensional visualization plot tool. However, some target species or subtypes were difficult to discriminate by perfect match analysis due to nonspecific binding of conserved 16S rDNA‐derived capture probes with high sequence similarity. We noticed that the patterns of specific spots for each strain were somewhat different in the two‐dimensional gradation plot. Therefore, to discriminate subtle differences between phylogenetically related pathogens, a pattern‐mapping statistical model was established using an artificial neural network algorithm trained by experimental repeats. The oligonucleotide microarray system harboring doubly specific capture probes combined with the pattern‐mapping analysis tool resulted in successful detection of all target pathogens including even subtypes of two closely related species showing strong nonspecific binding. Collectively, the results indicate that our novel combined system of a 16S rDNA‐based DNA microarray and a pattern‐mapping statistical analysis tool is a simple and effective method for detecting multiple pathogens. Biotechnol. Bioeng. 2010;106: 183–192. © 2010 Wiley Periodicals, Inc.     

Human-blind probes and primers for dengue virus identification

Reliable detection and identification of pathogens in complex biological samples, in the presence of contaminating DNA from a variety of sources, is an important and challenging diagnostic problem for the development of field tests. The problem is compounded by the difficulty of finding a single, unique genomic sequence that is present simultaneously in all genomes of a species of closely related pathogens and absent in the genomes of the host or the organisms that contribute to the sample background. Here we describe 'host-blind probe design'- a novel strategy of designing probes based on highly frequent genomic signatures found in the pathogen genomes of interest but absent from the host genome. Upon hybridization, an array of such informative probes will produce a unique pattern that is a genetic fingerprint for each pathogen strain. This multiprobe approach was applied to 83 dengue virus genome sequences, available in public databases, to design and perform in silico microarray experiments. The resulting patterns allow one to unequivocally distinguish the four major serotypes, and within each serotype to identify the most similar strain among those that have been completely sequenced. In an environment where dengue is indigenous, this would allow investigators to determine if a particular isolate belongs to an ongoing outbreak or is a previously circulating version. Using our probe set, the probability that misdiagnosis at the serotype level would occur is approximately 1 : 10(150).     

A Single-Nucleotide-Polymorphism-Based Multilocus Genotyping Assay for Subtyping Lineage I Isolates of Listeria monocytogenes

Listeria monocytogenes is a facultative intracellular pathogen responsible for food-borne disease with high mortality rates in humans and is the leading microbiological cause of food recalls. Lineage I isolates of L. monocytogenes are a particular public health concern because they are responsible for most sporadic cases of listeriosis and the vast majority of epidemic outbreaks. Rapid, reproducible, and sensitive methods for differentiating pathogens below the species level are required for effective pathogen control programs, and the CDC PulseNet Task Force has called for the development and validation of DNA sequence-based methods for subtyping food-borne pathogens. Therefore, we developed a multilocus genotyping (MLGT) assay for L. monocytogenes lineage I isolates based on nucleotide variation identified by sequencing 23,251 bp of DNA from 22 genes distributed across seven genomic regions in 65 L. monocytogenes isolates. This single-well assay of 60 allele-specific probes captured 100% of the haplotype information contained in approximately 1.5 Mb of comparative DNA sequence and was used to reproducibly type a total of 241 lineage I isolates. The MLGT assay provided high discriminatory power (Simpson's index value, 0.91), uniquely identified isolates from the eight listeriosis outbreaks examined, and differentiated serotypes 1/2b and 4b as well as epidemic clone I (ECI), ECIa, and ECII. In addition, the assay included probes for a previously characterized truncation mutation in inlA, providing for the identification of a specific virulence-attenuated subtype. These results demonstrate that MLGT represents a significant new tool for use in pathogen surveillance, outbreak detection, risk assessment, population analyses, and epidemiological investigations.     

Development and application of an oligonucleotide microarray for the detection of food-borne bacterial pathogens

The rapid and accurate detection and identification of food-borne pathogenic bacteria is critical for food safety. In this paper, we describe a rapid (<4 h) high-throughput detection and identification system that uses universal polymerase chain reaction (PCR) primers to amplify a variable region of bacterial the 16S rRNA gene, followed by reverse hybridization of the products to species-specific oligonucleotide probes on a chip. This procedure was successful in discriminating 204 strains of bacteria from pure culture belonging to 13 genera of bacteria. When this method was applied directly to 115 strains of bacteria isolated from foods, 112/115 (97.4%) were correctly identified; two strains were indistinguishable due to weak signal, while one failed to produce a PCR product. The array was used to detect and successfully identify two strains of bacteria from food poisoning outbreak samples, giving results through hybridization that were identical to those obtained by traditional methods. The sensitivity of the microarray assay was 10² CFU of bacteria. Thus, the oligonucleotide microarray is a powerful tool for the detection and identification of pathogens from foods. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

In situ-synthesized virulence and marker gene biochip for detection of bacterial pathogens in water

Pathogen detection tools with high reliability are needed for various applications, including food and water safety and clinical diagnostics. In this study, we designed and validated an in situ-synthesized biochip for detection of 12 microbial pathogens, including a suite of pathogens relevant to water safety. To enhance the reliability of presence/absence calls, probes were designed for multiple virulence and marker genes (VMGs) of each pathogen, and each VMG was targeted by an average of 17 probes. Hybridization of the biochip with amplicon mixtures demonstrated that 95% of the initially designed probes behaved as predicted in terms of positive/negative signals. The probes were further validated using DNA obtained from three different types of water samples and spiked with pathogen genomic DNA at decreasing relative abundance. Excellent specificity for making presence/absence calls was observed by using a cutoff of 0.5 for the positive fraction (i.e., the fraction of probes yielding a positive signal for a given VMG). A split multiplex PCR design for simultaneous amplification of the VMGs resulted in a detection limit of between 0.1 and 0.01% relative abundance, depending on the type of pathogen and the VMG. Thermodynamic analysis of the hybridization patterns obtained with DNA from the different water samples demonstrated that probes with a hybridization Gibbs free energy of approximately -19.3 kcal/mol provided the best trade-off between sensitivity and specificity. The developed biochip may be used to detect the described bacterial pathogens in water samples when parallel and specific detection is required.     

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.     

The use of fluorescein for labeling genomic probes in the checkerboard DNA-DNA hybridization method

Molecular methods that permit the simultaneous detection and quantification of a large number of microbial species are currently employed in the evaluation of complex ecosystems. The checkerboard DNA-DNA hybridization technique enables the simultaneous identification of distinct bacterial species in a large number of dental samples. The original technique employed digoxigenin-labeled whole genomic DNA probes which were detected by chemiluminescence. In this study, we present an alternative protocol for labeling and detecting whole genomic DNA probes in the Checkerboard DNA-DNA hybridization method. Whole genomic DNA was extracted from five bacterial species and labeled with fluorescein. The fluorescein labeled whole genomic DNA probes were hybridized against whole genomic DNA or subgingival plaque samples in a checkerboard hybridization format, followed by chemiluminescent detection. Our results reveal that fluorescein is a viable and adequate alternative labeling reagent to be employed in the checkerboard DNA-DNA hybridization technique.     

Impact of genomics on microbial food safety

Genome sequences are now available for many of the microbes that cause food-borne diseases. The information contained in pathogen genome sequences, together with the development of themed and whole-genome DNA microarrays and improved proteomics techniques, might provide tools for the rapid detection and identification of such organisms, for assessing their biological diversity and for understanding their ability to respond to stress. The genomic information also provides insight into the metabolic capacity and versatility of microbes; for example, specific metabolic pathways might contribute to the growth and survival of pathogens in a range of niches, such as food-processing environments and the human host. New concepts are emerging about how pathogens function, both within foods and in interactions with the host. The future should bring the first practical benefits of genome sequencing to the field of microbial food safety, including strategies and tools for the identification and control of emerging pathogens.     

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。     

A prototype taxonomic microarray targeting the rpsA housekeeping gene permits species identification within the rhizobial genus Ensifer

To develop a reliable tool for the identification and classification of the different Ensifer species, without the need for sequencing, a prototype DNA microarray that targets the rpsA housekeeping gene was designed and tested. Internal segments of the rpsA gene from 34 reference strains, representing the different Ensifer species, were sequenced and the sequences were used to select 44 diagnostic oligonucleotides that served as probes for the identification microarray. Both, genomic DNA and specific rpsA PCR-products were tested as a target in hybridisation experiments. Experimental conditions were optimised and the diagnostic oligonucleotides were validated. Hybridisation results with the rpsA PCR-products showed reliable identification of the reference strains to species and genomovar level. Our data indicate that a microarray targeting housekeeping genes is a promising, accurate and relatively simple genotyping technique that would also be applicable for the identification and characterization of other bacterial groups of interest.     

Extensive set of mitochondrial LSU rDNA-based oligonucleotide probes for the detection of common airborne fungi

Fungi exist in every indoor and outdoor environment. Many fungi are toxigenic or pathogens that may cause various public health concerns. Rapid and accurate detection and identification of fungi require specific markers. In this study, partial mitochondrial large subunit rDNA was amplified and sequenced from 32 fungal strains representing 31 species from 14 genera. Based on the sequence variation pattern, 26 oligonucleotide probes were designed for their discrimination. The specificity of the probes was evaluated through homology search against GenBank database and hybridization examination on 38 fungal strains. The 26 probes were verified as highly specific to 20 fungal species. A two-step detection procedure through PCR followed by probe hybridization gave ten-fold increase in detection sensitivity than single-step PCR assay and would be a practical approach for environmental sample screening. The probes developed in this study can be applied in clinical diagnosis and environmental monitoring of fungal agents.     

Waterborne Pathogen Detection by Use of Oligonucleotide-Based Microarrays

A small-oligonucleotide microarray prototype was designed with probes specific for the universal 16S rRNA and cpn60 genes of several pathogens that are usually encountered in wastewaters. In addition to these two targets, wecE-specific oligonucleotide probes were included in the microarray to enhance its discriminating power within the Enterobacteriaceae family. Universal PCR primers were used to amplify variable regions of 16S rRNA, cpn60, and wecE genes directly in Escherichia coli and Salmonella enterica serovar Typhimurium genomic DNA mixtures (binary); E. coli, S. enterica serovar Typhimurium, and Yersinia enterocolitica genomic DNA mixtures (ternary); or wastewater total DNA. Amplified products were fluorescently labeled and hybridized on the prototype chip. The detection sensitivity for S. enterica serovar Typhimurium was estimated to be on the order of 0.1% (10⁴ S. enterica genomes) of the total DNA for the combination of PCR followed by microarray hybridization. The sensitivity of the prototype could be increased by hybridizing amplicons generated by PCR targeting genes specific for a bacterial subgroup, such as wecE genes, instead of universal taxonomic amplicons. However, there was evidence of PCR bias affecting the detection limits of a given pathogen as increasing amounts of a different pathogen were spiked into the test samples. These results demonstrate the feasibility of using DNA microarrays in the detection of waterborne pathogens within mixed populations but also raise the problem of PCR bias in such experiments.     

Electronic microarray analysis of 16S rDNA amplicons for bacterial detection

Electronic microarray technology is a potential alternative in bacterial detection and identification. However, conditions for bacterial detection by electronic microarray need optimization. Using the NanoChip electronic microarray, we investigated eight marine bacterial species. Based on the 16S rDNA sequences of these species, we constructed primers, reporter probes, and species-specific capture probes. We carried out two separate analyses for longer (533 bp) and shorter (350 and 200 bp) amplified products (amplicons). To detect simultaneously the hybridization signals for the 350- and 200-bp amplicons, we designed a common reporter probe from an overlapping sequence within both fragments. We developed methods to optimize detection of hybridization signals for processing the DNA chips. A matrix analysis was performed for different bacterial species and complementary capture probes on electronic microarrays. Results showed that, when using the longer amplicon, not all bacterial targets hybridized with the complementary capture probes, which was characterized by the presence of false-positive signals. However, with the shorter amplicons, all bacterial species were correctly and completely detected using the constructed complementary capture probes.     

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.     

A single-nucleotide-polymorphism-based multilocus genotyping assay for subtyping lineage I isolates of Listeria monocytogenes

Listeria monocytogenes is a facultative intracellular pathogen responsible for food-borne disease with high mortality rates in humans and is the leading microbiological cause of food recalls. Lineage I isolates of L. monocytogenes are a particular public health concern because they are responsible for most sporadic cases of listeriosis and the vast majority of epidemic outbreaks. Rapid, reproducible, and sensitive methods for differentiating pathogens below the species level are required for effective pathogen control programs, and the CDC PulseNet Task Force has called for the development and validation of DNA sequence-based methods for subtyping food-borne pathogens. Therefore, we developed a multilocus genotyping (MLGT) assay for L. monocytogenes lineage I isolates based on nucleotide variation identified by sequencing 23,251 bp of DNA from 22 genes distributed across seven genomic regions in 65 L. monocytogenes isolates. This single-well assay of 60 allele-specific probes captured 100% of the haplotype information contained in approximately 1.5 Mb of comparative DNA sequence and was used to reproducibly type a total of 241 lineage I isolates. The MLGT assay provided high discriminatory power (Simpson's index value, 0.91), uniquely identified isolates from the eight listeriosis outbreaks examined, and differentiated serotypes 1/2b and 4b as well as epidemic clone I (ECI), ECIa, and ECII. In addition, the assay included probes for a previously characterized truncation mutation in inlA, providing for the identification of a specific virulence-attenuated subtype. These results demonstrate that MLGT represents a significant new tool for use in pathogen surveillance, outbreak detection, risk assessment, population analyses, and epidemiological investigations. DNA sequences were deposited in the GenBank database under accession numbers DQ 812146 to DQ 812517, DQ 843664 to DQ 844598, and AY 512391 to AY 512502.