Quantitative Detection of Microbial Genes by Using DNA Microarrays

To quantify target genes in biological samples using DNA microarrays, we employed reference DNA to normalize variations in spot size and hybridization. This method was tested using nitrate reductase (nirS), naphthalene dioxygenase (nahA), and Escherichia coli O157 O-antigen biosynthesis genes as model genes and lambda DNA as the reference DNA. We observed a good linearity between the log signal ratio and log DNA concentration ratio at DNA concentrations above the method's detection limit, which was approximately 10 pg. This approach for designing quantitative microarrays and the inferred equation from this study provide a simple and convenient way to estimate the target gene concentration from the hybridization signal ratio.     

Detection of Pathogenic Vibrio spp. in Shellfish by Using Multiplex PCR and DNA Microarrays

This study describes the development of a gene-specific DNA microarray coupled with multiplex PCR for the comprehensive detection of pathogenic vibrios that are natural inhabitants of warm coastal waters and shellfish. Multiplex PCR with vvh and viuB for Vibrio vulnificus, with ompU, toxR, tcpI, and hlyA for V. cholerae, and with tlh, tdh, trh, and open reading frame 8 for V. parahaemolyticus helped to ensure that total and pathogenic strains, including subtypes of the three Vibrio spp., could be detected and discriminated. For DNA microarrays, oligonucleotide probes for these targeted genes were deposited onto epoxysilane-derivatized, 12-well, Teflon-masked slides by using a MicroGrid II arrayer. Amplified PCR products were hybridized to arrays at 50°C and detected by using tyramide signal amplification with Alexa Fluor 546 fluorescent dye. Slides were imaged by using an arrayWoRx scanner. The detection sensitivity for pure cultures without enrichment was 10² to 10³ CFU/ml, and the specificity was 100%. However, 5 h of sample enrichment followed by DNA extraction with Instagene matrix and multiplex PCR with microarray hybridization resulted in the detection of 1 CFU in 1 g of oyster tissue homogenate. Thus, enrichment of the bacterial pathogens permitted higher sensitivity in compliance with the Interstate Shellfish Sanitation Conference guideline. Application of the DNA microarray methodology to natural oysters revealed the presence of V. vulnificus (100%) and V. parahaemolyticus (83%). However, V. cholerae was not detected in natural oysters. An assay involving a combination of multiplex PCR and DNA microarray hybridization would help to ensure rapid and accurate detection of pathogenic vibrios in shellfish, thereby improving the microbiological safety of shellfish for consumers.     

A New Short Oligonucleotide-Based Strategy for the Precursor-Specific Regulation of microRNA Processing by Dicer

The precise regulation of microRNA (miRNA) biogenesis seems to be critically important for the proper functioning of all eukaryotic organisms. Even small changes in the levels of specific miRNAs can initiate pathological processes, including carcinogenesis. Accordingly, there is a great need to develop effective methods for the regulation of miRNA biogenesis and activity. In this study, we focused on the final step of miRNA biogenesis; i.e., miRNA processing by Dicer. To test our hypothesis that RNA molecules can function not only as Dicer substrates but also as Dicer regulators, we previously identified by SELEX a pool of RNA oligomers that bind to human Dicer. We found that certain of these RNA oligomers could selectively inhibit the formation of specific miRNAs. Here, we show that these specific inhibitors can simultaneously bind both Dicer and pre-miRNAs. These bifunctional riboregulators interfere with miRNA maturation by affecting pre-miRNA structure and sequestering Dicer. Based on these observations, we designed a set of short oligomers (12 nucleotides long) that were capable of influencing pre-miRNA processing in vitro, both in reactions involving recombinant human Dicer and in cytosolic extracts. We propose that the same strategy may be used to develop effective and selective regulators to control the production of any miRNA. Overall, our findings indicate that the interactions between pre-miRNAs and other RNAs may form very complex regulatory networks that modulate miRNA biogenesis and consequently gene expression.     

苹果炭疽菌的分子鉴定与检测

测定苹果炭疽菌rDNA全序列,比对苹果炭疽菌和其它炭疽菌ITS序列以及构建系统关系树,发现苹果炭疽菌与胶孢炭疽菌的ITS序列相似性高达99．8％,并与胶孢炭疽菌聚在一起,可以明确苹果炭疽菌应属于胶孢炭疽菌。进一步的序列比对发现,苹果炭疽菌的18S rDNA3’端比其它胶孢炭疽菌多出一段379bp的序列,根据这一特有片段设计引物CgF1与通用引物ITS4配对,结果仅能从苹果炭疽菌中扩增出1232bp的特异性条带。用苹果炭疽菌接种离体苹果,以接种发病的病组织总DNA为模板,利用引物CgF1／ITS4进行PCR扩增,同样可以扩增出1232bp的特异性条带,而健康苹果组织DNA中未能扩增出任何条带,表明该方法可用于苹果炭疽菌的鉴定和快速检测。     

寡核苷酸探针悬浮芯片在病原体检测中的应用

悬浮芯片与固体芯片、荧光定量PCR并列成为核酸序列鉴定中的重要的分子生物学工具,并在病原菌检测方面显示出不同的应用领域.悬浮芯片能同时检测多种病原菌,具有处理多样本能力、使用灵活、低成本等特点,适合对未知样本检测及环境监控.能够在生物安全、公共卫生、工农业生产中发挥重要作用;而固体芯片能耦联成千上百个探针,但由于在多样本处理、成本方面欠缺,因此适合于对重要的未知病原体的鉴定;荧光定量PCR具较好特异性、灵敏度,以及多样本处理能力,但在高通量方面欠缺.适合有目的地检测已知病原体.目前已建立三种基于悬浮芯片的检测方法:多重PCR扩增、通用引物扩增16S/23S rDNA、直接对实际样本杂交检测.多重PCR具较好特异性,但其多重能力还难以满足悬浮芯片的高通量的需要;通用引物具较好灵敏度及扩增多靶分子能力,但也存在交叉反应等缺陷.同时,采用PCR扩增方法,悬浮芯片检测的是PCR产物,不能客观反应实际样本中存在病原菌数量及是否具生命力.直接杂交环境样本尽管避免了PCR的缺陷,但在灵敏度方面非常欠缺.目前,在环境样本处理上,仍然缺乏有效的、高通量、自动化的方法,不能满足PCR与悬浮芯片多样本检测的需要.     

水中病原微生物分子检测技术研究进展

基于PCR方法的多种分子检测技术已广泛的应用于水体病原微生物的检测中。而以DNA芯片为代表的微型化、快速化手段将是未来检测技术的发展方向,可实现对病原微生物实时和快速的检测。新检测技术的发展有利于建立水体污染早期预警机制,同时,可靠的病原微生物检测方法可降低有害微生物对人类健康的影响。对水体病原微生物分子检测方法及其在水污染相关疾病风险控制中所扮演的重要角色进行阐述。     

Detection and Characterizationof Cellular Immune Responses Using Peptide–MHC Microarrays

The detection and characterization of antigen-specific T cell populations is critical for understanding the development and physiology of the immune system and its responses in health and disease. We have developed and tested a method that uses arrays of peptide–MHC complexes for the rapid identification, isolation, activation, and characterization of multiple antigen-specific populations of T cells. CD4⁺ or CD8⁺ lymphocytes can be captured in accordance with their ligand specificity using an array of peptide–MHC complexes printed on a film-coated glass surface. We have characterized the specificity and sensitivity of a peptide–MHC array using labeled lymphocytes from T cell receptor transgenic mice. In addition, we were able to use the array to detect a rare population of antigen-specific T cells following vaccination of a normal mouse. This approach should be useful for epitope discovery, as well as for characterization and analysis of multiple epitope-specific T cell populations during immune responses associated with viral and bacterial infection, cancer, autoimmunity, and vaccination.     

Comparative Genomics Using Microarrays Reveals Divergence and Loss of Virulence-Associated Genes in Host-Specific Strains of the Insect Pathogen Metarhizium anisopliae

Many strains of Metarhizium anisopliae have broad host ranges, but others are specialists and adapted to particular hosts. Patterns of gene duplication, divergence, and deletion in three generalist and three specialist strains were investigated by heterologous hybridization of genomic DNA to genes from the generalist strain Ma2575. As expected, major life processes are highly conserved, presumably due to purifying selection. However, up to 7% of Ma2575 genes were highly divergent or absent in specialist strains. Many of these sequences are conserved in other fungal species, suggesting that there has been rapid evolution and loss in specialist Metarhizium genomes. Some poorly hybridizing genes in specialists were functionally coordinated, indicative of reductive evolution. These included several involved in toxin biosynthesis and sugar metabolism in root exudates, suggesting that specialists are losing genes required to live in alternative hosts or as saprophytes. Several components of mobile genetic elements were also highly divergent or lost in specialists. Exceptionally, the genome of the specialist cricket pathogen Ma443 contained extra insertion elements that might play a role in generating evolutionary novelty. This study throws light on the abundance of orphans in genomes, as 15% of orphan sequences were found to be rapidly evolving in the Ma2575 lineage.It is difficult to trace and reconstruct the evolutionary processes of diversification and radiation of species. In particular, genes that contribute to ecological diversification and the nature of the evolutionary forces acting during this process are poorly understood, partly because genes directly involved in ecological attributes are hard to identify (13). This is not the case with fungi, as they have genes encoding secreted products with specific environmental adaptations, e.g., scavenging nutrients and penetrating host barriers. During its pathogenic life cycle the ubiquitous insect pathogen Metarhizium anisopliae secretes a formidable array of hydrolytic enzymes, antimicrobial compounds, and toxins. These properties, plus its experimental tractability, have made M. anisopliae a common research subject and model system for studying pathogenicity and for developing useful products for medicine, agriculture, and biotechnology (33).The phylogeny of the Metarhizium genus has been well characterized (12). It is a largely clonal organism (4), containing subtypes with wide host ranges (e.g., M. anisopliae var. anisopliae Ma2575) and subtypes that, like M. anisopliae var. acridum Ma324 (used for locust control), show specificity for certain locusts, beetles, crickets, homopterans, etc., and are unable to infect other insects (5). While some specialized lineages, such as M. anisopliae var. acridum, are phylogenetically distant from generalist strains, implying evolutionarily conserved host use patterns, closely related strains can also differ greatly in host range and requirements for germination (16, 40, 42). Evidence that most specialists arose from generalists includes the following: (i) the vast majority of isolates found in nature belong to the genetically very diverse M. anisopliae var. anisopliae and typically demonstrate wide host ranges; (ii) specialist strains are scattered among generalists in phylogenies and have independently adapted to different insects; (iii) specialization is associated with conditions that are assumed to be derived, including reduced diet breadth (2, 35, 40). Specialist and generalist strains are often closely linked in phylogenies, indicating that there are genetic mechanisms allowing rapid adaptation (40).We are using genetic variation to explore the evolutionary history and pathogenic adaptations of M. anisopliae. The goal is to provide a detailed molecular classification of multiple strains and address the origins of intraspecific differences (gene loss/gain/divergence or modulation of gene expression). Correlation of strain differences with adaptations to specific hosts will identify the underlying regulatory, metabolic, and biosynthetic differences that define host preferences. To initiate this study, we used expressed sequence tag (EST) approaches to compare gene expression patterns between Ma2575 and Ma324 (17). These are two of the most distantly related strains and essentially span the range of variation within M. anisopliae (12, 40). About 60% of the ESTs expressed by Ma2575 during growth on insect cuticle encode secreted enzymes and toxins. We speculated that the large number and diversity of these effectors may be the key to Ma2575''s ability to infect a wide variety of insects. In contrast, Ma324 ESTs revealed fewer hydrolytic enzymes and very few toxins. This relates to life-styles. Strain Ma2575 kills hosts quickly via toxins and grows saprophytically in the cadaver. In contrast, Ma324 causes a systemic infection of host tissues before the host dies. This study showed that ESTs allow different pathogenic strategies to be understood from a broad perspective.Patterns of gene duplication, divergence, and deletion can be specifically determined by heterologous hybridization of total genomic DNA to microarrays (11, 20, 27). Heterologous hybridization has provided a fast and powerful tool facilitating the merging of functional genomics with physiology, ecology, and evolution (7, 31, 38) in species of yeast (22, 27), fish (9, 24), mammals (23, 25), and plants (1, 15). We have already verified that an array of Ma2575 ESTs can be used for heterologous hybridization with cDNAs. Thus, Ma2575 arrays were used to probe the causes of sectorization (production of nonsporulating cultures) in two commercial strains of M. anisopliae var. anisopliae. Probes from both strains cross-reacted strongly with the arrays, although with different expression profiles (46). We also used Ma2575 arrays to identify hundreds of genes differentially regulated by Ma324 in response to host or nonhost cuticles (45). Although only 8% of paralogous Ma2575 genes have greater than 80% identity, we expected cross-hybridization would potentially overestimate the overlap in genes expressed by different strains. However, individual genes within gene families were distinguished, revealing processes unique to Ma324 (45). In this study we exploit the fact that heterologous cDNA can provide information on physiological processes to allow us to gain a mechanistic perspective on the different life-styles that exist in insect-fungus interactions.     

Dual-Priming Oligonucleotide-Based Multiplex PCR for the Detection of Helicobacter pylori and Determination of Clarithromycin Resistance with Gastric Biopsy Specimens

Background:  Assessment of Helicobacter pylori ( H. pylori ) clarithromycin resistance has rarely been performed routinely despite an increasing resistance rate. Our aim was to develop and evaluate the use of dual-priming oligonucleotide (DPO)-based multiplex polymerase chain reaction (PCR) to detect point mutations in the 23S rRNA gene responsible for clarithromycin resistance of H. pylori.
Materials and Methods:  Gastric biopsy specimens from 212 untreated patients with dyspepsia were examined by culture, histology, and DPO-based multiplex PCR. A disk diffusion test and E-test were used for performing phenotypic antibiotic susceptibility tests.
Results:  Among the biopsy specimens tested, 22.2% (47/212), 42.5% (90/212), and 41.5% (88/212) of the specimens were classified as H. pylori positive by culture, histology, and DPO-based multiplex PCR, respectively. Among 96 strains identified by either culture or DPO-based multiplex PCR, 80 strains were clarithromycin-susceptible and 16 strains (16.7%) were clarithromycin-resistant. There was 94.1% (32/34) concordance between phenotypic susceptibility tests and DPO-based multiplex PCR. In two patients with discrepant results, only DPO-based multiplex PCR detected clarithromycin-resistant strains. DPO-based multiplex PCR identified additional 49 clarithromycin-resistant or clarithromycin-susceptible H. pylori among 165 culture-negative specimens.
Conclusions:  DPO-based multiplex PCR can be used as a practical method for the detection of H. pylori infection and the determination of clarithromycin susceptibility in addition to phenotypic antimicrobial susceptibility tests.     

Nucleic Acid Amplification Strategies for DNA Microarray-Based Pathogen Detection

DNA microarray-based screening and diagnostic technologies have long promised comprehensive testing capabilities. However, the potential of these powerful tools has been limited by front-end target-specific nucleic acid amplification. Despite the sensitivity and specificity associated with PCR amplification, the inherent bias and limited throughput of this approach constrain the principal benefits of downstream microarray-based applications, especially for pathogen detection. To begin addressing alternative approaches, we investigated four front-end amplification strategies: random primed, isothermal Klenow fragment-based, 29 DNA polymerase-based, and multiplex PCR. The utility of each amplification strategy was assessed by hybridizing amplicons to microarrays consisting of 70-mer oligonucleotide probes specific for enterohemorrhagic Escherichia coli O157:H7 and by quantitating their sensitivities for the detection of O157:H7 in laboratory and environmental samples. Although nearly identical levels of hybridization specificity were achieved for each method, multiplex PCR was at least 3 orders of magnitude more sensitive than any individual random amplification approach. However, the use of Klenow-plus-Klenow and 29 polymerase-plus-Klenow tandem random amplification strategies provided better sensitivities than multiplex PCR. In addition, amplification biases among the five genetic loci tested were 2- to 20-fold for the random approaches, in contrast to >4 orders of magnitude for multiplex PCR. The same random amplification strategies were also able to detect all five diagnostic targets in a spiked environmental water sample that contained a 63-fold excess of contaminating DNA. The results presented here underscore the feasibility of using random amplification approaches and begin to systematically address the versatility of these approaches for unbiased pathogen detection from environmental sources.     

Pathotyping Escherichia coli by Using Miniaturized DNA Microarrays

The detection of virulence determinants harbored by pathogenic Escherichia coli is important for establishing the pathotype responsible for infection. A sensitive and specific miniaturized virulence microarray containing 60 oligonucleotide probes was developed. It detected six E. coli pathotypes and will be suitable in the future for high-throughput use.     

柑橘黄龙病病原快速检测技术研究进展

柑橘黄龙病是一种毁灭性的病害,一旦感染若不能及时发现,就会迅速传播蔓延,殃及整个果园。在尚无有效药剂根治的情况下及时的发现,并铲除病原是十分重要的防止措施。因此,研究开发出精确、快速、方便、易操作的检测方法显得尤为重要。本文针对柑橘黄龙病在田间诊断、血清学检测、分子诊断、光谱检测等方面的快速检测技术进行了综述,并对各种检测技术进行比较分析,以期为未来柑橘黄龙病快速检测技术研究提供参考依据。     

Sequence versus Structure for the Direct Detection of 16S rRNA on Planar Oligonucleotide Microarrays

A two-probe proximal chaperone detection system consisting of a species-specific capture probe for the microarray and a labeled, proximal chaperone probe for detection was recently described for direct detection of intact rRNAs from environmental samples on oligonucleotide arrays. In this study, we investigated the physical spacing and nucleotide mismatch tolerance between capture and proximal chaperone detector probes that are required to achieve species-specific 16S rRNA detection for the dissimilatory metal and sulfate reducer 16S rRNAs. Microarray specificity was deduced by analyzing signal intensities across replicate microarrays with a statistical analysis-of-variance model that accommodates well-to-well and slide-to-slide variations in microarray signal intensity. Chaperone detector probes located in immediate proximity to the capture probe resulted in detectable, nonspecific binding of nontarget rRNA, presumably due to base-stacking effects. Species-specific rRNA detection was achieved by using a 22-nt capture probe and a 15-nt detector probe separated by 10 to 14 nt along the primary sequence. Chaperone detector probes with up to three mismatched nucleotides still resulted in species-specific capture of 16S rRNAs. There was no obvious relationship between position or number of mismatches and within- or between-genus hybridization specificity. From these results, we conclude that relieving secondary structure is of principal concern for the successful capture and detection of 16S rRNAs on planar surfaces but that the sequence of the capture probe is more important than relieving secondary structure for achieving specific hybridization.     

Detection and Quantification of the Coral Pathogen Vibrio coralliilyticus by Real-Time PCR with TaqMan Fluorescent Probes

A real-time quantitative PCR-based detection assay targeting the dnaJ gene (encoding heat shock protein 40) of the coral pathogen Vibrio coralliilyticus was developed. The assay is sensitive, detecting as little as 1 CFU per ml in seawater and 10⁴ CFU per cm² of coral tissue. Moreover, inhibition by DNA and cells derived from bacteria other than V. coralliilyticus was minimal. This assay represents a novel approach to coral disease diagnosis that will advance the field of coral disease research.Vibrio coralliilyticus has recently emerged as a coral pathogen of concern on reefs throughout the Indo-Pacific. It was first implicated as the etiological agent responsible for bleaching and tissue lysis of the coral Pocillopora damicornis on Zanzibar reefs (2). More recently, V. coralliilyticus has been identified as the causative agent of white syndrome (WS) outbreaks on several Pacific reefs (14). WS is a collective term describing coral diseases characterized by a spreading band of tissue loss exposing white skeleton on Indo-Pacific scleractinian corals (16). V. coralliilyticus is an emerging model pathogen for understanding the mechanisms linking bacterial infection and coral disease (13) and therefore provides an ideal model for the development of diagnostic assays to detect coral disease. Current coral disease diagnostic methods, which are based primarily upon field-based observations of macroscopic disease signs, often detect disease only at the latest stages of infection, when control measures are least effective. The development of diagnostic tools targeting pathogens underlying coral disease pathologies may provide early indications of infection, aid the identification of disease vectors and reservoirs, and assist managers in developing strategies to prevent the spread of coral disease outbreaks. In this paper, we describe the development and validation of a TaqMan-based real-time quantitative PCR (qPCR) assay that targets a segment of the V. coralliilyticus heat shock protein 40-encoding gene (dnaJ).Nucleotide sequences of the dnaJ gene were retrieved from relevant Vibrio species, including V. coralliilyticus (LMG 20984), using the National Center for Biotechnology Information''s (NCBI) Entrez Nucleotide Database search tool (http://www.ncbi.nlm.nih.gov/). Gene sequences of strains not available in public databases (V. coralliilyticus strains LMG 21348, LMG 21349, LMG 21350, LMG 10953, LMG 20538, LMG 23696, LMG 23691, LMG 23693, LMG 23692, and LMG 23694) were obtained through extraction of total DNA using a Promega Wizard Prep DNA Purification Kit (Promega, Sydney, Australia), PCR amplification, and sequencing using primers and thermal cycling parameters described by Nhung et al. (8). A 128-bp region (nucleotides 363 to 490) containing high concentrations of single nucleotide polymorphisms (SNPs), which were conserved within V. coralliilyticus strains but differed from non-V. coralliilyticus strains, was identified, and oligonucleotide primers Vc_dnaJ_F1 (5′-CGG TTC GYG GTG TTT CAA AA-3′) and Vc_dnaJ_R1 (5′-AAC CTG ACC ATG ACC GTG ACA-3′) and a TaqMan probe, Vc_dnaJ_TMP (5′-6-FAM-CAG TGG CGC GAA G-MGBNFQ-3′; 6-FAM is 6-carboxyfluorescein and MGBNFQ is molecular groove binding nonfluorescent quencher), were designed to target this region. The qPCR assay was optimized and validated using DNA extracted from V. coralliilyticus isolates, nontarget Vibrio species, and other bacterial species grown in marine broth (MB) (Table ​(Table1),1), under the following optimal conditions: 1× TaqMan buffer A, 0.5 U of AmpliTaq Gold DNA polymerase, 200 μM deoxynucleotide triphosphates (with 400 μM dUTP replacing deoxythymidine triphosphate), 0.2 U of AmpErase uracil N-glycosylase (UNG), 3 mM MgCl₂, 0.6 μM each primer, 0.2 μM fluorophore-labeled TaqMan, 1 μl of template, and sterile MilliQ water for a total reaction volume to 20 μl. All assays were conducted on a RotoGene 300 (Corbett Research, Sydney, Australia) real-time analyzer with the following cycling parameters: 50°C for 120 s (UNG activation) and 95°C for 10 min (AmpliTaq Gold DNA polymerase activation), followed by 40 cycles of 95°C for 15 s (denaturation) and 60°C for 60 s (annealing/extension). During the annealing/extension phase of each thermal cycle, fluorescence was measured in the FAM channel (470-nm excitation and 510-nm detection).

TABLE 1.

Species, strain, and threshold cycle for all bacterial strains tested^a

Development and Evaluation of a Panel of Filovirus Sequence Capture Probes for Pathogen Detection by Next-Generation Sequencing

A detailed understanding of the circulating pathogens in a particular geographic location aids in effectively utilizing targeted, rapid diagnostic assays, thus allowing for appropriate therapeutic and containment procedures. This is especially important in regions prevalent for highly pathogenic viruses co-circulating with other endemic pathogens such as the malaria parasite. The importance of biosurveillance is highlighted by the ongoing Ebola virus disease outbreak in West Africa. For example, a more comprehensive assessment of the regional pathogens could have identified the risk of a filovirus disease outbreak earlier and led to an improved diagnostic and response capacity in the region. In this context, being able to rapidly screen a single sample for multiple pathogens in a single tube reaction could improve both diagnostics as well as pathogen surveillance. Here, probes were designed to capture identifying filovirus sequence for the ebolaviruses Sudan, Ebola, Reston, Taï Forest, and Bundibugyo and the Marburg virus variants Musoke, Ci67, and Angola. These probes were combined into a single probe panel, and the captured filovirus sequence was successfully identified using the MiSeq next-generation sequencing platform. This panel was then used to identify the specific filovirus from nonhuman primates experimentally infected with Ebola virus as well as Bundibugyo virus in human sera samples from the Democratic Republic of the Congo, thus demonstrating the utility for pathogen detection using clinical samples. While not as sensitive and rapid as real-time PCR, this panel, along with incorporating additional sequence capture probe panels, could be used for broad pathogen screening and biosurveillance.