scholarly journals DNA microarrays for pathogen detection and characterisation

2006 ◽  
Vol 27 (2) ◽  
pp. 68
Author(s):  
Philippa Jack ◽  
David Boyle
Keyword(s):  
Dna Microarray ◽  
Clinical Microbiology ◽  
Pathogen Detection ◽  
Dna Microarrays ◽  
Pcr Amplification ◽  
Specific Primer ◽  
Pathogen Discovery ◽  
Primer Sets ◽  
Major Factors ◽  
Diagnostic Microbiology

DNA microarrays have three main potential diagnostic uses in clinical microbiology: detection of known pathogens, pathogen typing and novel pathogen discovery. Although DNA microarray platforms offer the ability to screen for a large number of agents in parallel, sensitivity is dependent on the ability to obtain adequate amounts of pathogen nucleic acids from collected samples. In general, high levels of sensitivity require a PCR amplification step using specific primer sets, subsequently reducing the overall scope of the microarray assay. At present, relatively high costs, restricted sample throughput capabilities and validation difficulties are also major factors limiting the implementation of DNA microarray assays in diagnostic microbiology laboratories.

scholarly journals Detection and Genotyping of Arcobacter and Campylobacter Isolates from Retail Chicken Samples by Use of DNA Oligonucleotide Arrays

2007 ◽  
Vol 73 (11) ◽  
pp. 3645-3655 ◽  
Author(s):  
Beatriz Quiñones ◽  
Craig T. Parker ◽  
John M. Janda ◽  
William G. Miller ◽  
Robert E. Mandrell
Keyword(s):  
Dna Microarray ◽  
Membrane Filtration ◽  
Pathogen Detection ◽  
Dna Microarrays ◽  
Pcr Amplification ◽  
Detection Sensitivity ◽  
Sensitivity Threshold ◽  
Campylobacter Coli ◽  
Oligonucleotide Arrays ◽  
High Level

ABSTRACT To explore the use of DNA microarrays for pathogen detection in food, we produced DNA oligonucleotide arrays to simultaneously determine the presence of Arcobacter and the presence of Campylobacter in retail chicken samples. Probes were selected that target housekeeping and virulence-associated genes in both Arcobacter butzleri and thermotolerant Campylobacter jejuni and Campylobacter coli. These microarrays showed a high level of probe specificity; the signal intensities detected for A. butzleri, C. coli, or C. jejuni probes were at least 10-fold higher than the background levels. Specific identification of A. butzleri, C. coli, and C. jejuni was achieved without the need for a PCR amplification step. By adapting an isolation method that employed membrane filtration and selective media, C. jejuni isolates were recovered from package liquid from whole chicken carcasses prior to enrichment. Increasing the time of enrichment resulted in the isolation of A. butzleri and increased the recovery of C. jejuni. C. jejuni isolates were further classified by using an additional subset of probes targeting the lipooligosaccharide (LOS) biosynthesis locus. Our results demonstrated that most of the C. jejuni isolates likely possess class B, C, or H LOS. Validation experiments demonstrated that the DNA microarray had a detection sensitivity threshold of approximately 10,000 C. jejuni cells. Interestingly, the use of C. jejuni sequence-specific primers to label genomic DNA improved the sensitivity of this DNA microarray for detection of C. jejuni in whole chicken carcass samples. C. jejuni was efficiently detected directly both in package liquid from whole chicken carcasses and in enrichment broths.

scholarly journals Nucleic Acid Amplification Strategies for DNA Microarray-Based Pathogen Detection

2004 ◽  
Vol 70 (5) ◽  
pp. 3047-3054 ◽  
Author(s):  
Gary J. Vora ◽  
Carolyn E. Meador ◽  
David A. Stenger ◽  
Joanne D. Andreadis
Keyword(s):  
Nucleic Acid ◽  
Multiplex Pcr ◽  
Dna Microarray ◽  
Pathogen Detection ◽  
Pcr Amplification ◽  
Environmental Water ◽  
Nucleic Acid Amplification ◽  
Klenow Fragment ◽  
Front End ◽  
Random Amplification

ABSTRACT 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, φ29 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 φ29 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.

Design of specific primer sets for SARS-CoV-2 variants using evolutionary algorithms

2021 ◽  
Author(s):  
Alejandro Lopez Rincon ◽  
Carmina A. Perez Romero ◽  
Lucero Mendoza Maldonado ◽  
Eric Claassen ◽  
Johan Garssen ◽  
...  
Keyword(s):  
Evolutionary Algorithms ◽  
Specific Primer ◽  
Primer Sets

scholarly journals Induction of Multiple Prophages from a Marine Bacterium: a Genomic Approach

2006 ◽  
Vol 72 (7) ◽  
pp. 4995-5001 ◽  
Author(s):  
Feng Chen ◽  
Kui Wang ◽  
Jeneen Stewart ◽  
Robert Belas
Keyword(s):  
Mitomycin C ◽  
Marine Bacterium ◽  
Pcr Amplification ◽  
Pulsed Field Gel Electrophoresis ◽  
Bacterial Genomes ◽  
Dominant Type ◽  
Transmission Electron ◽  
Viral Particles ◽  
Genomic Approach ◽  
Primer Sets

ABSTRACT Approximately 70% of sequenced bacterial genomes contain prophage-like structures, yet little effort has been made to use this information to determine the functions of these elements. The recent genomic sequencing of the marine bacterium Silicibacter sp. strain TM1040 revealed five prophage-like elements in its genome. The genomes of these prophages (named prophages 1 to 5) are approximately 74, 30, 39, 36, and 15 kb long, respectively. To understand the function of these prophages, cultures of TM1040 were treated with mitomycin C to induce the production of viral particles. A significant increase in viral counts and a decrease in bacterial counts when treated with mitomycin C suggested that prophages were induced from TM1040. Transmission electron microscopy revealed one dominant type of siphovirus, while pulsed-field gel electrophoresis demonstrated two major DNA bands, equivalent to 35 and 75 kb, in the lysate. PCR amplification with primer sets specific to each prophage detected the presence of prophages 1, 3, and 4 in the viral lysate, suggesting that these prophages are inducible, but not necessarily to the same level, while prophages 2 and 5 are likely defective or non-mitomycin C-inducible phages. The combination of traditional phage assays and modern microbial genomics provides a quick and efficient way to investigate the functions and inducibility of prophages, particularly for a host harboring multiple prophages with similar sizes and morphological features.

scholarly journals Development of PCR Assays for the Identification of Species and Pathotypes of Elsinoë Causing Scab on Citrus

Plant Disease ◽  
2007 ◽  
Vol 91 (7) ◽  
pp. 865-870 ◽  
Author(s):  
J. W. Hyun ◽  
N. A. Peres ◽  
S.-Y. Yi ◽  
L. W. Timmer ◽  
K. S. Kim ◽  
...  
Keyword(s):  
Internal Transcribed Spacer ◽  
Sweet Orange ◽  
Random Amplified Polymorphic Dna ◽  
Its Region ◽  
The United States ◽  
Specific Primer ◽  
Identification Of Species ◽  
Orange Fruit ◽  
Pcr Assays ◽  
Primer Sets

Two scab pathogens of citrus, Elsinoë fawcettii and E. australis, cause citrus scab and sweet orange scab, respectively, and pathotypes of each species have been described. The two species cannot be readily distinguished by morphological or cultural characteristics and can be distinguished only by host range and the sequence of the internal transcribed spacer (ITS) region. In this study, random amplified polymorphic DNA (RAPD) assays clearly distinguished E. fawcettii and E. australis, and the sweet orange and natsudaidai pathotypes within E. australis also could be differentiated. We developed specific primer sets, Efaw-1 for E. fawcettii; Eaut-1, Eaut-2, Eaut-3, and Eaut-4 for E. australis; and EaNat-1 and EaNat-2 for the natsudaidai pathotype within E. australis using RAPD products unique to each species or pathotype. Other primer sets, Efaw-2 and Eaut-5, which were specific for E. fawcettii and E. australis, respectively, were designed from previously determined ITS sequences. The Efaw-1 and Efaw-2 primer sets successfully identified E. fawcettii isolates from Korea, Australia, and the United States (Florida) and the Eaut-1 to Eaut-5 primer sets identified both the sweet orange pathotype isolates of E. australis from Argentina and the natsudaidai pathotype isolates from Korea. The EaNat-1 and EaNat-2 primer sets were specific for isolates of the natsudaidai pathotype. The Efaw-1 and Efaw-2 primer sets successfully detected E. fawcettii from lesions on diseased leaves and fruit from Korea and primer pairs Eaut-1, Eaut-2, Eaut-3, Eaut-4, and Eaut-5 detected E. australis from lesions on sweet orange fruit from Brazil.

scholarly journals Molecular Verification of Bloom-forming Aphanizomenon flos-aquae and Their Secondary Metabolites in the Nakdong River

2018 ◽  
Vol 15 (8) ◽  
pp. 1739 ◽  
Author(s):  
Hae-Kyung Park ◽  
Mi-Ae Kwon ◽  
Hae-Jin Lee ◽  
Jonghee Oh ◽  
Su-Heon Lee ◽  
...  
Keyword(s):  
Secondary Metabolites ◽  
Water Samples ◽  
Its Region ◽  
Cyanobacterial Blooms ◽  
Specific Primer ◽  
Nakdong River ◽  
Off Flavors ◽  
Primer Sets ◽  
The Nakdong River ◽  
Harmful Cyanobacterial Blooms

Aphanizomenon spp. have formed harmful cyanobacterial blooms in the Nakdong River during spring, autumn, and now in winter, and the expansion of blooming period and area, associated with the global warming is predicted. The genus Aphanizomenon has been described to produce harmful secondary metabolites such as off-flavors and cyanotoxins. Therefore, the production of harmful secondary metabolites from the Aphanizomenon blooms in the Nakdong River needs to be monitored to minimize the risk to both water quality and public health. Here, we sampled the cyanobacterial blooms in the Nakdong River and isolated ten Aphanizomenon strains, morphologically classified as Aphanizomenon flos-aquae Ralfs ex Bornet et Flahault 1888. Phylogenetic analysis using 16S rRNA and internal transcribed spacer (ITS) region nucleotide sequences confirmed this classification. We further verified the harmful secondary metabolites-producing potential of A. flos-aquae isolates and water samples containing cyanobacterial blooms using PCR with specific primer sets for genes involved in biosynthesis of off-flavor metabolites (geosmin) and toxins (microcystins, saxitoxins and cylindrospermopsins). It was confirmed that these metabolite biosynthesis genes were not identified in all isolates and water samples containing only Aphanizomenon spp. Thus, it is likely that there is a low potential for the production of off-flavor metabolites and cyanotoxins in Aphanizomenon blooms in the Nakdong River.

scholarly journals How to copy and paste DNA microarrays

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Stefan D. Krämer ◽  
Johannes Wöhrle ◽  
Philipp A. Meyer ◽  
Gerald A. Urban ◽  
Günter Roth
Keyword(s):  
Real Time ◽  
Dna Microarray ◽  
Dna Microarrays ◽  
Label Free ◽  
Binding Assays ◽  
Single Stranded Dna ◽  
Whole Process ◽  
Double Stranded Dna ◽  
Cost Efficient ◽  
Copy And Paste

Abstract Analogous to a photocopier, we developed a DNA microarray copy technique and were able to copy patterned original DNA microarrays. With this process the appearance of the copied DNA microarray can also be altered compared to the original by producing copies of different resolutions. As a homage to the very first photocopy made by Chester Charlson and Otto Kornei, we performed a lookalike DNA microarray copy exactly 80 years later. Those copies were also used for label-free real-time kinetic binding assays of apo-dCas9 to double stranded DNA and of thrombin to single stranded DNA. Since each DNA microarray copy was made with only 5 µl of spPCR mix, the whole process is cost-efficient. Hence, our DNA microarray copier has a great potential for becoming a standard lab tool.

Sign in / Sign up

Export Citation Format

Share Document