scholarly journals Novel Genomic Tools for Specific and Real-Time Detection of Biothreat and Frequently Encountered Foodborne Pathogens

2012 ◽  
Vol 75 (4) ◽  
pp. 660-670 ◽  
Author(s):  
ABDELA WOUBIT ◽  
TESHOME YEHUALAESHET ◽  
TSEGAYE HABTEMARIAM ◽  
TEMESGEN SAMUEL
Keyword(s):  
Real Time ◽  
Foodborne Pathogens ◽  
Yersinia Pseudotuberculosis ◽  
Bacterial Species ◽  
Preliminary Test ◽  
Shigella Dysenteriae ◽  
Whole Genome ◽  
Tularensis Subsp ◽  
Primer Sets

The bacterial genera Escherichia, Salmonella, Shigella, Vibrio, Yersinia, and Francisella include important food safety and biothreat agents. By extensive mining of the whole genome and protein databases of diverse, closely and distantly related bacterial species and strains, we have identified novel genome regions, which we utilized to develop a rapid detection platform for these pathogens. The specific genomic targets we have identified to design the primers in Francisella tularensis subsp. tularensis, F. tularensis subsp. novicida, Shigella dysenteriae, Salmonella enterica serovar Typhimurium, Vibrio cholerae, Yersinia pestis, and Yersinia pseudotuberculosis contained either known genes or putative proteins. Primer sets were designed from the target regions for use in real-time PCR assays to detect specific biothreat pathogens at species or strain levels. The primer sets were first tested by in silico PCR against whole-genome sequences of different species, subspecies, or strains and then by in vitro PCR against genomic DNA preparations from 23 strains representing six biothreat agents (Escherichia coli O157:H7 strain EDL 933, Shigella dysenteriae, S. enterica serovar Typhi, F. tularensis subsp. tularensis, V. cholerae, and Y. pestis) and six foodborne pathogens (Salmonella Typhimurium, Salmonella Saintpaul, Shigella sonnei, F. tularensis subsp. novicida, Vibrio parahaemolyticus, and Y. pseudotuberculosis). Each pathogen was specifically identifiable at the genus and species levels. Sensitivity assays performed with purified DNA showed the lowest detection limit of 128 fg of DNA/μl for F. tularensis subsp. tularensis. A preliminary test to detect Shigella organisms in a milk matrix also enabled the detection of 6 to 60 CFU/ml. These new tools could ultimately be used to develop platforms to simultaneously detect these pathogens.

scholarly journals Pseudomonas aeruginosa as a Potential Contaminant of Packed Fresh-Cut Lettuce in a Controlled Atmosphere. The Role of Phenotypes muc+/muc–

2020 ◽  
Vol 11 (2) ◽  
pp. 8716-8724
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Real Time ◽  
Foodborne Pathogens ◽  
Rrna Gene ◽  
Modified Atmosphere ◽  
Fresh Cut ◽  
Time Specific ◽  
Sanitation Systems ◽  
Biofilm Development

In order to shed light on contamination risks along the ready-to-eat chain of fresh commodities by emerging foodborne pathogens, we investigated the biofilm development in vitro of two Pseudomonas aeruginosa strains on fresh-cut lettuce (Lactuca sativa L. var. Iceberg). The experiment was performed employing a floating bioreactor system where modified atmosphere package conditions were mimicked, and fresh-cut lettuce disks of 2 cm2 were put into contact with a 106 CFU/mL of a phenotypic mucoid P. aeruginosa phenotype (muc+) or a non-mucoid one (muc-). Following a simulated 2-day refrigerated-shelf quantitative Real-Time PCR, designed on a target gene region of the 16S rRNA gene, defined the different muc phenotypes behavior on biofilm in lettuce phyllo-plane. Between the two strains, a development difference of nearly 1.0 log CFU/cm2 occurred, with the muc+ phenotype being the most settled and adherent. This result clearly showed a distinct contamination risk according to P. aeruginosa phenotype and the need to develop real-time, specific, fast, and easy to use detection protocols along with specific sanitation systems for modified atmosphere package ready-to-eat commodities.

scholarly journals Customizable PCR-Microplate Array for Differential Identification of Multiple Pathogens

2013 ◽  
Vol 76 (11) ◽  
pp. 1948-1957 ◽  
Author(s):  
ABDELA WOUBIT ◽  
TESHOME YEHUALAESHET ◽  
SHERRELLE ROBERTS ◽  
MARTHA GRAHAM ◽  
MOONIL KIM ◽  
...  
Keyword(s):  
Salmonella Typhimurium ◽  
Francisella Tularensis ◽  
Yersinia Pseudotuberculosis ◽  
Salmonella Typhi ◽  
Rapid Identification ◽  
Shigella Dysenteriae ◽  
Tularensis Subsp ◽  
Differential Identification ◽  
Hot Dog ◽  
Simultaneous Identification

Customizable PCR-microplate arrays were developed for the rapid identification of Salmonella Typhimurium, Salmonella Saintpaul, Salmonella Typhi, Shigella dysenteriae, Escherichia coli O157:H7, Francisella tularensis subsp. tularensis, Francisella tularensis subsp. novicida, Vibrio cholerae, Vibrio parahaemolyticus, Yersinia pestis, and Yersinia pseudotuberculosis. Previously, we identified highly specific primers targeting each of these pathogens. Here, we report the development of customizable PCR-microplate arrays for simultaneous identification of the pathogens using the primers identified. A mixed aliquot of genomic DNA from 38 strains was used to validate three PCR-microplate array formats. Identical PCR conditions were used to run all the samples on the three formats. Specific amplifications were obtained on all three custom plates. In preliminary tests performed to evaluate the sensitivity of these assays in samples inoculated in the laboratory with Salmonella Typhimurium, amplifications were obtained from 1 g of beef hot dog inoculated at as low as 9 CFU/ml or from milk inoculated at as low as 78 CFU/ml. Such microplate arrays could be valuable tools for initial identification or secondary confirmation of contamination by these pathogens.

scholarly journals Development and validation of SYBR Green- and probe-based reverse-transcription real-time PCR assays for detection of the S and M segments of Schmallenberg virus

2020 ◽  
Vol 32 (5) ◽  
pp. 710-717
Author(s):  
Ahmet Kursat Azkur ◽  
Wim H. M. van der Poel ◽  
Emel Aksoy ◽  
Renate Hakze-van der Honing ◽  
Murat Yildirim ◽  
...  
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Real Time Pcr ◽  
Sybr Green ◽  
Schmallenberg Virus ◽  
Hypervariable Regions ◽  
Field Samples ◽  
Primer Sets ◽  
Development And Validation

Schmallenberg virus (SBV), discovered in Germany in 2011, causes congenital malformations in ruminants. Reverse-transcription real-time PCR (RT-rtPCR) assays based on various segments of SBV have been developed for molecular detection. We developed alternative RT-rtPCR assays for SBV detection to avoid earlier reported mutations and hypervariable regions of the S and M segments of the viral genome. For SYBR Green-based detection of the S segment, the R2 value and efficiency of the developed assay were 0.99 and 99%, respectively. For probe-based S segment detection, 2 assays were developed; the first had an R2 value of 0.99 and 102% efficiency, and the second had a R2 value of 0.98 and 86% efficiency. The probe-based M segment assay had an R2 value of 1.00 and 103% efficiency. Detection limits of the RT-rtPCR assays with new primer sets were 102 and 101 copies/µL for the S and M segments, respectively. Field samples from cattle and sheep were also used for primary validation of the developed assays. Our assays should be suitable for SBV detection in ruminants and for in vitro studies of various SBV strains.

scholarly journals DzyNA-PCR: Use of DNAzymes to Detect and Quantify Nucleic Acid Sequences in a Real-Time Fluorescent Format

2000 ◽  
Vol 46 (5) ◽  
pp. 625-630 ◽  
Author(s):  
Alison V Todd ◽  
Caroline J Fuery ◽  
Helen L Impey ◽  
Tanya L Applegate ◽  
Margaret A Haughton
Keyword(s):  
Real Time ◽  
Genomic Dna ◽  
Clinical Utility ◽  
General Strategy ◽  
Dye Molecules ◽  
Antisense Sequence ◽  
Genetic Sequences ◽  
Human Genomic ◽  
Primer Sets

Abstract Background: DzyNA-PCR is a general strategy for the detection and quantification of specific genetic sequences associated with disease or the presence of foreign agents. The method allows homogeneous gene amplification coupled with signal detection in a single closed vessel. Methods: The strategy involves in vitro amplification of genetic sequences using a DzyNA primer that harbors the complementary (antisense) sequence of a 10-23 DNAzyme. During amplification, amplicons are produced that contain active (sense) copies of DNAzymes that cleave a reporter substrate included in the reaction mixture. The accumulation of amplicons during PCR can be monitored in real time by changes in fluorescence produced by separation of fluoro/quencher dye molecules incorporated into opposite sides of a DNAzyme cleavage site within the reporter substrate. The DNAzyme and reporter substrate sequences can be generic and hence can be adapted for use with primer sets targeting various genes or transcripts. Results: Experiments using K-ras plasmid as template demonstrated that DzyNA-PCR allows quantification of DNA over at least six orders of magnitude (r = 0.992). Studies with human genomic DNA demonstrated the ability to resolve as little as twofold differences in the amount of starting template. DzyNA-PCR allowed the detection of 10 or fewer copies of the target. The clinical utility of the assay was demonstrated using DzyNA-PCR to analyze DNA that was isolated from human serum. Conclusion: DzyNA-PCR is a simple, rapid, and sensitive technique for homogeneous amplification and quantification of nucleic acids in clinical specimens.

scholarly journals Method comparison for the direct enumeration of bacterial species using a chemostat model of the human colon

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Ines B. Moura ◽  
Charmaine Normington ◽  
Duncan Ewin ◽  
Emma Clark ◽  
Mark H. Wilcox ◽  
...  
Keyword(s):  
Gut Microbiota ◽  
Real Time ◽  
Quantitative Methods ◽  
Pearson Correlation ◽  
Bacterial Species ◽  
Method Comparison ◽  
Recurrent Infection ◽  
Human Colon ◽  
Chemostat Model

Abstract Background Clostridioides difficile infection (CDI) has a high recurrent infection rate. Faecal microbiota transplantation (FMT) has been used successfully to treat recurrent CDI, but much remains unknown about the human gut microbiota response to replacement therapies. In this study, antibiotic-mediated dysbiosis of gut microbiota and bacterial growth dynamics were investigated by two quantitative methods: real-time quantitative PCR (qPCR) and direct culture enumeration, in triple-stage chemostat models of the human colon. Three in vitro models were exposed to clindamycin to induce simulated CDI. All models were treated with vancomycin, and two received an FMT. Populations of total bacteria, Bacteroides spp., Lactobacillus spp., Enterococcus spp., Bifidobacterium spp., C. difficile, and Enterobacteriaceae were monitored using both methods. Total clostridia were monitored by selective culture. Using qPCR analysis, we additionally monitored populations of Prevotella spp., Clostridium coccoides group, and Clostridium leptum group. Results Both methods showed an exacerbation of disruption of the colonic microbiota following vancomycin (and earlier clindamycin) exposure, and a quicker recovery (within 4 days) of the bacterial populations in the models that received the FMT. C. difficile proliferation, consistent with CDI, was also observed by both qPCR and culture. Pearson correlation coefficient showed an association between results varying from 98% for Bacteroides spp., to 62% for Enterobacteriaceae. Conclusions Generally, a good correlation was observed between qPCR and bacterial culture. Overall, the molecular assays offer results in real-time, important for treatment efficacy, and allow the monitoring of additional microbiota groups. However, individual quantification of some genera (e.g. clostridia) might not be possible without selective culture.

scholarly journals Salmonella entericaPhylogeny Based on Whole-Genome Sequencing Reveals Two New Clades and Novel Patterns of Horizontally Acquired Genetic Elements

mBio ◽  
2018 ◽  
Vol 9 (6) ◽  
Author(s):  
Jay Worley ◽  
Jianghong Meng ◽  
Marc W. Allard ◽  
Eric W. Brown ◽  
Ruth E. Timme
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Foodborne Pathogens ◽  
Bacterial Species ◽  
Whole Genome Sequence ◽  
Nucleotide Sequencing ◽  
Whole Genome ◽  
Nucleotide Polymorphisms ◽  
Content Type ◽  
Genetic Elements

ABSTRACTUsing whole-genome sequence (WGS) data from the GenomeTrakr network, a globally distributed network of laboratories sequencing foodborne pathogens, we present a new phylogeny ofSalmonella entericacomprising 445 isolates from 266 distinct serovars and originating from 52 countries. This phylogeny includes two previously unidentifiedS. entericasubsp.entericaclades. Serovar Typhi is shown to be nested within clade A. Our findings are supported by both phylogenetic support, based on a core genome alignment, and Bayesian approaches, based on single-nucleotide polymorphisms. Serovar assignments were refined byin silicoanalysis using SeqSero. More than 10% of serovars were either polyphyletic or paraphyletic. We found variable genetic content in these isolates relating to gene mobilization and virulence factors which have different distributions within clades. Gifsy-1- and Gifsy-2-like phages appear more prevalent in clade A; other viruses are more evenly distributed. Our analyses reveal IncFII is the predominant plasmid replicon inS. enterica. Few core or clade-defining virulence genes are observed, and their distributions appear probabilistic in nature. Together, these patterns demonstrate that genetic exchange withinS. entericais more extensive and frequent than previously realized, which significantly alters how we view the genetic structure of the bacterial species.IMPORTANCERapid improvements in nucleotide sequencing access and affordability have led to a drastic increase in availability of genetic information. This information will improve the accuracy of molecular descriptions, including serovars, withinS. enterica. Although the concept of serovars continues to be useful, it may have more significant limitations than previously understood. Furthermore, the discrete absence or presence of specific genes can be an unstable indicator of phylogenetic identity. Whole-genome sequencing provides more rigorous tools for assessing the distributions of these genes. Our phylogenetic and genetic content analyses reveal how active genetic elements are dynamically distributed within a species, allowing us to better understand genetic reservoirs and underlying bacterial evolution.

Microbiological Flora of Aquacultured Hybrid Striped Bass

1993 ◽  
Vol 56 (12) ◽  
pp. 1054-1060 ◽  
Author(s):  
PATTI C. NEDOLUHA ◽  
DENNIS WESTHOFF
Keyword(s):  
Striped Bass ◽  
Foodborne Pathogens ◽  
Yersinia Pseudotuberculosis ◽  
Wild Fish ◽  
Shigella Dysenteriae ◽  
Hybrid Striped Bass ◽  
Bacillus Spp ◽  
Plate Counts ◽  
Aerobic And Anaerobic

Aquacultured hybrid striped bass were examined for quantity and quality of microflora. Plate counts were performed at three temperatures (7, 22, and 35°C) under aerobic and anaerobic conditions. Bacterial loads on the skin, gills, and intestines were similar to those reported for wild fish. Plate counts performed at 22°C yielded the highest counts and the greatest variety of species. The predominant groups of bacteria isolated were Aeromonas spp. (27%), coryneforms (14%), Pseudomonas spp. (12%), Flavobacterium/Cytophaga/Sphingobacterium group (8%), Plesiomonas shigelloides (7%), Bacillus spp. (7%), and Enterobacteriaceae (6%). Human foodborne pathogens Listeria monocytogenes, Staphylococcus aureus, Shigella dysenteriae, Vibrio spp., and Yersinia pseudotuberculosis were also isolated. There were no indications, however, that pond-raised fish present a higher risk of foodborne illness than do wild fish.

scholarly journals The Response Regulator PhoP Is Important for Survival under Conditions of Macrophage-Induced Stress and Virulence in Yersinia pestis

2000 ◽  
Vol 68 (6) ◽  
pp. 3419-3425 ◽  
Author(s):  
Petra C. F. Oyston ◽  
Nick Dorrell ◽  
Kerstin Williams ◽  
Shu-Rui Li ◽  
Michael Green ◽  
...  
Keyword(s):  
Gene Expression ◽  
Yersinia Pestis ◽  
Pathogenic Bacteria ◽  
Yersinia Pseudotuberculosis ◽  
Bacterial Species ◽  
Lethal Dose ◽  
Wild Type ◽  
Virulence Gene Expression

ABSTRACT The two-component regulatory system PhoPQ has been identified in many bacterial species. However, the role of PhoPQ in regulating virulence gene expression in pathogenic bacteria has been characterized only in Salmonella species. We have identified, cloned, and sequenced PhoP orthologues from Yersinia pestis,Yersinia pseudotuberculosis, and Yersinia enterocolitica. To investigate the role of PhoP in the pathogenicity of Y. pestis, an isogenic phoPmutant was constructed by using a reverse-genetics PCR-based strategy. The protein profiles of the wild-type and phoP mutant strains, grown at either 28 or 37°C, revealed more than 20 differences, indicating that PhoP has pleiotrophic effects on gene expression in Y. pestis. The mutant showed a reduced ability to survive in J774 macrophage cell cultures and under conditions of low pH and oxidative stress in vitro. The mean lethal dose of the phoP mutant in mice was increased 75-fold in comparison with that of the wild-type strain, indicating that the PhoPQ system plays a key role in regulating the virulence of Y. pestis.

scholarly journals Using a Genome-Based PCR Primer Prediction Pipeline to Develop Molecular Diagnostics for the Turfgrass Pathogen Acidovorax avenae

Plant Disease ◽  
2018 ◽  
Vol 102 (11) ◽  
pp. 2224-2232 ◽  
Author(s):  
Paul R. Giordano ◽  
Jie Wang ◽  
Joseph M. Vargas ◽  
Janette Jacobs ◽  
Martin I. Chilvers ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Bacterial Species ◽  
Creeping Bentgrass ◽  
Pcr Assay ◽  
Putting Greens ◽  
Specificity And Sensitivity ◽  
A Genome ◽  
Polymerase Chain ◽  
Primer Sets

Acidovorax avenae is the causal agent of bacterial etiolation and decline (BED) of creeping bentgrass, a poorly understood and often misdiagnosed disease that can result in considerable aesthetic and functional damage to golf course putting greens. Current diagnostics of BED are based on laborious culture-based methods. In this work, we employed a novel alignment-free primer prediction pipeline to design diagnostic primers for turfgrass-pathogenic A. avenae using 15 draft genomes of closely related target and nontarget Acidovorax spp. as input. Twenty candidate primer sets specific to turfgrass-pathogenic A. avenae were designed. The specificity and sensitivity of these primer sets were validated via a traditional polymerase chain reaction (PCR) and a real-time PCR assay. Primer sets 0017 and 0019 coupled with an internal oligo probe showed optimal sensitivity and specificity when evaluated with the target pathogen, closely related bacterial species, and microorganisms that inhabit the same host and soil environment. Finally, the accuracy of the newly developed real-time PCR assay was evaluated to detect BED pathogens from BED-symptomatic and asymptomatic turfgrass samples. The diagnostic results produced by the real-time PCR assay were consistent with results of a cultural-based method. This assay will allow quicker and more effective detection of the BED pathogen, thus potentially reducing misdiagnoses and unnecessary usage of fungicides.

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