scholarly journals Development of a DNA Microarray for Molecular Identification of All 46 Salmonella O Serogroups

2013 ◽  
Vol 79 (11) ◽  
pp. 3392-3399 ◽  
Author(s):  
Dan Guo ◽  
Bin Liu ◽  
Fenxia Liu ◽  
Boyang Cao ◽  
Min Chen ◽  
...  
Keyword(s):  
Genomic Dna ◽  
Bacterial Species ◽  
Rapid Identification ◽  
Detection Sensitivity ◽  
Epidemiological Surveillance ◽  
Disease Assessment ◽  
Content Type ◽  
Detection And Identification ◽  
Food Borne

ABSTRACTSalmonellais a major cause of food-borne disease in many countries. Serotype determination ofSalmonellais important for disease assessment, infection control, and epidemiological surveillance. In this study, a microarray system that targets the O antigen-specific genes was developed for simultaneously detecting and identifying all 46SalmonellaO serogroups. Of these, 40 serogroups can be confidently identified, and the remaining 6, in three pairs (serogroups O67 and B, E1 and E4, and A and D1), need to be further distinguished from each other using PCR methods or conventional serotyping methods. The microarray was shown to be highly specific when evaluated against 293Salmonellastrains, 186Shigellastrains, representativeEscherichia colistrains, and 10 strains of other bacterial species. The assay correctly identified 288 (98%) of theSalmonellastrains. The detection sensitivity was determined to be 50 ng genomic DNA per sample. By testing simulated samples in a tomato background, 2 to 8 CFU per gram inoculated could be detected after enrichment. This newly developed microarray assay is the first molecular protocol that can be used for the comprehensive detection and identification of all 46SalmonellaO serogroups. Compared to the traditional serogrouping method, the microarray provides a reliable, high-throughput, and sensitive approach that can be used for rapid identification of multipleSalmonellaO serogroups simultaneously.

scholarly journals Use of a DNA Microarray for Detection and Identification of Bacterial Pathogens Associated with Fishery Products

2011 ◽  
Vol 77 (23) ◽  
pp. 8219-8225 ◽  
Author(s):  
Boyang Cao ◽  
Rongrong Li ◽  
Songjin Xiong ◽  
Fangfang Yao ◽  
Xiangqian Liu ◽  
...  
Keyword(s):  
Dna Microarray ◽  
Bacterial Species ◽  
Simultaneous Detection ◽  
Detection Sensitivity ◽  
23S Rrna ◽  
Rrna Gene ◽  
Bacterial Strains ◽  
Content Type ◽  
Detection And Identification ◽  
Fishery Products

ABSTRACTWe established a microarray for the simultaneous detection and identification of diverse putative pathogens often associated with fishery products by targeting specific genes ofListeria monocytogenes,Salmonella,Shigella,Staphylococcus aureus,Streptococcus pyogenes,Vibrio cholerae,Vibrio parahaemolyticus,Vibrio vulnificus, andYersinia enterocoliticaand the 16S-23S rRNA gene internal transcribed spacer (ITS) region ofProteus mirabilisandProteus vulgaris. The microarray contained 26 specific probes and was tested against a total of 123 target bacterial strains that included 55 representative strains, 68 clinical isolates, and 45 strains of other bacterial species that belonged to 8 genera and 34 species, and it was shown to be specific and reproducible. A detection sensitivity of 10 ng DNA or 10 CFU/ml for pure cultures of each target organism demonstrated that the assay was highly sensitive and reproducible. Mock and real fishery product samples were tested by the microarray, and the accuracy was 100%. The DNA microarray method described in this communication is specific, sensitive, and reliable and has several advantages over traditional methods of bacterial culture and antiserum agglutination assays.

scholarly journals Validation and Implementation of a Diagnostic Algorithm for DNA Detection ofBordetella pertussis,B. parapertussis, andB. holmesiiin a Pediatric Referral Hospital in Barcelona, Spain

2018 ◽  
Vol 57 (1) ◽  
Author(s):  
Ana Valero-Rello ◽  
Desiree Henares ◽  
Lesly Acosta ◽  
Mireia Jane ◽  
Iolanda Jordan ◽  
...  
Keyword(s):  
Internal Control ◽  
Whooping Cough ◽  
Diagnostic Algorithm ◽  
Clinical Samples ◽  
Analytical Validation ◽  
Content Type ◽  
Detection And Identification ◽  
Ofbordetella Pertussis ◽  
Lower Limits

ABSTRACTThis study aimed to validate a comprehensive diagnostic protocol based on real-time PCR for the rapid detection and identification ofBordetella pertussis,Bordetella parapertussis, andBordetella holmesii, as well as its implementation in the diagnostic routine of a reference children’s hospital. The new algorithm included a triplex quantitative PCR (qPCR) targeting IS481gene (inB. pertussis,B. holmesii, and someBordetella bronchisepticastrains), pIS1001(B. parapertussis-specific) andrnaseP as the human internal control. Two confirmatory singleplex tests forB. pertussis(ptxA-Pr) andB. holmesii(hIS1001) were performed if IS481was positive. Analytical validation included determination of linear range, linearity, efficiency, precision, sensitivity, and a reference panel with clinical samples. Once validated, the new algorithm was prospectively implemented in children with clinical suspicion of whooping cough presenting to Hospital Sant Joan de Deu (Barcelona, Spain) over 12 months. Lower limits of detection obtained were 4.4, 13.9, and 27.3 genomic equivalents/ml of sample for IS481(onB. pertussis), pIS1001and hIS1001, and 777.9 forptxA-Pr. qPCR efficiencies ranged from 86.0% to 96.9%. Intra- and interassay variabilities were <3% and <5%, respectively. Among 566 samples analyzed,B. pertussis,B. holmesii, andB. parapertussiswere detected in 11.1%, 0.9% (only in females >4 years old), and 0.2% of samples, respectively. The new algorithm proved to be a useful microbiological diagnostic tool for whooping cough, demonstrating a low rate of other non-pertussisBordetellaspecies in our surveilled area.

scholarly journals Development of a DNA Microarray Method for Detection and Identification of All 15 Distinct O-Antigen Forms of Legionella pneumophila

2013 ◽  
Vol 79 (21) ◽  
pp. 6647-6654 ◽  
Author(s):  
Boyang Cao ◽  
Fangfang Yao ◽  
Xiangqian Liu ◽  
Lu Feng ◽  
Lei Wang
Keyword(s):  
Dna Microarray ◽  
Legionella Pneumophila ◽  
Detection Sensitivity ◽  
Immunological Methods ◽  
Clinical Samples ◽  
Content Type ◽  
O Antigen ◽  
Detection And Identification ◽  
Legionnaires Disease ◽  
Reference Strains

ABSTRACTLegionellais ubiquitous in many environments. At least 50 species and 70 serogroups of the Gram-negative bacterium have been identified. Of the 50 species, 20 are pathogenic, andLegionella pneumophilais responsible for the great majority (approximately 90%) of the Legionnaires' disease cases that occur. Furthermore, of the 15L. pneumophilaserogroups identified, O1 alone causes more than 84% of the Legionnaires' disease cases that occur worldwide. Rapid and reliable assays for the detection and identification ofL. pneumophilain water, environmental, and clinical samples are in great demand.L. pneumophilabacteria are traditionally identified by their O antigens by immunological methods. We have recently developed an O serogroup-specific DNA microarray for the detection of all 15 distinct O-antigen forms ofL. pneumophila, including serogroups O1 to O15. A total of 35 strains were used to verify the specificity of the microarray, including 15L. pneumophilaO-antigen standard reference strains and sevenL. pneumophilaclinical isolates as target strains, seven reference strains of other non-pneumophila Legionellaspecies as closely related strains, and six non-Legionellabacterial species as nonrelated strains. The detection sensitivity was 1 ng of genomic DNA or 0.4 CFU/ml in water samples with filter enrichment and plate culturing. This study demonstrated that the microarray allows specific, sensitive, and reproducible detection ofL. pneumophilaserogroups. To the best of our knowledge, this is the first report of a microarray serotyping method for all 15 distinct O-antigen forms ofL. pneumophila.

scholarly journals Use of Bacteroidales Microbial Source Tracking To Monitor Fecal Contamination in Fresh Produce Production

2013 ◽  
Vol 80 (2) ◽  
pp. 612-617 ◽  
Author(s):  
Kruti Ravaliya ◽  
Jennifer Gentry-Shields ◽  
Santos Garcia ◽  
Norma Heredia ◽  
Anna Fabiszewski de Aceituno ◽  
...  
Keyword(s):  
Fresh Produce ◽  
Fecal Contamination ◽  
Microbial Source Tracking ◽  
Source Tracking ◽  
Rrna Gene ◽  
Specific Marker ◽  
Production Environment ◽  
Content Type ◽  
Food Borne

ABSTRACTIn recent decades, fresh and minimally processed produce items have been associated with an increasing proportion of food-borne illnesses. Most pathogens associated with fresh produce are enteric (fecal) in origin, and contamination can occur anywhere along the farm-to-fork chain. Microbial source tracking (MST) is a tool developed in the environmental microbiology field to identify and quantify the dominant source(s) of fecal contamination. This study investigated the utility of an MST method based onBacteroidales16S rRNA gene sequences as a means of identifying potential fecal contamination, and its source, in the fresh produce production environment. The method was applied to rinses of fresh produce, source and irrigation waters, and harvester hand rinses collected over the course of 1 year from nine farms (growing tomatoes, jalapeño peppers, and cantaloupe) in Northern Mexico. Of 174 samples, 39% were positive for a universalBacteroidalesmarker (AllBac), including 66% of samples from cantaloupe farms (3.6 log10genome equivalence copies [GEC]/100 ml), 31% of samples from tomato farms (1.7 log10GEC/100 ml), and 18% of samples from jalapeño farms (1.5 log10GEC/100 ml). Of 68 AllBac-positive samples, 46% were positive for one of three human-specific markers, and none were positive for a bovine-specific marker. There was no statistically significant correlation betweenBacteroidalesand genericEscherichia coliacross all samples. This study provides evidence thatBacteroidalesmarkers may serve as alternative indicators for fecal contamination in fresh produce production, allowing for determination of both general contamination and that derived from the human host.

scholarly journals A General Method for Rapid Determination of Antibiotic Susceptibility and Species in Bacterial Infections

2014 ◽  
Vol 53 (2) ◽  
pp. 425-432 ◽  
Author(s):  
Anja Mezger ◽  
Erik Gullberg ◽  
Jenny Göransson ◽  
Anna Zorzet ◽  
David Herthnek ◽  
...  
Keyword(s):  
Antibiotic Susceptibility ◽  
Bacterial Infections ◽  
Pathogenic Bacteria ◽  
Rapid Determination ◽  
Bacterial Species ◽  
Content Type ◽  
Tract Infections ◽  
Susceptibility Profiles ◽  
General Method

To ensure correct antibiotic treatment and reduce the unnecessary use of antibiotics, there is an urgent need for new rapid methods for species identification and determination of antibiotic susceptibility in infectious pathogenic bacteria. We have developed a general method for the rapid identification of the bacterial species causing an infection and the determination of their antibiotic susceptibility profiles. An initial short cultivation step in the absence and presence of different antibiotics was combined with sensitive species-specific padlock probe detection of the bacterial target DNA to allow a determination of growth (i.e., resistance) and no growth (i.e., susceptibility). A proof-of-concept was established for urinary tract infections in which we applied the method to determine the antibiotic susceptibility profiles ofEscherichia colifor two drugs with 100% accuracy in 3.5 h. The short assay time from sample to readout enables fast appropriate treatment with effective drugs and minimizes the need to prescribe broad-spectrum antibiotics due to unknown resistance profiles of the treated infection.

scholarly journals OmpT Outer Membrane Proteases of Enterohemorrhagic and Enteropathogenic Escherichia coli Contribute Differently to the Degradation of Human LL-37

2011 ◽  
Vol 80 (2) ◽  
pp. 483-492 ◽  
Author(s):  
Jenny-Lee Thomassin ◽  
John R. Brannon ◽  
Bernard F. Gibbs ◽  
Samantha Gruenheid ◽  
Hervé Le Moual
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Time Course ◽  
Dependent Manner ◽  
Content Type ◽  
Reading Frame ◽  
E Coli ◽  
Food Borne ◽  
Mutant Strains

ABSTRACTEnterohemorrhagicEscherichia coli(EHEC) and enteropathogenicE. coli(EPEC) are food-borne pathogens that cause serious diarrheal diseases. To colonize the human intestine, these pathogens must overcome innate immune defenses such as antimicrobial peptides (AMPs). Bacterial pathogens have evolved various mechanisms to resist killing by AMPs, including proteolytic degradation of AMPs. To examine the ability of the EHEC and EPEC OmpT outer membrane (OM) proteases to degrade α-helical AMPs,ompTdeletion mutants were generated. Determination of MICs of various AMPs revealed that both mutant strains are more susceptible than their wild-type counterparts to α-helical AMPs, although to different extents. Time course assays monitoring the degradation of LL-37 and C18G showed that EHEC cells degraded both AMPs faster than EPEC cells in an OmpT-dependent manner. Mass spectrometry analyses of proteolytic fragments showed that EHEC OmpT cleaves LL-37 at dibasic sites. The superior protection provided by EHEC OmpT compared to EPEC OmpT against α-helical AMPs was due to higher expression of theompTgene and, in turn, higher levels of the OmpT protein in EHEC. Fusion of the EPECompTpromoter to the EHECompTopen reading frame resulted in decreased OmpT expression, indicating that transcriptional regulation ofompTis different in EHEC and EPEC. We hypothesize that the different contributions of EHEC and EPEC OmpT to the degradation and inactivation of LL-37 may be due to their adaptation to their respective niches within the host, the colon and small intestine, respectively, where the environmental cues and abundance of AMPs are different.

scholarly journals A PCR Detection Method for Rapid Identification ofMelissococcus pluton in Honeybee Larvae

1998 ◽  
Vol 64 (5) ◽  
pp. 1983-1985 ◽  
Author(s):  
V. A. Govan ◽  
V. Br�zel ◽  
M. H. Allsopp ◽  
S. Davison
Keyword(s):  
Sequence Data ◽  
Bacterial Species ◽  
Pcr Primers ◽  
Rapid Identification ◽  
Rrna Gene ◽  
16S Rrna Sequence ◽  
Growth Requirements ◽  
Detection And Identification ◽  
Pcr Products ◽  
Pure Cultures

ABSTRACT Melissococcus pluton is the causative agent of European foulbrood, a disease of honeybee larvae. This bacterium is particularly difficult to isolate because of its stringent growth requirements and competition from other bacteria. PCR was used selectively to amplify specific rRNA gene sequences of M. pluton from pure culture, from crude cell lysates, and directly from infected bee larvae. The PCR primers were designed from M. pluton 16S rRNA sequence data. The PCR products were visualized by agarose gel electrophoresis and confirmed as originating from M. plutonby sequencing in both directions. Detection was highly specific, and the probes did not hybridize with DNA from other bacterial species tested. This method enabled the rapid and specific detection and identification of M. pluton from pure cultures and infected bee larvae.

scholarly journals Pyogranulomatous Pneumonia in Goats Caused by an Undescribed Porphyromonas Species, “Porphyromonas katsikii”

2014 ◽  
Vol 53 (3) ◽  
pp. 795-798 ◽  
Author(s):  
George Filioussis ◽  
Evanthia Petridou ◽  
Emmanouel Karavanis ◽  
Joachim Frey
Keyword(s):  
Agar Medium ◽  
Bacterial Species ◽  
Pcr Amplification ◽  
Rrna Gene ◽  
Undescribed Species ◽  
Acute Respiratory Disease ◽  
Anaerobic Conditions ◽  
Specific Primers ◽  
Content Type

A yet-undescribed bacterial species, tentatively named “Porphyromonaskatsikii,” was isolated from individuals of a small goat herd with pyogranulomatous pneumonia during an outbreak of acute respiratory disease. The isolated bacteria grew in the form of black-pigmented colonies after 14 days of incubation under anaerobic conditions at 37°C on a tryptic soy blood agar medium. The bacteria were identified as a yet-undescribedPorphyromonasspecies by determination of the nucleotide sequence of therrs16S rRNA gene, and this species was tentatively namedPorphyromonaskatsikii. PCR amplification with specific primers for this yet-undescribed species revealed the presence ofP. katsikiiin the lung tissue of all affected animals, while no PCR signals were evidenced from the lungs of healthy goats or from goats with pasteurellosis caused byMannheimia haemolytica. These data indicateP. katsikiias the causative agent of acute respiratory distress.P. katsikiiis phylogenetically related toPorphyromonassomeraeandPorphyromonas levii, which cause pathologies in humans and animals, respectively.P. katsikiiwas not detected by PCR from samples of the gingival pockets or of the faces of healthy goats.

scholarly journals Highly Sensitive Loop-Mediated Isothermal Amplification for the Detection of Leptospira

2015 ◽  
Vol 2015 ◽  
pp. 1-6 ◽  
Author(s):  
Hua-Wei Chen ◽  
Giulia Weissenberger ◽  
Erin Atkins ◽  
Chien-Chung Chao ◽  
Yupin Suputtamongkol ◽  
...  
Keyword(s):  
Genomic Dna ◽  
Bacterial Species ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Detection Sensitivity ◽  
Detection Methods ◽  
Rickettsia Conorii ◽  
Bartonella Bacilliformis ◽  
Loop Mediated Isothermal Amplification ◽  
Different Strains

Leptospirosis is a worldwide zoonosis caused by an infection with the pathogenic species of Leptospira. We have developed a loop-mediated isothermal amplification (LAMP) assay to detect the DNA of Leptospira spp. Six sets of primers targeting the gene of the subsurface protein, lipL32, were evaluated for their detection sensitivity. The best primer set detected less than 25 copies of lipL32 per reaction of both plasmid DNA template and purified leptospiral genomic DNA. By combining primers targeting lipL32 with the previously published primer set targeting lipL41, the sensitivity of the assay was improved to 12 copies of L. interrogans. The specificity of the LAMP assay was evaluated by using the genomic DNA from other clinically encountered bacterial species such as different strains of Orientia tsutsugamushi, Rickettsia typhi, Rickettsia conorii, Rickettsia rickettsii, Coxiella burnetii, and Bartonella bacilliformis. These genomic DNA samples were all negative in our LAMP assay. The sensitivity of the LAMP assay was very similar to that of quantitative real time PCR. Several detection methods for the amplified product of LAMP assay were performed to demonstrate the simplicity of the assay. In summary, our results have suggested that this assay is rapid, robust, and easy to perform and has the potential to be used in endemic locations.

scholarly journals High-Throughput Quantitative Analysis of the Human Intestinal Microbiota with a Phylogenetic Microarray

2009 ◽  
Vol 75 (11) ◽  
pp. 3572-3579 ◽  
Author(s):  
Oleg Paliy ◽  
Harshavardhan Kenche ◽  
Frank Abernathy ◽  
Sonia Michail
Keyword(s):  
Gut Microbiota ◽  
Genomic Dna ◽  
Bacterial Species ◽  
Detection Sensitivity ◽  
Rrna Gene ◽  
Healthy Children ◽  
Data Set ◽  
Fecal Samples ◽  
Human Intestinal Microbiota ◽  
Validation Experiments

ABSTRACT Gut microbiota carry out key functions in health and participate in the pathogenesis of a growing number of diseases. The aim of this study was to develop a custom microarray that is able to identify hundreds of intestinal bacterial species. We used the Entrez nucleotide database to compile a data set of bacterial 16S rRNA gene sequences isolated from human intestinal and fecal samples. Identified sequences were clustered into separate phylospecies groups. Representative sequences from each phylospecies were used to develop a microbiota microarray based on the Affymetrix GeneChip platform. The designed microbiota array contains probes to 775 different bacterial phylospecies. In our validation experiments, the array correctly identified genomic DNA from all 15 bacterial species used. Microbiota array has a detection sensitivity of at least 1 pg of genomic DNA and can detect bacteria present at a 0.00025% level of overall sample. Using the developed microarray, fecal samples from two healthy children and two healthy adults were analyzed for bacterial presence. Between 227 and 232 species were detected in fecal samples from children, whereas 191 to 208 species were found in adult stools. The majority of identified phylospecies belonged to the classes Clostridia and Bacteroidetes. The microarray revealed putative differences between the gut microbiota of healthy children and adults: fecal samples from adults had more Clostridia and less Bacteroidetes and Proteobacteria than those from children. A number of other putative differences were found at the genus level.

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