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Results for “pathogen detection”

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At least 163 records · Page 9Linked to original sources

PubMed1993

Detection of pathogenic Yersinia enterocolitica in foods and water by immunomagnetic separation, nested polymerase chain reactions, and colorimetric detection of amplified DNA.

A two-step polymerase chain reaction (PCR) procedure with two nested pairs of primers specific for the yadA gene of Yersinia enterocolitica was developed. The PCR assay identified all common pathogenic serogroups (O:3, O:5,27, O:8, O:9, O:13, and O:21) from three continents and differentiated pathogenic Y. enterocolitica from Y. pseudotuberculosis and from a variety of nonpathogenic yersiniae representing 25 serogroups and four species. The performance of the method was evaluated with seeded food and water samples. We compared two procedures for sample preparation prior to PCR: one was based on immunomagnetic separation of the target bacteria from the sample, using magnetic particles coated with immunoglobulin antibodies to Y. enterocolitica serogroup O:3, and the other method consisted of a series of centrifugation steps combined with proteinase treatment. Regardless of the method used, the PCR assay was capable of detecting 10 to 30 CFU/g of meat in 10(6)-fold excess of indigenous bacteria. When the samples were enriched overnight in a nonselective medium, the sensitivity was increased to approximately 2 CFU/g, except for samples with an extremely high background flora (> 10(7) CFU/g). We compared gel electrophoretic detection of PCR products with a colorimetric detection method designated DIANA (detection of immobilized amplified nucleic acids), which enabled easy visualization of amplified fragments in a microtiter plate format with an optical density reader. DIANA and gel electrophoresis showed complete concordance in their discrimination between positive and negative samples. The combination of immunomagnetic separation, nested PCR, and DIANA makes possible the development of a fully automated analytic process which requires a minimum of laboratory manipulations.

Adhesins, Bacterial↗

PubMed2000

[Development and approbation of polymerase chain reaction for detection of pathogen in Listeria infection].

A system of primers for detection of Listeria by polymerase chain reaction (PCR) has been developed. Specificity and sensitivity of the method was evaluated by analyses of reference clinical and abiotic samples. Clinical trials of PCR were carried by detecting latent (asymptomatic) carriers of Listeria among healthy women of epidemiologically significant professions. PCR is characterized by numerous advantages in comparison with the traditional bacteriological analysis.

DNA, Bacterial↗

PubMed2005

Multiplexed detection of pathogen DNA with DNA-based fluorescence nanobarcodes.

Rapid, multiplexed, sensitive and specific molecular detection is of great demand in gene profiling, drug screening, clinical diagnostics and environmental analysis. One of the major challenges in multiplexed analysis is to identify each specific reaction with a distinct label or 'code'. Two encoding strategies are currently used: positional encoding, in which every potential reaction is preassigned a particular position on a solid-phase support such as a DNA microarray, and reaction encoding, where every possible reaction is uniquely tagged with a code that is most often optical or particle based. The micrometer size, polydispersity, complex fabrication process and nonbiocompatibility of current codes limit their usability. Here we demonstrate the synthesis of dendrimer-like DNA-based, fluorescence-intensity-coded nanobarcodes, which contain a built-in code and a probe for molecular recognition. Their application to multiplexed detection of the DNA of several pathogens is first shown using fluorescence microscopy and dot blotting, and further demonstrated using flow cytometry that resulted in detection that was sensitive (attomole) and rapid.

Bacillus anthracis↗

PubMed2004

Detection of pathogenic bacteria in skin lesions of patients with chiclero's ulcer. Reluctant response to antimonial treatment.

We investigated the bacterial flora present in skin lesions of patients with chiclero's ulcer from the Yucatan peninsula of Mexico using conventional culture methods (11 patients), and an immunocolorimetric detection of pathogenic Streptococcus pyogenes (15 patients). Prevalence of bacteria isolated by culture methods was 90.9% (10/11). We cultured, from chiclero's ulcers (60%), pathogenic bacterial such as Staphylococcus aureus (20%), S. pyogenes (1.6%), Pseudomonas aeruginosa (1.6%), Morganella morganii (1.6%), and opportunist pathogenic bacteria such as Klebsiella spp. (20.0%), Enterobacter spp. (20%), and Enterococcus spp. (20%). We also cultured coagulase-negative staphylococci in 40% (4/10) of the remaining patients. Micrococcus spp. and coagulase-negative staphylococci constituted the bacterial genuses more frequently isolated in the normal skin of patients with chiclero's ulcer and healthy individuals used as controls. We also undertook another study to find out the presence of S. pyogenes by an immunocolorimetric assay. This study indicated that 60% (9/15) of the ulcerated lesions, but not normal controls, were contaminated with S. pyogenes. Importantly, individuals with purulent secretion and holding concomitant infections with S. pyogenes, S. aureus, P. aeruginosa, M. morganii, and E. durans took longer to heal Leishmania (L.) mexicana infections treated with antimonial drugs. Our results suggest the need to eliminate bacterial purulent infections, by antibiotic treatment, before starting antimonial administration to patients with chiclero's ulcer.

Antiprotozoal Agents↗

PubMed2005

Application of a spotting sample preparation technique for the detection of pathogens in woody plants by RT-PCR and real-time PCR (TaqMan).

An extraction technique for reverse transcription-PCR (RT-PCR) detection of plant pathogens including viruses, bacteria and phytoplasma is described. The total nucleic acid of these plant pathogens was obtained by direct spotting of crude sap derived from infected leaf, petiole or cambial tissue onto two different types of membranes, positively charged Hybond N(+) Nylon and FTA membranes, and processed for use in PCR. Thirteen different plant viruses, Xylella fastidiosa (causal agent of Pierce's disease) and phytoplasmas were included in the experiment. A thermal treatment (95 degrees C for 10 min) of the Hybond N(+) Nylon discs in a buffered solution improved the detection, but for FTA membrane discs the thermal treatment was not required and the discs were directly placed in the PCR reaction cocktail. Specific amplification of genomic or ribosomal RNA fragments of these pathogens was obtained by one-step RT-PCR except for X. fastidiosa in which a fragment of the genomic DNA was used for amplification. The same sample preparation methods also worked well for real-time RT-PCR (TaqMan). The sample preparation techniques reported here could be used to store samples for future PCR test or for long distance shipment to a detection laboratory.

Edetic Acid↗

PubMed2002

Indirect immunofluorescence colony staining method for detecting bacterial pathogens of tomato.

An indirect immunofluorescence colony staining method was developed for the detection of important seed-borne bacterial pathogens of tomato. The method involves the use of specific antiserum for initial binding of target bacteria and visualization of positive colonies with a commercially available secondary antiserum conjugated with FITC and observed under a fluorescence microscope. The indirect method is especially suitable for laboratories, seed companies, and quarantine stations which have no facilities for conjugation of primary antiserum. It is more economical and overcomes the problems generally encountered with variable conjugate quality in new batches of conjugates prepared from the same stock of primary antiserum. The assay is easy to perform and results can be easily assessed by visual scoring or image analyser. Results are available in 4-5 days as compared to 30-45 days in traditional methods. The resulting bacterial culture can be tested by PCR or host infectivity and a culture can be stored for future reference. Used in combination with highly specific antibodies (commercially available monoclonal and recombinant antibodies) it can be used as a very sensitive detection tool and has application potential in localization studies as well. Choosing the right secondary conjugate is however necessary to get best results in the assay.

Actinomycetales↗

PubMed1998

A simplified sample preparation method from various foods for PCR detection of pathogenic Yersinia enterocolitica: a possible model for other food pathogens.

A simplified method for the direct application of multiplex polymerase chain reaction (PCR) was developed to detect plasmid-bearing virulent serotypes of Yersinia enterocolitica (YEP+) in a variety of foods. Strains of YEP+ representing five serotypes were detected in enriched swab samples of artificially contaminated pork chops, ground pork, cheese and zucchini using multiplex PCR analysis. The method was also effective for identifying YEP+ strains in naturally contaminated porcine tongues. The use of swabs eliminated time-consuming extraction of DNA from food, inhibition of PCR by food-derived DNA, interference by background flora and reduced the time needed for processing samples. The detection of other food pathogens should be feasible by this technique.

Animals↗

PubMed2003

Detection of pathogenic bacteria in shellfish using multiplex PCR followed by CovaLink NH microwell plate sandwich hybridization.

Outbreak of diseases associated with consumption of raw shellfish especially oysters is a major concern to the seafood industry and public health agencies. A multiplex PCR amplification of targeted gene segments followed by DNA-DNA sandwich hybridization was optimized to detect the etiologic agents. First, a multiplex PCR amplification of hns, spvB, vvh, ctx and tl was developed enabling simultaneous detection of total Salmonella enterica serotype Typhimurium, Vibrio vulnificus, Vibrio cholerae and Vibrio parahaemolyticus from both pure cultures and seeded oysters. Amplicons were then subjected to a colorimetric CovaLink NH microwell plate sandwich hybridization using phosphorylated and biotinlylated oligonucleotide probes, the nucleotide sequences of which were located internal to the amplified DNA. The results from the hybridization with the multiplexed PCR amplified DNA exhibited a high signal/noise ratio ranging between 14.1 and 43.2 measured at 405 nm wavelength. The sensitivity of detection for each pathogen was 10(2) cells/g of oyster tissue homogenate. The results from this study showed that the combination of the multiplex PCR with a colorimetric microwell plate sandwich hybridization assay permits a specific, sensitive, and reproducible system for the detection of the microbial pathogens in shellfish, thereby improving the microbiological safety of shellfish to consumers.

Animals↗

PubMed2003

Detection of pathogenic Vibrio parahaemolyticus in oyster enrichments by real time PCR.

A real time polymerase chain reaction (PCR) assay was developed and evaluated to detect the presence of the thermostable direct hemolysin gene (tdh), a current marker of pathogenicity in Vibrio parahaemolyticus. The real time PCR fluorogenic probe and primer set was tested against a panel of numerous strains from 13 different bacterial species. Only V. parahaemolyticus strains possessing the tdh gene generated a fluorescent signal, and no cross-reaction was observed with tdh negative Vibrio or non-Vibrio spp. The assay detected a single colony forming unit (CFU) per reaction of a pure culture template. This sensitivity was achieved when the same template amount per reaction was tested in the presence of 2.5 microl of a tdh negative oyster:APW enrichment (oyster homogenate enriched in alkaline peptone water overnight at 35 degrees C). This real time technique was used to test 131 oyster:APW enrichments from an environmental survey of Alabama oysters collected between March 1999 and September 2000. The results were compared to those previously obtained using a streak plate procedure for culture isolation from the oyster:APW enrichment combined with use of a non-radioactive DNA probe for detection of the tdh gene. Real time PCR detected tdh in 61 samples, whereas the streak plate/probe method detected tdh in 15 samples. Only 24 h was required for detection of pathogenic V. parahaemolyticus in oyster:APW enrichments by real time PCR, whereas the streak plate/probe method required 3 days and was more resource intensive. This study demonstrated that real time PCR is a rapid and reliable technique for detecting V. parahaemolyticus possessing the tdh gene in pure cultures and in oyster enrichments.

Animals↗

PubMed2025

Development of a multiplex PCR for detection of pathogenic Mycobacterium orygis in cattle tissues harboring tuberculous-like lesions.

Mycobacterium orygis, a recently defined member species of Mycobacterium tubercuolsis complex (MTBC), is emerging as a major threat to zoonotic tuberculosis control, especially in the Asian Subcontinent. The dearth of low-cost diagnostic assay to differentiate M. orygis from other members of the MTBC leads to unavailability of information about the actual burden of this species in human and animal population. In this study, we developed a multiplex PCR for distinguishing M. orygis from other MTBC based on two M. orygis-specific nonsynonymous point mutations in mbtG and fadD23 genes identified by comparative genome analysis. The specificity of the assay shows that a 434 bp IS1081 fragment was amplified from common MTBC species including M. orygis while 240 bp and 181 bp mbtG and fadD23 gene fragments were amplified only from M. orygis. No amplification was observed for nontuberculous Mycobacterium (NTM) and non-Mycobacterial pathogens. The multiplex PCR assay showed a detection limit of 32 pg of M. orygis DNA. Furthermore, a total of 85 tuberculous-like lesions in the different tissues of slaughtered cattle were tested for identification of the M. orygis, and the results showed IS1081, mbtG and fadD23 amplicons in three tissue DNA extracts confirming they contain M. orygis DNA. Also, a single IS1081 amplicon was amplified from one tissue sample signifying presence of DNA of any MTBC species other than M. orygis. An established TaqMan real time PCR assay targeting region of differences (RD) in M. orygis genome was carried out to validate the result of the assay. This showed 100 % accuracy of the in-house developed multiplex PCR.

Mycobacterium orygis↗

PubMedDate not supplied

Lipid peroxidation products in human spermatozoa: detection and pathogenic significance.

Products of lipid peroxidation (Schiff-base products were detected in normal human spermatozoa. During aerobic incubation of the spermatozoa with an oxidant (ferrous sulphate) the peroxides became detectable in increased concentration. Coincidentally with the formation of peroxides the sperm motility declined.

Adult↗

PubMed2003

Comparative genomics tools applied to bioterrorism defence.

Rapid advances in the genomic sequencing of bacteria and viruses over the past few years have made it possible to consider sequencing the genomes of all pathogens that affect humans and the crops and livestock upon which our lives depend. Recent events make it imperative that full genome sequencing be accomplished as soon as possible for pathogens that could be used as weapons of mass destruction or disruption. This sequence information must be exploited to provide rapid and accurate diagnostics to identify pathogens and distinguish them from harmless near-neighbours and hoaxes. The Chem-Bio Non-Proliferation (CBNP) programme of the US Department of Energy (DOE) began a large-scale effort of pathogen detection in early 2000 when it was announced that the DOE would be providing bio-security at the 2002 Winter Olympic Games in Salt Lake City, Utah. Our team at the Lawrence Livermore National Lab (LLNL) was given the task of developing reliable and validated assays for a number of the most likely bioterrorist agents. The short timeline led us to devise a novel system that utilised whole-genome comparison methods to rapidly focus on parts of the pathogen genomes that had a high probability of being unique. Assays developed with this approach have been validated by the Centers for Disease Control (CDC). They were used at the 2002 Winter Olympics, have entered the public health system, and have been in continual use for non-publicised aspects of homeland defence since autumn 2001. Assays have been developed for all major threat list agents for which adequate genomic sequence is available, as well as for other pathogens requested by various government agencies. Collaborations with comparative genomics algorithm developers have enabled our LLNL team to make major advances in pathogen detection, since many of the existing tools simply did not scale well enough to be of practical use for this application. It is hoped that a discussion of a real-life practical application of comparative genomics algorithms may help spur algorithm developers to tackle some of the many remaining problems that need to be addressed. Solutions to these problems will advance a wide range of biological disciplines, only one of which is pathogen detection. For example, exploration in evolution and phylogenetics, annotating gene coding regions, predicting and understanding gene function and regulation, and untangling gene networks all rely on tools for aligning multiple sequences, detecting gene rearrangements and duplications, and visualising genomic data. Two key problems currently needing improved solutions are: (1) aligning incomplete, fragmentary sequence (eg draft genome contigs or arbitrary genome regions) with both complete genomes and other fragmentary sequences; and (2) ordering, aligning and visualising non-colinear gene rearrangements and inversions in addition to the colinear alignments handled by current tools.

Amino Acid Sequence↗

PubMed2004

Detection of pathogen Escherichia coli O157:H7 using self-excited PZT-glass microcantilevers.

Composite self-excited PZT-glass cantilevers (5 and 3 mm in length, 1.8 and 2.0 mm wide) were fabricated and their resonance characteristics were determined in air and at 1 mm liquid immersion. In air, resonance occurred at 65.8 and 63.4 kHz for the two cantilevers used in this paper. Monoclonal antibody (MAb) specific to the pathogen Escherichia coli (E. coli) O157:H7 was immobilized at the cantilever glass tip, and then exposed to pathogen in the concentration range of 7x10(2) to 7x10(7)bacteria/mL. Resonance of the second mode decreased due to pathogen attachment in accordance with a proposed kinetic model. The specific attachment rate constant was found to be 3x10(-9) to 5x10(-9) min-1 (cell/mL)-1. Exposure to a mixed population containing both a pathogenic and non-pathogenic strain showed that the antibody-immobilized cantilever is highly selective, thus demonstrating its usefulness for detecting water-borne pathogens.

Antibodies↗

PubMed2006

Developing nucleic acid-based electrical detection systems.

Development of nucleic acid-based detection systems is the main focus of many research groups and high technology companies. The enormous work done in this field is particularly due to the broad versatility and variety of these sensing devices. From optical to electrical systems, from label-dependent to label-free approaches, from single to multi-analyte and array formats, this wide range of possibilities makes the research field very diversified and competitive. New challenges and requirements for an ideal detector suitable for nucleic acid analysis include high sensitivity and high specificity protocol that can be completed in a relatively short time offering at the same time low detection limit. Moreover, systems that can be miniaturized and automated present a significant advantage over conventional technology, especially if detection is needed in the field. Electrical system technology for nucleic acid-based detection is an enabling mode for making miniaturized to micro- and nanometer scale bio-monitoring devices via the fusion of modern micro- and nanofabrication technology and molecular biotechnology. The electrical biosensors that rely on the conversion of the Watson-Crick base-pair recognition event into a useful electrical signal are advancing rapidly, and recently are receiving much attention as a valuable tool for microbial pathogen detection. Pathogens may pose a serious threat to humans, animal and plants, thus their detection and analysis is a significant element of public health. Although different conventional methods for detection of pathogenic microorganisms and their toxins exist and are currently being applied, improvements of molecular-based detection methodologies have changed these traditional detection techniques and introduced a new era of rapid, miniaturized and automated electrical chip detection technologies into pathogen identification sector. In this review some developments and current directions in nucleic acid-based electrical detection are discussed.

Journal Article↗

PubMed2003

Innovative tools for detection of plant pathogenic viruses and bacteria.

Detection of harmful viruses and bacteria in plant material, vectors or natural reservoirs is essential to ensure safe and sustainable agriculture. The techniques available have evolved significantly in the last few years to achieve rapid and reliable detection of pathogens, extraction of the target from the sample being important for optimising detection. For viruses, sample preparation has been simplified by imprinting or squashing plant material or insect vectors onto membranes. To improve the sensitivity of techniques for bacterial detection, a prior enrichment step in liquid or solid medium is advised. Serological and molecular techniques are currently the most appropriate when high numbers of samples need to be analysed. Specific monoclonal and/or recombinant antibodies are available for many plant pathogens and have contributed to the specificity of serological detection. Molecular detection can be optimised through the automatic purification of nucleic acids from pathogens by columns or robotics. New variants of PCR, such as simple or multiplex nested PCR in a single closed tube, co-operative-PCR and real-time monitoring of amplicons or quantitative PCR, allow high sensitivity in the detection of one or several pathogens in a single assay. The latest development in the analysis of nucleic acids is micro-array technology, but it requires generic DNA/RNA extraction and pre-amplification methods to increase detection sensitivity. The advances in research that will result from the sequencing of many plant pathogen genomes, especially now in the era of proteomics, represent a new source of information for the future development of sensitive and specific detection techniques for these microorganisms.

Bacteria↗

PubMed2006

A study of the role of multiple site blood cultures in the evaluation of neonatal sepsis.

BACKGROUND: The optimal number of blood cultures needed to document sepsis in an ill neonate has undergone little critical evaluation. Multiple site cultures may improve pathogen detection if intermittent bacteremia occurs, or if a low density of bacteria is present in the blood. We hypothesized, however, that bacterial clearance is slower and bacteremia more continuous in septic neonates, so that a single site blood culture should be sufficient to accurately document true septicemia. OBJECTIVE: To determine the need for multiple site blood cultures in the evaluation of neonates for sepsis. DESIGN/METHODS: Clinical data were prospectively collected for 216 neonates who had 269 pairs of blood cultures taken from two different peripheral sites for the evaluation of possible sepsis. A minimum of 1 ml of blood was obtained from the two peripheral sites within 15-30 min of each other. Based on prior retrospective data, we determined that 203 infants would need to have two site blood cultures to demonstrate a significant improvement in pathogen detection at an alpha of 0.05 and a beta of 0.20 (80%) power. RESULTS: A total of 186 culture pairs were taken for evaluation of early-onset sepsis in 186 neonates, while 83 pairs were drawn for evaluation of late-onset sepsis in 43 neonates. In all, 21 neonates from the late-onset group were evaluated more than once, and 12 neonates were evaluated for both early- and late-onset sepsis. In all, 20 (9.2%) of 216 neonates had 22 episodes of culture-proven sepsis at a median age of 18 days. All neonates with positive cultures had the same organism with a similar sensitivity pattern obtained from the two different peripheral sites. The other 196 study neonates had negative blood cultures from both sites. The single episode of early-onset sepsis was caused by Listeria monocytogenes, while all remaining episodes were late-onset with the following organisms: Staphylococcus epidermidis (7), methicillin-resistant Staphylococcus aureus (MRSA) (3), combined MRSA and Candida albicans (2), Candida albicans alone (2), late-onset Group B beta-hemolytic Streptococcus (GBS) (2), Klebsiella pneumoniae (2), Enterococcus fecalis (1), Escherichia coli (1), and Serratia marcescens (1). Since no infant grew organisms from only one of the two sites, the data indicate that the diagnosis of sepsis would have been made correctly in all infants with a single site culture. CONCLUSIONS: Two site blood cultures for the initial evaluation of neonatal sepsis do not have a better yield in pathogen detection. Sepsis in neonates can be detected with no loss of accuracy with a single site blood culture with blood volume of>or=1 ml.

Bacteremia↗

PubMed2005

Multiplex PCR-DNA probe assay for the detection of pathogenic Escherichia coli.

A multiplex PCR-DNA probing assay was developed to detect four major Escherichia coli virotypes. Six highly specific polymerase chain reaction (PCR) primer sets and DIG-labeled chemiluminescent probes were designed to target the Shiga-like toxin I and II genes (stxI and stxII) of verotoxigenic E. coli (VTEC), heat-stable and heat-labile toxin genes of enterotoxigenic E. coli (ETEC), adherence factor (EAF) of enteropathogenic E. coli (EPEC) and a fragment of the invasiveness plasmid (IAL) of enteroinvasive E. coli (EIEC). The primer pairs generate products of 350, 262, 170, 322, 293 and 390 bp in length, respectively. The multiplex primers and probes were tested for specificity against 31 pathogenic E. coli strains, nine nonpathogenic E. coli and non-E.coli enteric and environmental bacterial strains. The results showed a high degree of specificity of the primers and probes for strains from corresponding virotypes and no reaction with the nontarget bacterial strains. The proposed multiplex PCR-DNA probing assay provides rapid and specific detection of four major virotypes of E. coli.

Adhesins, Bacterial↗

PubMed2003

New biochip technology for label-free detection of pathogens and their toxins.

microSERS is a new biochip technology that uses surface-enhanced Raman scattering (SERS) microscopy for label-free transduction. The biochip itself comprises pixels of capture biomolecules immobilized on a SERS-active metal surface. Once the biochip has been exposed to the sample and the capture biomolecules have selectively bound their ligands, a Raman microscope is used to collect SERS fingerprints from the pixels on the chip. SERS, like other whole-organism fingerprinting techniques, is very specific. Our initial studies have shown that the Gram-positive Listeria and Gram-negative Legionella bacteria, Bacillus spores and Cryptosporidium oocysts can often be identified at the subspecies/strain level on the basis of SERS fingerprints collected from single organisms. Therefore, pathogens can be individually identified by microSERS, even when organisms that cross-react with the capture biomolecules are present in a sample. Moreover, the SERS fingerprint reflects the physiological state of a bacterial cell, e.g., when pathogenic Listeria and Legionella were cultured under conditions known to affect virulence, their SERS fingerprints changed significantly. Similarly, nonviable (e.g., heat- or UV-killed) microorganisms could be differentiated from their viable counterparts by SERS fingerprinting. Finally, microSERS is also capable of the sensitive and highly specific detection of toxins. Toxins that comprised as little as 0.02% by weight of the biomolecule-toxin complex produced strong, unique fingerprints when spectra collected from the complexes were subtracted from the spectra of the uncomplexed biomolecules. For example, aflatoxins B(1) and G(1) could be detected and individually identified when biochips bearing pixels of antibody or enzyme capture biomolecules were incubated in samples containing one or both aflatoxins, and the spectra were then collected for 20 s from an area of the biomolecule pixel approximately 1 microm in diameter. In the future, we plan to investigate the use of hyperspectral imaging Raman microscopy for collecting fingerprints from all the pixels on the biochip, individually yet simultaneously, to enable the rapid detection of diverse pathogens and their toxins in a sample, using a single biochip.

Aflatoxins↗