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PubMedDate not supplied

Surface plasmon resonance biosensors for detection of pathogenic microorganisms: strategies to secure food and environmental safety.

This review describes the exploitation of exclusively optical surface plasmon resonance (SPR) biosensors for the direct and indirect detection of pathogenic microorganisms in food chains and the environment. Direct detection is, in most cases, facilitated by the use of defined monoclonal or polyclonal antibodies raised against (a part of) the target pathogenic microorganisms. The antibodies were immobilized to a solid phase of the sensor to capture the microbe from the sample. Alternatively, antibodies were used in an inhibition-like assay involving incubation with the target organism prior to analysis of nonbound antibodies. The free immunoglobins were screened on a sensor surface coated with either purified antigens or with Fc or Fab binding antibodies. Discussed examples of these approaches are the determination of Escherichia coli O1 57:H7, Salmonella spp., and Listeria monocytogenes. Another direct detection strategy involved SPR analysis of polymerase chain reaction products of Shiga toxin-2 genes reporting the presence of E. coli O157:H7 in human stool. Metabolic products have been exploited as biomarkers for the presence of a microbial agent, such as enterotoxin B and a virulence factor for the occurrence of Staphylococcus aureus and Streptococcus suis, respectively. Indirect detection, on the other hand, is performed by analysis of a humoral immune response of the infected animal or human. By immobilization of specific antigenic structures, infections with Herpes simplex and human immunodeficiency viruses, Salmonella and Treponema pallidum bacteria, and Schistosoma spp. parasites were revealed using human, avian, and porcine sera and avian eggs. Bound antibodies were easily isotyped using an SPR biosensor to reveal the infection history of the individual. Discussed studies show the recent recognition of the suitability of this type of instrument for (rapid) detection of health-threatening microbes to food and environmental microbial safety.

Biosensing Techniques↗

PubMed1998

Detection of pathogenic Yersinia enterocolitica in enrichment media and pork by a multiplex PCR: a study of sample preparation and PCR-inhibitory components.

A multiplex PCR assay including sample preparation was developed to detect viable pathogenic strains of Yersinia enterocolitica in PCR-inhibitory samples, such as pork and enrichment media. The method developed was used to simultaneously detect the plasmid-borne virulence gene yadA and a Yersinia-specific region of the 16S rRNA gene. According to an auto-agglutination test for virulence-plasmid-bearing strains of Y. enterocolitica, all potential pathogenic strains tested were detected by the assay. A DNA extraction procedure, an aqueous two-phase system composed of polyethylene glycol 4000 and dextran 40 and a buoyant density centrifugation method, based on Percoll, were compared with regard to their efficiency in separating Yersinia enterocolitica from PCR inhibitors originating from enrichment media and pork. Using the density gradient centrifugation method resulted in a detection level of 4.0 x 10(2) CFU Y. enterocolitica per ml enrichment media. To ensure detection of viable bacteria a short enrichment step was included in the sample preparation together with the density gradient centrifugation. When this sample treatment method was evaluated with a selective enrichment medium together with a background flora inoculated with approximately 1.0 x 10(1) CFU per ml of Y. enterocolitica and incubated at 25 degrees C, a positive PCR result was obtained after 6 to 8 h. Our results indicate that selective enrichment followed by buoyant density gradient centrifugation provides a convenient and user-friendly sample preparation method prior to PCR.

Agglutination Tests↗

PubMed2006

Mass spectrometry-based proteomics for the detection of plant pathogens.

Plant diseases caused by fungi, oomycetes, viruses, and bacteria are devastating both to the economy and to the food supply of a nation. Therefore, the development of new, rapid methods to identify these pathogens is a highly important area of research that is of international concern. MS-based proteomics has become a powerful and increasingly popular approach to not only identify these pathogens, but also to better understand their biology. However, there is a distinction between identifying a pathogen protein and identifying a pathogen based upon the detection of one of its proteins and this must be considered before the general application of MS for plant pathogen detection is made. There has been a recent push in the proteomics community to make data from large-scale proteomics experiments publicly available in the form of a centralized repository. Such a resource could enable the use of MS as a universal plant pathogen detection technology.

Bacterial Proteins↗

PubMed2003

Laboratory diagnosis of Mycoplasma pneumoniae infection.

Diagnosis of Mycoplasma pneumoniae infection is challenging due to the fastidious nature of the pathogen, the considerable seroprevalence, and the possibility of transient asymptomatic carriage. During recent years, various new techniques have been adapted for the diagnosis of M. pneumoniae infection, notably in the field of molecular biology. Standard polymerase chain reaction (PCR) is currently the method of choice for direct pathogen detection, but several PCR-related methods provide enhanced sensitivity or more convenient handling procedures, and have been successfully applied for research purposes. Among these techniques are real-time PCR, nested PCR, reverse transcriptase PCR (RT-PCR) and multiplex PCR. Generally, amplification-based methods have replaced hybridization assays and direct antigen detection. Serology, which is the basic strategy for mycoplasma diagnosis in routine clinical practice, has been improved by the widespread availability of sensitive assays for separate detection of different antibody classes. For the diagnosis of mycoplasma pneumonia, serology and direct pathogen detection should be combined. Extrapulmonary diseases may be diagnosed by direct pathogen detection alone, but the value of this diagnostic approach is limited by the probably immunologically mediated pathogenesis of some manifestations. This review summarizes the current state of Mycoplasma pneumoniae diagnosis, with special reference to molecular techniques. The value of different methods for routine diagnosis and research purposes is discussed.

Culture Media↗

PubMed2006

Electrochemical detection of pathogen infection using cell chip.

The immobilized cell using self-assembled synthetic oligopeptide was applied to the electrochemical detection of pathogen infection. Thin films based on cysteine-terminated synthetic oligopeptides were fabricated for the immobilization of HeLa cell on gold (Au) substrate. Layer formation and immobilization of the cell were investigated with surface plasmon resonance (SPR) and electrochemical impedance spectroscopy (EIS). Experimental results showed that the thin film of cysteine-terminated synthetic oligopeptide was successfully fabricated and it could be applied for the immobilization of HeLa cells. The adhered living cell was exposed to E. coli O157:H7, which induced the change of SPR angle and electrochemical impedance signal. The proposed cell immobilization method using self-assembly technique can be applied to construct the cell microarray for on-site pathogen monitoring.

Carcinoma↗

PubMed2006

Nonantibody-based recognition: alternative molecules for detection of pathogens.

Immunoassays have been well established for many years as the cornerstone of detection technologies. These assays are sensitive, selective and, in general, highly resistant to interference from complex sample matrices when compared with nucleic acid-based tests. However, both antibody- and nucleic acid-based detection systems require a priori knowledge of the target and development of specific reagents; multiplexed assays can become increasingly problematic when attempting to detect a plethora of different targets, the identities of which are unknown. In an effort to circumvent many of the limitations inherent in these conventional assays, other recognition reagents are being explored as alternatives, or indeed as adjuncts, to antibodies for pathogen and toxin detection. This article will review a number of different recognition systems ranging in complexity from small molecules, such as nucleic-acid aptamers, carbohydrates and peptides, to systems as highly complicated as whole cells and organisms. All of these alternative systems have tremendous potential to achieve superior sensitivity, selectivity, and stability, but are also subject to their own limitations, which are also discussed. In short, while in its infancy, this field holds great promise for the development of rapid, fieldable assays that are highly complementary to existing antibody- and nucleic acid-based technologies.

Bacteria↗

PubMed1997

[Infection with Helicobacter pylori and its serologic detection].

Pathogenicity of Helicobacter pylori is based partially on the production of toxic substances. On the other hand the gastric mucosal injury may be caused by the Helicobacter pylori-induced host inflammatory response. Considerable antibody response during Helicobacter pylori infection provides a ground for indirect non-invasive serological detection. Appropriate clinical applications of serological tests are based on a good understanding of the antibody response characteristics during Helicobacter pylori infection.

Antibodies, Bacterial↗

PubMed1989

The use of the Listeria monocytogenes DTH gene for the detection of pathogenic biovars in food.

A gene probe encoding a Listeria monocytogenes delayed type hypersensitivity factor (DTH) was used for the detection of pathogenic biovars/serovars of the genus Listeria isolated from soft cheese. A total of 187 strains, isolated by plating techniques, were screened. The probe reacted with all isolated serovars of L. monocytogenes (except one 4b strain) and did not react with any of the Listeria innocua strains. Using the mouse bio-assay, as described by Kaufmann, a significant correlation was found between probe reaction and pathogenicity.

Animals↗

PubMed1994

Specific detection of pathogenic Yersinia enterocolitica by two-step PCR using hot-start and DMSO.

A pair of polymerase chain reaction (PCR) primers, YC1 and YC2, selected from the sequence of the invasin locus (inv) of Y. enterocolitica, has been evaluated for specific detection of pathogenic Y. enterocolitica by PCR. The primers were hybridized at high stringency conditions to DNA from 65 pathogenic Y. enterocolitica, 16 non-pathogenic Y. enterocolitica, 18 other Yersinia and 124 non-Yersinia strains. YC2 hybridized to the pathogenic Y. enterocolitica only, while YC1 hybridized weakly to nine non-Yersinia as well. In a PCR with annealing at 64 degrees C all Y. enterocolitica, pathogenic and non-pathogenic, were positive. However, DNA from 60 non-Y. enterocolitica was amplified. With annealing at 72 degrees C, 10 non-pathogenic Y. enterocolitica and 41 non-Y. enterocolitica were positive. When a two-step PCR assay with annealing at 72 degrees C, hot-start and 1% dimethylsulphoxide (DMSO) were used, only DNA from the pathogenic Y. enterocolitica were amplified. The limit of detection was shown for four different strains to be less than 10 cells per PCR tube.

Base Sequence↗

PubMed1999

Molecular detection of pathogen DNA in ticks (Acari: Ixodidae): a review.

Ticks play an important role in human and veterinary medicine, in particular due to their ability to transmit a wide spectrum of pathogenic micro-organisms of protozoal, rickettsial, bacterial and viral origin. Pathogens in ticks can be identified by conventional methods such as indirect immunofluorescence, isolation in cell culture or by using histological staining techniques. However, the advent of the polymerase chain reaction (PCR) has resulted in tremendous improvements in the specific and sensitive detection of pathogen DNA in ticks. In this paper, literature on DNA extraction methods, PCR protocols, primers and probes, which are in use for the successful detection and identification of pathogens in ticks, are critically reviewed. Some recommendations are also given towards the end of this review.

Anaplasma↗

PubMed2004

High-throughput detection of pathogenic yeasts of the genus trichosporon.

The need for a rapid and accurate method for the detection of fungal pathogens has become imperative as the incidence of fungal infections has increased dramatically. Herein, we tested the Luminex 100, a novel flow cytometer, for the detection of the medically important genus Trichosporon. This genus was selected as our proof-of-concept model due to the close phylogenetic relationship between the species. The method, which is based on a nucleotide hybridization assay, consists of a combination of different sets of fluorescent beads covalently bound to species-specific capture probes. Upon hybridization, the beads bearing the target amplicons are classified by their spectral addresses with a 635-nm laser. Quantitation of the hybridized biotinylated amplicon is based on fluorescence detection with a 532-nm laser. We tested in various multiplex formats 48 species-specific and group-specific capture probes designed in the D1/D2 region of ribosomal DNA, internal transcribed spacer regions, and intergenic spacer region. Species-specific biotinylated amplicons were generated with three sets of primers to yield fragments from the three regions. The assay was specific and fast, as it discriminated species differing by 1 nucleotide and required less than 50 min following amplification to process a 96-well plate. The sensitivity of the assay allowed the detection of 10(2) genome molecules in PCRs and 10(7) to 10(8) molecules of biotinylated amplification product. This technology provided a rapid means of detection of Trichosporon species with the flexibility to identify species in a multiplex format by combining different sets of beads.

Animals↗

PubMed2004

Blinded application of microscopy, bacteriological culture, immunoassays and PCR to detect gastrointestinal pathogens from faecal samples of patients with community-acquired diarrhoea.

A blinded trial in two different laboratories was performed to compare the detection of selected enteric pathogens in 92 unselected faecal samples collected from patients with community-acquired diarrhoea by conventional and PCR-based techniques. Conventional techniques detected a single potential etiological agent in 15% of the samples, whereas results of PCR detected evidence of at least one agent in 41% of the samples. Overall, the detection rates for the different pathogens were as follows: adenovirus serogroup F, 1%; Campylobacter spp., 7.6%; Salmonella spp., 4%; enteroaggregative Escherichia coli, 9.8%; enteropathogenic E. coli, 6.5%; enterotoxigenic Clostridium perfringens, 3%; Cryptosporidium spp., 13%; and Giardia spp., 11%. Results for the detection of Salmonella spp., Campylobacter spp. and C. perfringens were similar by both techniques, whereas Cryptosporidium and Giardia spp. were detected 22 times more often by PCR than by conventional microscopy. It was not possible to compare the results for detection of enteroaggregative E. coli and enteropathogenic E. coli since these were only investigated by PCR. The results of this small study clearly demonstrate the advantages of PCR-based methods compared to conventional techniques for the detection of gastrointestinal pathogens.

Clinical Laboratory Techniques↗

PubMed2005

Detection of pathogenic bacteria in food samples using highly-dispersed carbon particles.

There is an unmet need for detection methods that can rapidly and sensitively detect food borne pathogens. A flow through immunoassay system utilizing highly dispersed carbon particles and an amperometric technique has been developed and optimized. A sandwich immunoassay format was utilized in which pathogenic cells were captured by antibodies immobilized onto activated carbon particles, and labeled with horseradish peroxidase (HRP) conjugated antibodies. Flow of the peroxidase substrates resulted in an amperometric signal that is proportional to the number of captured cells. Factors influencing the analytical performance of the system, such as the quantity of carbon particles and concentrations of capture antibody, enzyme labeled antibody, and enzyme substrates, were investigated and optimized. Detection and quantification of Escherichia coli, Listeria monocytogenes and Campylobacter jejuni were demonstrated with low detection limits of 50, 10, and 50 cells/ml, respectively, and an overall assay time of 30 min. Milk and chicken extract samples were spiked with various concentrations of these pathogens and were used to challenge the system. The system design is flexible enough to allow its application to the detection of viruses and proteins.

Bacteria↗

PubMedDate not supplied

[Value of antigen, antibody and pathogen-specific lymphocyte detection in diagnosis of pathogen-induced arthritis].

In the differential diagnosis of infection-related arthritis (infectious arthritis, viral arthritis, reactive arthritis or Reiter's syndrome, Lyme disease) various laboratory methods are applied for the detection of the inciting antigen, specific antibodies or microbe-specific T-lymphocytes. In infectious (septic) bacterial or fungal arthritis, the definitive diagnosis can be made only by recovering the organism from the synovial fluid or membrane. Also, in reactive arthritis following extraarticular infection with Yersinia, Salmonella, Shigella, Campylobacter, or Chlamydia, one of the major shifts in perception of disease pathogenesis has been the detection of bacterial determinants by immunological methods and polymerase chain reaction (PCR) actually within the joint. In sexually acquired reactive arthritis, the etiologic diagnosis should be based on the direct detection of the pathogen (mainly C. trachomatis) from the urogenital smear specimen. For clinical routine, serological tests for bacteria specific antibodies (IgM and IgA class) are often necessary to show recent or persistent infection with the triggering pathogen. However, a cautionary note regarding the diagnostic significance of antibacterial antibody profiles has been sounded in several studies because of the high prevalence of bacteria-specific antibodies in the healthy population. The same problem may arise in the interpretation of virus-specific antibodies in the differential diagnosis of acute polyarthritis. Antigen-specific proliferation of synovial fluid lymphocytes can confirm the clinical diagnosis in patients with reactive arthritis and Lyme disease, although unspecific proliferation to several bacteria can also be observed in reactive arthritis as well as in many other arthritis.

Antibodies↗

PubMed2004

Multiplex PCR allows simultaneous detection of pathogens in ships' ballast water.

There is enormous potential for global transfer of microorganisms, including pathogens, in ships' ballast water. We contend that a major advancement in the study of ballast-water microorganisms in particular, and of aquatic pathogens in general, will be expedited sample analysis, such as provided by the elegant technology of DNA microarrays. In order to use DNA microarrays, however, one must establish the appropriate conditions to bind target sequences in samples to multiple probes on the microarrays. We conducted proof-of-concept experiments to optimize simultaneous detection of multiple microorganisms using polymerase chain reaction (PCR) and Southern hybridization. We chose three target organisms, all potentially found in ballast water: a calicivirus, the bacterium Vibrio cholerae, and the photosynthetic protist Aureococcus anophagefferens. Here, we show simultaneous detection of multiple pathogens is possible, a result supporting the promising future use of microarrays for simultaneous detection of pathogens in ballast water.

Blotting, Southern↗

PubMed1997

Detection of pathogenic Yersinia enterocolitica in milk and pork using a DIG-labelled probe targeted against the yst gene.

A PCR-amplified, digoxigenin (DIG)-labelled yst probe was evaluated in naturally and artificially contaminated pork and milk samples for its ability to detect and differentiate between pathogenic and nonpathogenic strains of Yersinia enterocolitica following enrichment in irgasan-ticarcillin-chlorate (ITC) enrichment broth. A total of 44 raw pork samples were obtained from one meat-processing factory and two local retail stores. Y. enterocolitica serogroup O:3/biovar 4 was isolated from 6 of the 44 (14%) pork samples tested. The hybridization results were in good agreement when compared with those from standard biochemical and serological tests. The lowest concentration detectable by a colony hybridization technique in the milk samples inoculated with Y. enterocolitica O:8 PA-A corresponded to 10 cfu/ml in the original sample. No pathogenic serovars were detected in 6 raw milk and 3 pasteurized milks tested. The results of this investigation demonstrate that the DIG-labelled yst probe is a reliable tool for rapid detection of pathogenic Y. enterocolitica in naturally and artificially contaminated food samples.

Animals↗

PubMed2006

Plant NBS-LRR proteins in pathogen sensing and host defense.

Plant proteins belonging to the nucleotide-binding site-leucine-rich repeat (NBS-LRR) family are used for pathogen detection. Like the mammalian Nod-LRR protein 'sensors' that detect intracellular conserved pathogen-associated molecular patterns, plant NBS-LRR proteins detect pathogen-associated proteins, most often the effector molecules of pathogens responsible for virulence. Many virulence proteins are detected indirectly by plant NBS-LRR proteins from modifications the virulence proteins inflict on host target proteins. However, some NBS-LRR proteins directly bind pathogen proteins. Association with either a modified host protein or a pathogen protein leads to conformational changes in the amino-terminal and LRR domains of plant NBS-LRR proteins. Such conformational alterations are thought to promote the exchange of ADP for ATP by the NBS domain, which activates 'downstream' signaling, by an unknown mechanism, leading to pathogen resistance.

Amino Acid Sequence↗

PubMed1989

The specificity of pilin DNA sequences for the detection of pathogenic Neisseria.

A nucleic acid hybridization assay for the detection of the pilin gene of Neisseria gonorrhoeae has been devised. The method involves solution hybridization of pilin specific synthetic oligonucleotide probes to genomic DNA in crude cell lysates. This is followed by capture of the probe-target complex onto a microtitre dish well, signal amplification and labelling based on horseradish peroxidase conjugated to oligonucleotides. Detection is achieved with a chemiluminescent enzyme substrate. With a detection limit of about 20,000 cells, the 4-h assay is as sensitive as a radioactive dot-blot method. Over 150 strains of Neisseria gonorrhoeae collected from a variety of sources were detected with the assay. Several N. meningitidis serogroups were also found to react positively. No reactivity was observed with non-pathogenic Neisseria spp. or with other known pathogenic or normal microbial inhabitants of the human urogenital tract.

Bacterial Outer Membrane Proteins↗