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PubMed2004

Low-level detection of a bacillus anthracis simulant using Love-wave biosensors on 36 degrees YX LiTaO3.

We present an acoustic Love-wave biosensor for detection of the Bacillus anthracis simulant, Bacillus thuringiensis at or below inhalational infectious levels. The present work is an experimental study of 36 degrees YX cut LiTaO3 based Love-wave devices for detection of pathogenic spores in aqueous conditions. Given that the detection limit (D1) of Love-wave-based sensors is a strong function of the overlying waveguide, two waveguide materials have been investigated, which are polyimide and polystyrene. To determine the mass sensitivity of Love-wave sensor, bovine serum albumin (BSA) protein was injected into the Love-wave test cell while recording the magnitude and phase shift across each sensor. Polyimide had the lowest mass detection limit with an estimated value of 1.0-2.0 ng/cm2, as compared to polystyrene where D1 = 2.0 ng/cm2. Suitable chemistries were used to orient antibodies on the Love-wave sensor using protein G. The thickness of each biofilm was measured using ellipsometry from which the surface concentrations were calculated. The monoclonal antibody BD8 with a high degree of selectivity for anthrax spores was used to capture the non-pathogenic simulant B. thuringiensis B8 spores. Bacillus subtilis spores were used as a negative control to determine whether significant non-specific binding would occur. Spore aliquots were prepared using an optical counting method, which permitted removal of background particles for consistent sample preparation. This work demonstrates that Love-wave biosensors are promising for low-level detection for whole-cell biological pathogens.

Bacillus anthracis↗

PubMed2005

Migration of Caenorhabditis elegans to manure and manure compost and potential for transport of Salmonella newport to fruits and vegetables.

A study was done to determine if a free-living, bacterivorous nematode, Caenorhabditis elegans, migrates to bovine manure, turkey manure, composted bovine manure, composted turkey manure, and manure-amended soil inoculated with Salmonella Newport. Movement of the worm to lettuce, strawberries, and carrots was also studied. C. elegans moved most rapidly to turkey manure and strawberries, with 35% and 60% of worms, respectively, associating with samples within 30 min. Survival and reproduction of C. elegans in test materials were not affected by the presence of S. newport. Bovine manure and bovine manure compost inoculated with S. newport (8.6 log10 CFU/g) were separately placed in the bottom of a glass jar and covered with a layer of soil (5 cm) inoculated (50 worms/g) or not inoculated with C. elegans. A piece of lettuce, strawberry, or carrot was placed on top of the soil before jars were sealed and held at 20 degrees C for up to 10 days. In the system using soil inoculated with C. elegans, S. newport initially in bovine manure was detected on the surface of lettuce, strawberry, and carrot samples within 3, 1, and 1 days, respectively. The pathogen was detected on lettuce, strawberry, and carrot within 1, 7, and 1 days, respectively, when initially present in bovine manure compost. With one exception, the pathogen was not detected on the produce over the 10-day incubation period when C. elegans was not present in the soil. Results indicate that C. elegans has the potential for transporting S. newport in soil to the surface of preharvest fruits and vegetables in contact with soil.

Animals↗

PubMed2000

Molecular detection of Marteilia sydneyi, pathogen of Sydney rock oysters.

The life cycle of Marteilia sydneyi, the aetiological agent of QX disease in the Sydney rock oyster Saccostrea commercialis, is not known. We have developed and optimised 2 diagnostic assays, the polymerase chain reaction (PCR) and in situ hybridisation, for use in investigating the role of possible alternative hosts in the life cycle of this pathogen. PCR primers, designed within the ITS1 rDNA of M. sydneyi, amplified a 195 bp fragment. Sensitivity of the PCR assay was assessed using DNA extracted from known numbers of sporonts purified from infected oyster digestive gland. DNA equivalent to 0.01 sporonts was detectable following agarose gel electrophoresis. The potential inhibitory effect of the presence of host DNA on the PCR assay was tested by the addition of oyster genomic DNA during amplification. Concentrations of host DNA in excess of 50 ng per 20 microliters reaction reduced the sensitivity of the test. Environmental validation of the PCR assay was demonstrated by the amplification of M. sydneyi DNA from 50 ng of genomic DNA extracted from QX-infected oysters. A DNA probe was constructed using the M. sydneyi unique primers and was able to detect 10 pg of M. sydneyi PCR amplified DNA in dot-blot hybridisations. The probe hybridised with presporulating and sporulating M. sydneyi stages in paraffin sections of oyster digestive gland. No non-specific binding was observed. Hybridisation consistency and signal intensity decreased as sporonts matured. While the high sensitivity and specificity of the PCR test will allow rapid screening of large numbers of potential alternative hosts for the presence of parasite DNA, it does not actually identify infective stages. In situ hybridisation conducted on paraffin sections will determine the location of the parasite within the host for morphological characterisation.

Animals↗

PubMed2001

Fluorogenic PCR-based quantitative detection of a murine pathogen, Helicobacter hepaticus.

Helicobacter hepaticus infection in mice is being used as an animal model for elucidating the pathogenesis of gastrointestinal and biliary diseases in humans. H. hepaticus, which forms a spreading film on selective agar, is not amenable to routine quantitative counts of organisms in tissues using a CFU method. In this study, a fluorogenic PCR-based assay was developed to quantitatively detect H. hepaticus in mouse ceca and feces using the ABI Prism 7700 sequence detection system. A pair of primers and a probe for this assay were generated from the H. hepaticus cdtB gene (encoding subunit B of the H. hepaticus cytolethal distending toxin). Using this assay, the sensitivity for detection of H. hepaticus chromosomal DNA prepared from pure culture was 20 fg, which is equivalent to approximately 14 copies of the H. hepaticus genome based on an estimated genome size of approximately 1.3 Mb determined by pulsed-field gel electrophoresis. H. hepaticus present in feces and cecal samples from H. hepaticus-infected mice was readily quantified. The selected PCR primers and probe did not generate fluorescent signals from eight other helicobacters (H. canis, H. cineadi, H. felis, H. mustelae, H. nemestrinae, H. pullorum, H. pylori, and H. rodentium). A fluorescent signal was detected from 20 ng of H. bilis DNA but with much lower sensitivity (10(6)-fold) than from H. hepaticus DNA. Therefore, this assay can be used with high sensitivity and specificity to quantify H. hepaticus in experimentally infected mouse models as well as in naturally infected mice.

Animals↗

PubMed2003

Evaluation of the diagnostic accuracy of the stop codon (SC) assay for identifying protein-truncating mutations in the BRCA1and BRCA2genes in familial breast cancer.

Screening for protein-truncating mutations of the BRCA1 and BRCA2 genes is useful in genetic testing for familial breast cancer because, first, the methods are usually simple and not expensive, and second, the detected mutations indicate pathogenic mutations in general. We evaluated the diagnostic accuracy of the stop codon (SC) assay for detecting protein-truncating mutations in the BRCA1 and BRCA2 genes by comparing the results with DNA sequencing in samples from 29 patients with breast cancer from 24 Japanese families with a history of breast cancer. Protein-truncating mutations were detected in 5 of the 24 families (20.8%; two in the BRCA1 gene and three in the BRCA2 gene). Among the 176 DNA fragments examined using the SC assay, the existence of three protein-truncating mutations (one in the BRCA1 gene and two in the BRCA2gene) was predicted correctly by the assay. Only one reverse transcriptase-polymerase chain reaction fragment was positive for the SC assay but was negative using DNA sequencing. Our study showed clearly that the SC assay is sensitive (3 of 3, 100%) and specific (172 of 173, 99%) for detecting pathogenic protein-truncating mutations in the BRCA1 and BRCA2 genes, and that it could be useful for screening larger populations.

Biological Assay↗

PubMed2006

Very low prevalence of germline MSH6 mutations in hereditary non-polyposis colorectal cancer suspected patients with colorectal cancer without microsatellite instability.

Hereditary non-polyposis colorectal cancer (HNPCC) is caused by mutations in one of the mismatch repair genes MLH1, MSH2, MSH6, or PMS2 and results in high-level microsatellite instability (MSI-high) in tumours of HNPCC patients. The MSI test is considered reliable for indicating mutations in MLH1 and MSH2, but is questioned for MSH6. Germline mutation analysis was performed in 19 patients with an MSI-high tumour and absence of MSH2 and/or MSH6 protein as determined by immunohistochemistry (IHC), without an MLH1 or MSH2 mutation, and in 76 out of 295 patients suspected of HNPCC, with a non-MSI-high colorectal cancer (CRC). All 295 non-MSI-high CRCs were analysed for presence of MSH6 protein by IHC. In 10 patients with an MSI-high tumour without MSH2 and/or MSH6 expression, a pathogenic MSH6 mutation was detected, whereas no pathogenic MSH6 mutation was detected in 76 patients with a non-MSI-high CRC and normal MSH6 protein expression. In none of the 295 CRCs loss of MSH6 protein expression was detected. The prevalence of a germline MSH6 mutation is very low in HNPCC suspected patients with non-MSI-high CRC. Microsatellite instability analysis in CRCs is highly sensitive to select patients for MSH6 germline mutation analysis.

Adult↗

PubMed2005

Design and development of a DNA array for rapid detection and genotyping of seven kinds of pathogenic microbes.

A DNA array for rapid detection and genotyping of the pathogenic microbes of epidemic hemorrhagic fever, tsutsugamushi disease, leptospirosis, malaria, schistosomiasis, cholera, and hemorrhagic colitis was developed. The specific and relatively conserved PCR primers and DNA probes were screened from the characteristic genes of the pathogenic microbes. The PCR or RT-PCR methods were established for amplifying and labeling the DNA fragments of the pathogenic microbes. All the probes with the same Tm value were synthesized chemically and modified with an NH2 at their 5' terminus, they were printed on glass slides for fabrication of a oligonucleotide DNA array. The developed DNA array could be used for detecting and genotyping the pathogenic microbes simultaneously, and they had a high sensitivity and specificity.

Animals↗

PubMed2001

Detection of putative oral pathogens in acute periradicular abscesses by 16S rDNA-directed polymerase chain reaction.

A 16S rDNA-directed polymerase chain reaction method was used to assess the occurrence of four black-pigmented anaerobic rods, Treponema denticola, and Actinobacillus actinomycetemcomitans in acute periradicular abscesses. Pus was collected by aspiration from 10 cases diagnosed as acute abscesses of endodontic origin. DNA was extracted from the samples and analyzed using a polymerase chain reaction-based identification assay. The method allowed detecting black-pigmented anaerobes in 80% of the examined abscesses. Porphyromonas endodontalis was found in 70%, T. denticola in 50%, Porphyromonas gingivalis in 40%, and Prevotella intermedia in 10% of the cases. P. gingivalis was always found associated with P. endodontalis. Prevotella nigrescens and A. actinomycetemcomitans were not found in any pus sample. The high prevalence of P. endodontalis, T. denticola, and P. gingivalis suggests that they can play an important role in the etiology of acute periradicular abscesses.

Adult↗

PubMed2005

Electrohydrodynamic (EHD) dispensing of nanoliter DNA droplets for microarrays.

In the present paper, we first demonstrated the possibility of electrohydrodynamic (EHD) dispensing method for preparing nanoliter probe DNA droplets on surfaces in DNA microarrays. To study the effect of an electric field on the dynamic behavior of pendent DNA droplet, visualization experiments with three kinds of electrode shapes are performed. In the early stage of droplet dispensing, it is shown that applied electric field assists a gravitational force exerted on DNA droplet. The pendent droplet is elongated in the parallel direction of applied electric field. However, after making fluid bridge between electrodes, it is shown that the electric force accelerates the capillary breaking of droplet by assisting a surface tension force exerted on droplet surface. Specifically, nanoliter dispensing volume (2 nL) is obtained in the needle-type electrode configuration. In addition, for the case of hydrophobic electrode surface, it is shown that the dispensing volume and spot size are remarkably decreased. Under the high relative humidity condition, it is observed that spot size is rapidly increased because of reduction in evaporation rate on droplet surface during the dispensing procedure. On the other hand, it is obtained that the spot size is not changed significantly in the wide range of DNA concentration from 1 to 10,000 nM. To monitor the influence of high electric voltage on DNA stability, we prepared a silicon-based chip with five capture probes for pathogens related with respiratory infectious diseases by EHD dispensing method. From the examination, it is clearly confirmed that pathogens are detected and the effective signal levels of pathogenic bacteria after hybridization are retained. Consequently, it is found that EHD dispensing method can be used to make cost-effective DNA microarrays with no thermal and electrical influences on DNA properties.

DNA↗

PubMed2000

[Signal identification of potentially pathogenous Yersinia].

A four-tube method with diagnostic nutrient media is proposed for differentiating between pathogenic and apathogenic Yersinia. Yersinia cultures are detected by inoculating typical or suspected colonies from solid media to media with lactose, glucose, urea, and indicators. Subsequent culturing in three other media (with sucrose and sorbitol, with esculin, rhamnose, and salicin, and with pyrasinamide) and the use of appropriate indicators help detect potentially pathogenous pseudotuberculous and enteric Yersinia of 1B, 2, 3, and 5 biovars from apathogenic species and variants. The method is simple, rapid, economic, reliable, and is recommended for clinical laboratory diagnosis.

Bacterial Typing Techniques↗

PubMed1994

Update of the rapid diagnosis of infectious diseases. I: Bacteria, fungi and parasites.

The application of monoclonal antibodies and DNA probes in the clinical microbiology laboratory has resulted in an array of rapid diagnostic tests. The immunofluorescent assay or enzyme-linked immunoassay is widely used in the rapid diagnosis of bacteria eg Group A streptococcus, Legionella pneumophila, Mycoplasma pneumoniae, Bordetella pertussis; parasites eg Chlamydia tachomatis, Cryptosporidium species; and fungi eg Pneumocystis carinii. The BACTEC system was first introduced to detect bacteraemia pathogens. It has been further developed to detect Mycobacterium species in clinical specimens and this has greatly reduced turn-around time in the laboratory diagnosis of Mycobacterium species. The discovery of the polymerase chain reaction has led to hopes of using it as a potential diagnostic tool in the microbiology laboratory.

Bacterial Infections↗

PubMed2002

Detection and differentiation of pathogenicity of avian paramyxovirus serotype 1 from field cases using one-step reverse transcriptase-polymerase chain reaction.

Amplification of avian paramyxovirus serotype 1 (APMV-1)-specific nucleic acid fragments, followed by restriction endonuclease analysis (REA) using BglI, was carried out to type strains according to their virulence. Primer sequences were used to amplify a 202 base pair fragment, encompassing the fusion protein cleavage site, in a one-step reverse transcriptase-polymerase chain reaction (RT-PCR) test for detection of a range of field cases and reference strains of APMV-1. Subsequent REA of the amplified fragments enabled differentiation of low virulent lentogenic field and vaccine strains from more virulent mesogenic and velogenic field strains of APMV-1, including pigeon PMV-1. In the present paper, we report the development and application of a one-step RT-PCR test coupled with REA as a fast, specific method for both the detection and typing of APMV-1 from field samples.

Animals↗

PubMed2005

Simultaneous detection of four human pathogenic microsporidian species from clinical samples by oligonucleotide microarray.

Microsporidian species have been rapidly emerging as human enteric pathogens in immunocompromised and immunocompetent individuals in recent years. Routine diagnostic techniques for microsporidia in clinical laboratories are laborious and insensitive and tend to underestimate their presence. In most instances, they are unable to differentiate species of spores due to their small sizes and similar morphologies. In this study, we report the development of another protozoan oligonucleotide microarray assay for the simultaneous detection and identification to the species level of four major microsporidian species: Enterocytozoon bieneusi, Encephalitozoon cuniculi, Encephalitozoon hellem, and Encephalitozoon intestinalis. The 18S small-subunit rRNA gene was chosen as the amplification target, labeled with fluorescence dye, and hybridized to a series of species-specific oligonucleotide probes immobilized on a microchip. The specificity and sensitivity of the microarray were clearly demonstrated by the unique hybridization profiles exhibited by each species of microsporidian tested and its ability to detect as few as 10 spores. In order to assess the applicability of this microarray in a clinical setting, we conducted microarray assays of 20 fecal samples from AIDS patients. Twelve of these samples were positive for the presence of microsporidia and could be confidently identified; 11 of them were positive for more than one species. Our results suggested that this microarray-based approach represents an attractive diagnostic tool for high-throughput detection and identification of microsporidian species in clinical and epidemiological investigations.

Acquired Immunodeficiency Syndrome↗

PubMed2005

Simultaneous detection of two bacterial pathogens using bacteriophage amplification coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

The simultaneous analysis of multiple target microorganisms using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) with bacteriophage amplification is discussed. Following infection of target bacteria with specific bacteriophages, proteins contained in the progeny phage are utilized as a secondary biomarker for the target bacterium. Escherichia coli when mixed with MS2 and MPSS-1 phages specific for E. coli and Salmonella spp., respectively, at levels below their corresponding detection limits, produced only the protein (13.7 kDa) characteristic of the MS2. Likewise, Salmonella spp. when mixed with the two phages only produced a protein (13.5 kDa) characteristic of MPSS-1. When the two bacteria and the two phages were mixed together, proteins characteristic of MS2 and MPSS-1 were observed indicating that both bacteriophages had been amplified. Identification of each bacterium was made based on the presence of the secondary bacteriophage biomarkers. No deleterious effects on bacteriophage amplification were observed because of the presence of multiple bacteria or bacteriophages.

Bacteriophages↗

PubMed2004

Immunosensor for detection of Yersinia enterocolitica based on imaging ellipsometry.

An immunosensor for the detection of pathogens was developed using imaging ellipsometry (IE) as a detection method. Yersinia enterocolitica was selected as the target pathogen in this study. A gold surface deposited with a self-assembled layer of 11-mercaptoundecanoic acid (11-MUA) was used as a substrate. For the fabrication of the immunosensor, protein G spots were made on the substrate using an inkjet-type microarrayer, and monoclonal antibody (Mab) was adsorbed onto the protein G spots. Deposition of each layer onto the substrate was confirmed by the measurement of surface plasmon resonance. The ellipsometric image of the protein G spot and the Mab-adsorbed protein G spot were acquired using an off-null ellipsometry type of imaging ellipsometry system. By measuring the ellipsometric angles of the protein layers, the surface concentration of each protein layer was calculated. The change in the mean optical intensity of the protein spot to the various concentrations of Y.enterocolitica was estimated. The immunosensor using imaging ellipsometry could successfully detect Y. enterocolitica in concentrations varying from 10(3) to 10(7) cfu/mL. The proposed immunosensor system has the advantage of allowing label-free detection, high sensitivity, and operational simplicity.

Antibody Affinity↗

PubMed2006

Detection and differentiation of pathogenicity of avian paramyxovirus serotype 1 (APMV-1) from field cases using one-step real-time RT-PCR.

A real-time fluorescent RT-PCR assay was developed to amplify avian paramyxovirus serotype 1 (APMV-1)-specific nucleic acid fragments from field samples. Subsequent restriction endonuclease analysis (REA) using BglI was carried out to type strains according to their virulence. Primer sequences were used to amplify a 202 base-pair fragment, encompassing the fusion protein cleavage site, in a one-step RT-PCR test for detection of a range of field cases and reference strains of APMV-1. Subsequent restriction endonuclease analysis of the amplified fragments enabled differentiation of low virulent lentogenic field and vaccine strains from more virulent mesogenic and velogenic field strains of APMV-1, including pigeon PMV-1. In 2004, seven cases of pigeon PMV-1 in Northern Ireland were diagnosed and differentiated more rapidly using the fluorescent RT-PCR assay when compared with the use of virus isolation. We report the development and application of a one-step real-time RT-PCR test coupled with REA as a fast, specific method for both the detection and typing of APMV-1 from field samples.

Animals↗

PubMed1997

A universal protocol for PCR detection of 13 species of foodborne pathogens in foods.

A universal protocol for PCR detection of 13 species of foodborne pathogens in foods was developed. The protocol used a universal culture medium and the same PCR conditions with 13 sets of specific primers. The 13 species of foodborne pathogens examined were Escherichia coli, E. coli-ETEC, E. coli-O157:H7, Shigella spp., Salmonella spp., Yersinia enterocolitica, Y. pseudotuberculosis, Vibrio cholerae, V. parahaemolyticus, V. vulnificus, Listeria monocytogenes, Staphylococcus aureus and Bacillus cereus. No interference was observed using the PCR assay when food sample was artificially inoculated with each individual bacterial species. Twelve different seafood samples and two soft cheese samples without artificial inoculation were examined by this protocol. Vibrio vulnificus, Salmonella spp., E. coli, Listeria monocytogenes and Bacillus cereus were detected in some foods. Internal probe hybridization and nested PCR procedures were used to confirm the above findings.

Bacteria↗

PubMed1997

Development of PCR primers from internal transcribed spacer region 2 for detection of Phytophthora species infecting potatoes.

We developed PCR primers and assay methods to detect and differentiate three Phytophthora species which infect potatoes and cause late blight (Phytophthora infestans) and pink rot (P. erythroseptica and P. nicotianae) diseases. Primers based on sequence analysis of internal transcribed spacer region 2 of ribosomal DNA produced PCR products of 456 bp (P. infestans), 136 bp (P. erythroseptica), and 455 bp (P. nicotianae) and were used to detect the pathogens in potato leaf (P. infestans) and tuber (P. infestans, P. erythroseptica, and P. nicotianae) tissue with a sensitivity of 1 to 10 pg of DNA. Leaf and tuber tissue were processed for PCR by a rapid NaOH method as well as a method based on the use of commercially available ion-exchange columns of P. infestans primers and the rapid NaOH extraction method were used to detect late blight in artificially and naturally infected tubers of potato cultivar Red LaSoda. In sampling studies, P. infestans was detected by PCR from artificially infected tubers at 4 days postinoculation, before any visible symptoms were present. The PCR assay and direct tissue extraction methods provide tools which may be used to detect Phytophthora pathogens in potato seedlots and storages and thus limit the transmission and spread of new, aggressive strains of P. infestans in U.S. potato-growing regions.

DNA Primers↗