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Results for “pathogen detection”

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PubMed1994

Design and characterization of PCR primers for detection of pathogenic Neisseriae.

Oligonucleotide polymerase chain reaction (PCR) primers directed at a group of closely related Neisserial species were designed from 16S rDNA sequences even though only a single such sequence from the targeted group was known. The amplifiable group included all Neisserial species considered pathogenic for man, including Neisseria gonorrhoea and Neisseria meningitidis. None of 43 other bacterial DNA specimens were amplified, including five non-Neisserial Neisseriaceae and three non-pathogenic Neisseriae. Another non-pathogenic Neisserial species gave a signal only at high DNA concentrations. DNA specimens from the pathogenic Neisseriae were detectable in amounts as low as 0.01 pg per PCR reaction, the approximate equivalent of a single organism, with equal sensitivity in buffer or in simple extracts of human inflammatory synovial fluids to which Neisserial DNA had been added. Simultaneously studied control specimens lacking added DNA were negative. The approach used to design these group-directed primers using only a single rDNA sequence from the targeted group by exploiting known patterns of sequence conservation among the 16S rDNA genes may prove useful for designing other similar group-directed primers. Polymerase chain reaction primers prepared in this way should prove of value in a number of areas, both investigational and clinical.

Base Sequence↗

PubMed2003

Market analysis of biosensors for food safety.

This paper is presented as an overview of the pathogen detection industry. The review includes pathogen detection markets and their prospects for the future. Potential markets include the medical, military, food, and environmental industries. Those industries combined have a market size of $563 million for pathogen detecting biosensors and are expected to grow at a compounded annual growth rate of 4.5%. The food market is further segmented into different food product industries. The overall food-pathogen testing market is expected to grow to $192 million and 34 million tests by 2005. The trend in pathogen testing emphasizes the need to commercialize biosensors for the food safety industry as legislation creates new standards for microbial monitoring. With quicker detection time and reusable features, biosensors will be important to those interested in real time diagnostics of disease causing pathogens. As the world becomes more concerned with safe food and water supply, the demand for rapid detecting biosensors will only increase.

Biological Assay↗

PubMed1999

A simple extraction procedure for efficient routine detection of pathogenic bacteria in plant material by polymerase chain reaction.

A simple and rapid method for extracting DNA from plants based on the use of an extraction buffer and precipitation with isopropanol was assayed to see its usefulness in detecting pathogenic bacteria in plant material. The method was compared with a phenol-chloroform standard procedure obtaining higher sensitivity levels of detection. The protocol developed was efficient for detecting a Gram-positive bacterium, Clavibacter michiganensis subsp. sepedonicus and several Gram-negative pathogenic bacteria (Ralstonia solanacearum, Erwinia amylovora, Xanthomonas axonopodis pv. citri) with a sensitivity of 10(2)-10(3) cfu/ml in spiked samples. It was also efficient to specifically identify such bacteria in naturally infected plant material. This procedure is proposed as a routine tool for detection of plant pathogenic bacteria, as well as in environmental microbiology and biotechnology studies.

2-Propanol↗

PubMed2004

Detection of pathogens from periodontal lesions.

OBJECTIVE: To comparatively detect A. actinomycetemcomitans and F. nucleatum from periodontal and healthy sites. METHODS: Subgingival clinical samples from 50 periodontitis adult patients and 50 healthy subjects were analyzed. Both organisms were isolated using a trypticase soy agar-bacitracin-vancomycin (TSBV) medium and detected by PCR. Conventional biochemical tests were used for bacteria identification. RESULTS: A. actinomycetemcomitans and F. nucleatum were isolated in 18% and 20% of the patients, respectively, and in 2% and 24% of healthy subjects. Among A. actinomycetemcomitans isolates, biotype II was the most prevalent. Primer pair AA was 100% sensitive in the detection of A. actinomycetemcomitans from both subject groups. Primers ASH and FU were also 100% sensitive to detect this organism in healthy subject samples. Primer pair FN5047 was more sensitive to detect F. nucleatum in patients or in healthy samples than primer 5059S. Primers ASH and 5059S were more specific in the detection of A. actinomycetemcomitans and F. nucleatum, respectively, in patients and in healthy subject samples. CONCLUSIONS: PCR is an effective tool for detecting periodontal pathogens in subgingival samples, providing a faster and safer diagnostic tool of periodontal diseases. The method's sensitivity and specificity is conditioned by the choice of the set of primers used.

Adult↗

PubMed1995

Detection of pathogenic fungi in human blood by the polymerase chain reaction.

The ability of the polymerase chain reaction (PCR) to detect pathogenic fungi in human blood was investigated. A DNA fragment of about 300 bp from the 18S rDNA, highly conserved in all fungi, was amplified with target DNA from 18 different species of fungi commonly isolated from clinical samples. The presence of PCR products was confirmed by hybridization with a fluorescein-labelled internal probe (21-mer). The PCR assay described is sensitive enough to detect 125 fg of purified Candida albicans DNA and 10 to 100 yeast cells per millilitre of blood.

Base Sequence↗

PubMed1995

[Detection of pathogenic Entamoeba histolytica DNA in liver abscess aspirates with polymerase chain reaction].

Polymerase chain reaction (PCR) was used to diagnose amebic liver abscess by detecting pathogenic Entamoeba histolytica DNA in liver aspirates from patients with liver abscess. Oligonucleotide primers specific for gene encoding of the 30,000 molecule of pathogenic Entamoeba histolytica were used in the test. Liver aspirates from 23 cases with amebic liver abscess as evidenced by typical clinical findings or very high titres of anti-E. Histolytica antibodies with ELISA were found to be positive with PCR. Fourteen control samples (3 cases of bacterial liver abscess, 1 of liver cancer and 10 of abscess in other sites) were all negative to PCR. The results suggest that PCR is a sensitive and useful method for diagnosing amebic liver abscess.

Animals↗

PubMed2005

The fabrication of protein chip based on surface plasmon resonance for detection of pathogens.

Protein chip based on surface plasmon resonance (SPR) was developed for detection of pathogens existing in contaminated environment, such as Escherichia coli O157:H7, Salmonella typhimurium, Legionella pneumophila, and Yersinia enterocolitica. Protein G was immobilized to endow the orientation of antibody molecules on the SPR surface. The pathogen binding of the protein chip was investigated by SPR spectroscopy. Consequently, it was found that the four kinds of pathogen could be selectively detected by using SPR-based protein chip.

Bacteria↗

PubMed2006

Detection of pathogenic intestinal bacteria by Toll-like receptor 5 on intestinal CD11c+ lamina propria cells.

Toll-like receptors (TLRs) recognize distinct microbial components and induce innate immune responses. TLR5 is triggered by bacterial flagellin. Here we generated Tlr5-/- mice and assessed TLR5 function in vivo. Unlike other TLRs, TLR5 was not expressed on conventional dendritic cells or macrophages. In contrast, TLR5 was expressed mainly on intestinal CD11c+ lamina propria cells (LPCs). CD11c+ LPCs detected pathogenic bacteria and secreted proinflammatory cytokines in a TLR5-dependent way. However, CD11c+ LPCs do not express TLR4 and did not secrete proinflammatory cytokines after exposure to a commensal bacterium. Notably, transport of pathogenic Salmonella typhimurium from the intestinal tract to mesenteric lymph nodes was impaired in Tlr5-/- mice. These data suggest that CD11c+ LPCs, via TLR5, detect and are used by pathogenic bacteria in the intestinal lumen.

Animals↗

PubMed2006

A new MSPQC system for rapid detection of pathogens in clinical samples.

A new multi-channel series piezoelectric quartz crystal (MSPQC) system for detection of pathogens in clinical sample was proposed. Some factors, which affect the detection of pathogens by using MSPQC, were all investigated. A total of 650 clinical samples were detected by MSPQC and compared with licensed BACTEC 9120 system (Becton Dickinson Diagnostic Instrument Systems, Sparks, MD, USA) simultaneously in the Third Xiangya Hospital of Central South University, China. When the incubation period was 5 days, two systems had similar detected results: the MSPQC system detected 123 growth of 650 (18.92%) bottles while the BACTEC 9120 detected 125 growth of 650 (19.23%) bottles. The MSPQC had 2 false-positive signals and 2 false-negative signals. However, BACTEC 9120 had 3 false-positive signals and 0 false-negative signals. Further identifications of bacteria were run by VITEK-2 (bioMérieux China Ltd.), 5% sheep blood trypticase soy agar (SBA) and chocolate agar (CA). Comparing with BACTEC 9120, MSPQC system possesses following advantages: shorter average detection time, less blood volume needed, less false-positive results and low cost. It can also provide information in real time. So MSPQC has a wonderful perspective in clinical application.

Bacteremia↗

PubMed1996

Detection of pathogenic Yersinia enterocolitica in environmental waters by PCR.

The isolation of pathogenic strains of Yersinia enterocolitica from food and water samples by culture is time-consuming and unreliable. A two-step PCR procedure has been developed which, after a period of bacterial enrichment, can detect and confirm the presence of pathogenic Y. enterocolitica within a single day. This PCR method works effectively for a range of environmental water types, including reticulated waters, reservoirs and creeks. A survey of environmental waters in Victoria, Australia, showed that the PCR method detected pathogenic Y. enterocolitica in water sampled from four separate sites (two creeks and two reservoirs). Repeat samplings of the two reservoirs yielded PCR-positive results on all but one occasion. Culture analysis of the same samples detected pathogenic Y. enterocolitica in only one sample, indicating that the PCR can detect pathogenic Y. enterocolitica which are undetectable by culture. Results from this study confirm that potentially pathogenic strains of Y. enterocolitica can exist in environmental waters.

Base Sequence↗

PubMed1995

Polymerase chain reaction (PCR) for detection of pathogenic microorganisms in bacteriological monitoring of dairy products.

The presence of pathogenic bacteria poses a serious problem in sustaining the safety of dairy products. Microbiological routine controls of these products make use of selective culture techniques. To detect pathogenic species, isolated colonies are characterized by specific metabolic activities and by serotyping. We present an alternative biochemical approach that does not require culture of bacteria. The total bacterial populations of food samples were isolated by centrifugation and analysed by PCRs specific for pathogenic species. A total of 90 raw milk samples and dairy products made from raw milk were screened by this method for the presence of Listeria monocytogenes, Escherichia coli, enterotoxigenic E. coli, Campylobacter jejuni and C. coli. Detection rates were 12/90 (13%) for L. monocytogenes, 41/90 (46%) for E. coli, 18/90 (20%) for enterotoxigenic E. coli producing heat-labile toxin type I or heat-stable toxin type I, and 6/90 (7%) for C. jejuni or C. coli. Except for the use of different amplification primers, this approach is identical for any bacterial species to be detected. Direct PCR analysis of food samples offers rapid screening for the presence of specific bacteria and enables selection of critical samples prior to culture.

Animals↗

PubMed2005

Comparison of primers for the detection of pathogenic Escherichia coli using real-time PCR.

AIMS: To evaluate PCR primers for the detection of pathogenic Escherichia coli in a real-time PCR assay and determine their utility in produce irrigation water testing. METHODS AND RESULTS: Three previously published PCR primer sets and one set designed for this study were tested for their ability to produce amplification products for several pathogenic E. coli serotypes from whole cells as template. Two of the previously published primer sets were chosen for real-time PCR detection limit determination. The coneaeA and PEH detection limit of E. coli O157:H7 was 10(0) and 10(1) CFU rxn(-1) in sterile water respectively. To detect E. coli O157:H7 in sprout irrigation water, the water required dilution due to PCR inhibitors. The detection limit of the coneaeA and PEH was 10(1) and between 10(2) and 10(3) CFU rxn(-1) in diluted sprout irrigation water respectively. CONCLUSIONS: The primer set coneaeA was able to produce an amplification product from each E. coli serotype, except O128:H7 and most sensitive for real-time PCR detection of pathogenic E. coli in diluted sprout irrigation water. SIGNIFICANCE AND IMPACT OF THE STUDY: The necessity of a dissociation analysis to distinguish positive samples from those with fluorescence of random dsDNA generation for real-time PCR in a complex background was established.

Adhesins, Bacterial↗

PubMed2006

Duplex PCR-hybridization based detection of pathogenic Leptospira in environmental water samples obtained from endemic areas in northeast region of Thailand.

Leptospirosis, a major health problem worldwide, is known to be endemic in the northeastern part of Thailand with the risk of infection by exposure to pathogenic Leptospira in contaminated aquatic environment. A method based on PCR-hybridization detection of pathogenic Leptospira in water was established. The method included filtration of water sample through membrane filters of two pore sizes, DNA extraction from filters using a guanidine thiocyanate extraction method, a duplex-PCR assay with two primer pairs, and hybridization with a synthetic LipL32 DNA probe. The duplex-PCR allowed detection of two products of 279 bp for LipL32 gene and 430 bp for 16S rRNA gene. In water samples artificially seeded with serovar bratislava, at least 10(3) cells could be analyzed by PCR-agarose gel electrophoresis and 1-10 cells by PCR-Southern blot hybridization. The protocol was applied to the detection of pathogenic Leptospira in environmental waters collected from endemic areas in the northeast region of Thailand. Of 100 water samples analyzed, 23 samples were positive for pathogenic Leptospira with PCR performed with Southern blot hybridization only, but none was detected by PCR-agarose gel-electrophoresis. However, PCR performed with the chemiluminescent LipL32 probe using the Fluorescein ULS labeling facilitated the detection of low numbers of pathogenic Leptospira in water. This method should prove useful for monitoring of pathogenic Leptospira pollution in environmental waters, and has the potential to become a valuable tool to the surveillance of leptospirosis in endemic areas, thus leading to enhanced public health protection.

Animals↗

PubMed2004

Association of colonic and endometrial carcinomas in Portuguese families with hereditary nonpolyposis colorectal carcinoma significantly increases the probability of detecting a pathogenic mutation in mismatch repair genes, primarily the MSH2 gene.

BACKGROUND: Hereditary nonpolyposis colorectal carcinoma (HNPCC) significantly raises the risk of developing colorectal carcinoma (CRC) and other extracolonic tumors. It is defined by the Amsterdam Criteria and is associated with germline mutations in mismatch repair genes, primarily MLH1 and MSH2. The objectives of the current study were to evaluate the presence of CRC (Type I) and other extracolonic tumors (Type II) in families with HNPCC and to analyze the findings for correlations with germline mutations in the MLH1 and MSH2 genes. METHODS: Seventy families with an HNPCC diagnosis were analyzed. Denaturing gradient gel electrophoresis and direct sequencing were used for germline mutation analysis in the MLH1 and MSH2 genes. RESULTS: Forty-three of 70 families (61%) presented with HNPCC Type II. In 21 of 30 families that had a complete genetic diagnosis, 16 pathogenic germline mutations (7 MLH1 mutations and 9 MSH2 mutations) and 5 mutations of unknown pathogenecity (all MLH1 mutations) were found. In the remaining nine families, no mutations were detected. Unequivocally pathogenic mutations were far more common in families with HNPCC Type II compared with families that had CRC only (P = 0.01). Families with endometrial carcinoma presented with the greatest probability of mutational detection (P = 0.005). MLH1 was only gene affected in families with HNPCC Type I, whereas mutations in both MLH1 and MSH2 were found in families with HNPCC Type II (P = 0.04). However, the MSH2 gene was more frequently involved in families with HNPCC in which endometrial carcinoma was present (P = 0.005). CONCLUSIONS: CRC and endometrial carcinoma were associated with a greater probability of detecting pathogenic mutations in mismatch repair genes, with MSH2 involvement predominating. The results support specific mutational screening strategies, based on observed phenotypes, for families with HNPCC.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Development of a duplex-polymerase chain reaction for rapid detection of pathogenic Leptospira.

A duplex-polymerase chain reaction (PCR) for the rapid detection of pathogenic leptospires was developed by using two sets of newly designed primers which amplified in the same reaction two different DNA fragments simultaneously: 279-bp of LipL32 and 430-bp of 16S rRNA. For DNA extraction from bacterial cultures, the silica-based spin column method was found to be more suitable and was selected for the extraction of DNAs from all 92 bacterial strains including 56 strains of pathogenic Leptospira, 15 strains of non-pathogenic Leptospira and 21 other strains of bacteria. The PCR products were analyzed by agarose gel-electrophoresis with confirmation by Southern and dot hybridization using synthetic DNA probe prepared from LipL32 gene of a pathogenic reference strain, L. interrogans serovar pyrogenes. The duplex-PCR allowed detection of two products of 279 bp and 430 bp in all pathogenic Leptospira. Non-pathogenic Leptospira generated a single product of 430 bp. Other bacterial strains failed to reveal any amplification products. As little as 1 pg of pure DNA corresponding to 100 cells could be detected by agarose gel-electrophoresis, and 1-10 fg of pure DNA by hybridization.

DNA Probes↗

PubMed1989

Development and testing of a synthetic oligonucleotide probe for the detection of pathogenic Yersinia strains.

A 24-base oligonucleotide probe specific for a region of the Yersinia enterocolitica virulence plasmid (pYV) associated with HEp-2 cell cytotoxicity and the Sereny reaction was constructed by using sequences flanking critical TnphoA insertions in a subcloned fragment of pYV. This probe, highly specific and sensitive for virulent yersiniae, detected pathogenic Y. enterocolitica isolates in artificially inoculated foods.

Base Sequence↗

PubMed2003

Detection of bovine torovirus and other enteric pathogens in feces from diarrhea cases in cattle.

The objectives of this study were to determine the prevalence of bovine torovirus (BoTV) in bovine fecal samples from diarrhea cases submitted to the Ohio Animal Disease Diagnostic Laboratory (ADDL) and to assess if a relationship exists between BoTV and the other enteric pathogens detected. From November 1999 to May 2001, 259 specimens from 53 calves (< or = 6 months old), 27 young adults (52 years), 125 adults (> or = 2 years), and 54 animals of unknown age were examined by an antigen-capture enzyme-linked immunosorbent assay (ELISA) and reverse transcriptase-polymerase chain reaction (RT-PCR) assay developed to detect BoTV. Testing for other enteric pathogens was performed by ADDL, and the results were analyzed with the BoTV data. The BoTV was detected using ELISA or RT-PCR in 9.7% (25/259) of the clinical samples, 56% (14/25) of which were from calves (P < 0.001) representing 26.4% (14/53) of the calves tested. Of the BoTV-positive calves, 71% (10/14) were less than 3 weeks of age. In 11/25 positive specimens, BoTV was the only pathogen detected among those examined. Other enteric organisms detected alone or in combination with BoTV in calf samples were rotavirus, coronavirus, Salmonella spp., Cryptosporidium spp., and Giardia spp.; but no consistent association between BoTV and these organisms was observed. In summary, BoTV was detected in fecal samples from cattle with diarrhea, principally in young calves less than 3 weeks of age. Future studies of infectious diarrhea in cattle should also include assays for this etiologic agent.

Aging↗

PubMed1996

Detection of pathogenic and non-pathogenic bacteria by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

The proteins isolated from the whole cells of bacterial pathogens and related non-pathogenic simulants were analyzed directly, with minimal sample preparation by matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry. Inspection of mass spectrometric profiles obtained from direct MALDI-MS analysis of the protein extracts revealed specific biomarkers for individual bacterial cells. The observed biomarkers enabled us not only to detect pathogenic bacteria (Bacillus anthracis, Yersinia pestis and Brucella meliteusis), but also to distinguish them from the corresponding non-pathogenic species. By examining a series of strains of several Bacillus species (anthracis, thuringiensis, cereus and subtilis), it was possible to derive genus, species and strain-specific biomarkers from the measured molecular masses of the intact proteins. Additional series of biomarkers were obtained from direct mass spectrometric analysis of tryptic digests of the protein extracts. The application of this technique for rapid chemotaxonomic classification of microorganisms is demonstrated.

Bacteria↗