SEARCH · PubMed Health

Results for “Pathogen detection”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 667 records · Page 37Linked to original sources

PubMed1998

Quantification of poliovirus in seawater and sewage by competitive reverse transcriptase--polymerase chain reaction.

Reverse transcriptase-polymerase chain reaction (RT-PCR) has been used extensively to detect enteric viruses in environmental samples. Advantages of RT-PCR include its high detection sensitivity and rapid turn-around time. However, unlike traditional cell culture, RT-PCR has not provided quantitation and infectivity information. In this study, we have developed a quantitative RT-PCR method that can be used to determine the amount of poliovirus RNA in environmental samples. An RNA internal standard for poliovirus RT-PCR was designed and obtained through genetic engineering. Serial dilutions of RNA internal standard templates were amplified with a 5'-carboxyfluorescein-labeled poliovirus downstream primer and a nonlabeled poliovirus upstream primer in the RT-PCR. The fluorescent light intensity of labeled RT-PCR products was quantified using an ABI DNA sequencer with GeneScan software. The internal standard was coamplified with poliovirus in the RT-PCR, allowing for enumeration of the poliovirus RNA present in the seawater and sewage samples. This method, using a cloned internal standard and specified primers in the PCR, may be applied to quantify other microorganisms in environmental samples. Although quantitative RT-PCR has begun to be used more extensively for detecting pathogens in clinical samples, the complex nature of many environmental samples has limited the sample range of the effectiveness of quantitative RT-PCR.

Environmental Microbiology↗

PubMed1998

[Hemolytic uremic syndrome in children of Mendoza, Argentina: association with Shiga toxin-producing Escherichia coli infection].

Shiga toxin-producing Escherichia coli (STEC) has been associated with pathogenesis of hemolytic uremic syndrome (HUS) worldwide. The aim of the present study was to characterize the HUS cases reported in Mendoza and to determine their association with STEC infection. From July 1994 through June 1996 thirty-six patients with HUS were admitted to Hospital Pediátrico "Dr. HJ Notti" (Mean age 22.8 +/- 14.9 months, 44% females). The children developed HUS following an acute diarrheal illness in 94.4% of the cases. Bloody diarrhea was observed in 83.3% of them. Antimicrobial therapy had been administered to 69.4% of the patients. Most of the patients were well-nourished (88.9%), belong to middle-low socioeconomical condition (91.7%), from urban areas (72.2%) and they were mostly assisted during summer and the beginning of autumn. The acute stage of the disease occurred with presentation of pallor (100%), edema (25%), anuria (38.9%), oliguria (41.7%), hemolytic anemia (97.2%), thrombocytopenia (86.1%) and neurological involvement (41.7%). Twenty-five of them presented the full clinical syndrome. Peritoneal dialysis were performed in 50% and packed blood cell transfusion in 88.9%. The mean days of hospitalization was 15.1 +/- 9.2 [range 1-32]. A 91.7% of the patients recovered renal function, two developed chronic renal failure and one died. Cumulative evidence of STEC infection was found in 19 (86.4%) of 22 HUS patients. STEC O157:H7, biotype C was found in 8 (36.4%). The prevalent Stx type was Stx2 in STEC, free fecal Stx (STMF) and Stx-neutralizing antibodies (a-Stx). In Mendoza, as in the rest of Argentina E. coli O157:H7, biotype C, Stx2 producer is the most frequently detected pathogen in HUS cases.

Argentina↗

PubMed2006

Microchip-based one step DNA extraction and real-time PCR in one chamber for rapid pathogen identification.

Optimal detection of a pathogen present in biological samples depends on the ability to extract DNA molecules rapidly and efficiently. In this paper, we report a novel method for efficient DNA extraction and subsequent real-time detection in a single microchip by combining laser irradiation and magnetic beads. By using a 808 nm laser and carboxyl-terminated magnetic beads, we demonstrate that a single pulse of 40 seconds lysed pathogens including E. coli and Gram-positive bacterial cells as well as the hepatitis B virus mixed with human serum. We further demonstrate that the real-time pathogen detection was performed with pre-mixed PCR reagents in a real-time PCR machine using the same microchip, after laser irradiation in a hand-held device equipped with a small laser diode. These results suggest that the new sample preparation method is well suited to be integrated into lab-on-a-chip application of the pathogen detection system.

DNA, Bacterial↗

PubMed2006

Novel lysine biosynthetic gene sequences (LYS1 and LYS5) used as PCR targets for the detection of the pathogenic Candida yeast.

We report here a sensitive and specific polymerase chain reaction (PCR) detection assay for the pathogenic Candida yeast based on the novel LYS1 [encoding saccharopine dehydrogenase (SDH)] and LYS5 [encoding phosphopantetheinyl transferase (PPTase)] gene sequences of the fungal unique lysine biosynthetic pathway. Both LYS1 and LYS5 DNA-specific PCR primers SG1, SG2 and SG3, SG4, respectively, amplified predicted 483 and 648-bp fragments from Candida albicans genomic DNA but not from other selected fungal, bacterial, or human DNA. The 18S rDNA control primers exhibited positive amplifications in all PCR assays. The LYS1-and LYS5-specific primers strongly amplified C. albicans and Candida tropicalis target sequences; however, the LYS1 primers also weakly amplified fragments from Candida kefyr and Candida lusitaniae DNA. Both sets of primers amplified target sequences from less than 10 pg of serially diluted C. albicans DNA, and the LYS1 specific primers also detected DNA isolated from serially diluted 50 C. albicans cells. The PCR primers reported here are sufficiently sensitive and specific for the potential early detection of Candida infections with no possibility of false positive results from cross-contamination with bacterial or human DNA.

Amino Acid Sequence↗

PubMed2005

Detection of periodontal pathogen Porphyromonas gingivalis by loop-mediated isothermal amplification method.

A method for nucleic acid amplification, loop-mediated isothermal amplification (LAMP) was employed to develop a rapid and simple detection system for periodontal pathogen, Porphyromonas gingivalis. A set of six primers was designed by targeting the 16S ribosomal RNA gene. By the detection system, target DNA was amplified and visualized on agarose gel within 30 min under isothermal condition at 64 degrees C with a detection limit of 20 cells of P. gingivalis. Without gel electrophoresis, the LAMP amplicon was directly visualized in the reaction tube by addition of SYBR Green I for a naked-eye inspection. The LAMP reaction was also assessed by white turbidity of magnesium pyrophosphate (a by-product of LAMP) in the tube. Detection limits of these naked-eye inspections were 20 cells and 200 cells, respectively. Although false-positive DNA amplification was observed from more than 10(7) cells of Porphyromonas endodontalis, no amplification was observed in other five related oral pathogens. Further, quantitative detection of P. gingivalis was accomplished by a real-time monitoring of the LAMP reaction using SYBR Green I with linearity over a range of 10(2)-10(6) cells. The real-time LAMP was then applied to clinical samples of dental plaque and demonstrated almost identical results to the conventional real-time PCR with an advantage of rapidity. These findings indicate the potential usefulness of LAMP for detecting and quantifying P. gingivalis, especially in its rapidity and simplicity.

Bacteriological Techniques↗

PubMed1995

Photonic detection of bacterial pathogens in living hosts.

The study of pathogenic processes is often limited to ex vivo assays and cell-culture correlates. A greater understanding of infectious diseases would be facilitated by in vivo analyses. Therefore, we have developed a method for detecting bacterial pathogens in a living host and used this method to evaluate disease processes for strains of Salmonella typhimurlum that differ in their virulence for mice. Three strains of Salmonella were marked with bioluminescence through transformation with a plasmid conferring constitutive expression of bacterial luciferase. Detection of photons transmitted through tissues of animals infected with bioluminescent Salmonella allowed localization of the bacteria to specific tissues. In this manner progressive infections were distinguished from those that were persistent or abortive. We observed patterns of bioluminescence that suggested the caecum may play a pivotal role in Salmonella pathogenesis. In vivo efficacy of an antibiotic was monitored using this optical method. This study demonstrates that real time non-invasive analyses of pathogenic events and pharmacological monitoring can be performed in vivo.

Animals↗

PubMed2003

Limitations of TaqMan PCR for detecting divergent viral pathogens illustrated by hepatitis A, B, C, and E viruses and human immunodeficiency virus.

Recent events illustrate the imperative to rapidly and accurately detect and identify pathogens during disease outbreaks, whether they are natural or engineered. Particularly for our primary goal of detecting bioterrorist releases, detection techniques must be both species-wide (capable of detecting all known strains of a given species) and species specific. Due to classification restrictions on the publication of data for species that may pose a bioterror threat, we illustrate the challenges of finding such assays using five nonthreat organisms that are nevertheless of public health concern: human immunodeficiency virus (HIV) and four species of hepatitis viruses. Fluorogenic probe-based PCR assays (TaqMan; Perkin-Elmer Corp., Applied Biosystems, Foster City, Calif.) may be sensitive, fast methods for the identification of species in which the genome is conserved among strains, such as hepatitis A virus. For species such as HIV, however, the strains are highly divergent. We use computational methods to show that nine TaqMan primer and probe sequences, or signatures, are needed to ensure that all strains will be detected, but this is an unfeasible number, considering the cost of TaqMan probes. Strains of hepatitis B, C, and E viruses show intermediate divergence, so that two to three TaqMan signatures are required to detect all strains of each virus. We conclude that for species such as hepatitis A virus with high levels of sequence conservation among strains, signatures can be found computationally for detection by the TaqMan assay, which is a sensitive, rapid, and cost-effective method. However, for species such as HIV with substantial genetic divergence among strains, the TaqMan assay becomes unfeasible and alternative detection methods may be required. We compare the TaqMan assay with some of the alternative nucleic acid-based detection techniques of microarray, chip, and bead technologies in terms of sensitivity, speed, and cost.

Computational Biology↗

PubMed1996

Detection of foodborne pathogens using DNA probes and a dipstick format.

The detection of foodborne microorganisms has traditionally been done using microbiologically based methods. Such "gold standard" methods are generally reliable but have the disadvantages of being labor intensive, subjective, and time consuming. Over the last several years, the development of DNA probe-based methods has simplified the methods used to detect organisms such as Salmonella, Listeria, and E. coli by targeting the unique DNA or RNA sequences of these organisms using DNA probes and nonradioactive detection.

DNA Probes↗

PubMed2003

Comparison of fluorescence and resonance light scattering for highly sensitive microarray detection of bacterial pathogens.

Microarrays have emerged as potential tools for bacterial detection and identification. Given their high parallelism, they might represent a breakthrough in current diagnostic methods, provided they can be coupled to simplified labeling protocols and detected with adequate sensitivities. We describe here a technique to directly label total bacterial RNA, thus avoiding the multiple steps and possible biases associated with enzymatic amplification (e.g. PCR). We have then compared the performances of one white-light source and two laser-based fluorescence scanners for detection reliability and sensitivity. Our study reveals that nanoparticle-labeled bacterial RNA generates reproducible resonance light scattering signals that are at least 50 times more intense than state-of-the-art confocal-based fluorescence signals.

Light↗

PubMed2005

Nucleic acid-based methods for the detection of bacterial pathogens: present and future considerations for the clinical laboratory.

BACKGROUND: Recent advances in nucleic acid-based methods to detect bacteria offer increased sensitivity and specificity over traditional microbiological techniques. The potential benefit of nucleic acid-based testing to the clinical laboratory is reduced time to diagnosis, high throughput, and accurate and reliable results. METHODS: Several PCR and hybridization tests are commercially available for specific organism detection. Furthermore, hundreds of nucleic acid-based bacterial detection tests have been published in the literature and could be adapted for use in the clinical setting. Contamination potential, lack of standardization or validation for some assays, complex interpretation of results, and increased cost are possible limitations of these tests, however, and must be carefully considered before implementing them in the clinical laboratory. CONCLUSIONS: A major area of advancement in nucleic acid-based assay development has been for specific and broad-range detection of bacterial pathogens.

Bacterial Infections↗

PubMed1998

Detection of microbial pathogens in shellfish with multiplex PCR.

Multiplex PCR amplification of uidA, cth, invA, ctx, and tl genes was developed enabling simultaneous detection in shellfish of Escherichia coli, an indicator of fecal contamination and microbial pathogens, Salmonella typhimurium, Vibrio vulnificus, V. cholerae, and V. parahaemolyticus, respectively. Each of the five pairs of oligonucleotide primers was found to support PCR amplifications of only its targeted gene. The optimized multiplex PCR reaction utilized a PCR reaction buffer containing 2.5 mM MgCl2 and primer annealing temperature of 55 degrees C. Oyster tissue homogenate seeded with these microbial pathogens was subjected to DNA purification by the Chelextrade mark 100 (BioRad) method. The sensitivity of detection for each of the microbial pathogens was </=10(1)-10(2) cells following a "double" multiplex PCR amplification approach. Amplified target genes in a multiplex PCR reaction were subjected to a colorimetric GeneCombtrade mark (BioRad) DNA-DNA hybridization assay. This assay was rapid and showed sensitivity of detection comparable to the agarose gel electrophoresis method. The colorimetric GeneCombtrade mark assay avoids use of hazardous materials inherent in conventional gel electrophoresis and radioactive-based hybridization methods. Multiplex PCR amplification, followed by colorimetric GeneCombtrade mark DNA-DNA hybridization, has been shown to be an effective, sensitive, and rapid method to detect microbial pathogens in shellfish.

Animals↗

PubMed2006

Sampling strategy for intraoral detection of periodontal pathogens before and following periodontal therapy.

BACKGROUND: The aim of this study was to identify a sampling strategy with high probability for detecting oral colonization by Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Eikenella corrodens, Tannerella forsythensis, Prevotella intermedia, Prevotella nigrescens, and Treponema denticola before and following mechanical periodontal therapy. METHODS: Samples were taken from the following intraoral sites in 35 patients with untreated chronic periodontitis before and 1.5, 3, and 6 months after non-surgical periodontal therapy: supra- and subgingival plaque from the deepest pockets in each sextant; pooled supra- and subgingival plaque from another six randomly selected, less affected teeth; mucosal swab samples from the tongue, tonsils, throat, and buccal mucosa; and stimulated and unstimulated saliva. Microbial species were identified by polymerase chain reaction (PCR). RESULTS: Of the sampling of all assessed sites, the highest probability for simultaneously detecting the tested pathogens was found in respect to the combination of supra- and subgingival plaque samples taken from the most affected tooth in each sextant in untreated patients (probability: 83% to 95% for the assessed bacteria). These results were consistently observed throughout the study period. CONCLUSIONS: For determining the intraoral carrier state of patients with periodontitis, a combined sample of supra- and subgingival plaque taken from the deepest periodontal pocket in each sextant may yield the most reliable result. This sampling strategy may be used in routine microbial testing and clinical research.

Bacteria, Anaerobic↗

PubMed2003

Bioelectronic DNA detection of human papillomaviruses using eSensor: a model system for detection of multiple pathogens.

BACKGROUND: We used human papillomaviruses (HPV) as a model system to evaluate the utility of a nucleic acid, hybridization-based bioelectronic DNA detection platform (eSensor) in identifying multiple pathogens. METHODS: Two chips were spotted with capture probes consisting of DNA oligonucleotide sequences specific for HPV types. Electrically conductive signal probes were synthesized to be complementary to a distinct region of the amplified HPV target DNA. A portion of the HPV L1 region that was amplified by using consensus primers served as target DNA. The amplified target was mixed with a cocktail of signal probes and added to a cartridge containing a DNA chip to allow for hybridization with complementary capture probes. RESULTS: Two bioelectric chips were designed and successfully detected 86% of the HPV types contained in clinical samples. CONCLUSIONS: This model system demonstrates the potential of the eSensor platform for rapid and integrated detection of multiple pathogens.

Biosensing Techniques↗

PubMed2004

Multiplex PCR assay for detection of bacterial pathogens associated with warm-water Streptococcosis in fish.

A multiplex PCR-based method was designed for the simultaneous detection of the main pathogens involved in warm-water streptococcosis in fish (Streptococcus iniae, Streptococcus difficilis, Streptococcus parauberis, and Lactococcus garvieae). Each of the four pairs of oligonucleotide primers exclusively amplified the targeted gene of the specific microorganism. The sensitivity of the multiplex PCR using purified DNA was 25 pg for S. iniae, 12.5 pg for S. difficilis, 50 pg for S. parauberis, and 30 pg for L. garvieae. The multiplex PCR assay was useful for the specific detection of the four species of bacteria not only in pure culture but also in inoculated fish tissue homogenates and naturally infected fish. Therefore, this method could be a useful alternative to the culture-based method for the routine diagnosis of warm-water streptococcal infections in fish.

Animals↗

PubMed2002

Density of total and pathogenic (tdh+) Vibrio parahaemolyticus in Atlantic and Gulf coast molluscan shellfish at harvest.

The densities of total and pathogenic Vibrio parahaemolyticus in 671 samples of molluscan shellfish harvested in 1999 and 2000 from 14 sites in seven Gulf and Atlantic coast states were determined at 2-week intervals over a period of 12 to 16 months in each state. Changes in V. parahaemolyticus densities in shellfish between harvest and sample analysis were minimized with time and temperature controls. Densities were measured by direct plating techniques, and gene probes were used for identification. Total and pathogenic V. parahaemolyticus organisms were identified with probes for the thermolabile direct hemolysin (tlh) gene and the thermostable direct hemolysin (tdh) gene, respectively. An enrichment procedure involving 25 g of shellfish was also used for the recovery of pathogenic V. parahaemolyticus. The densities of V. parahaemolyticus in shellfish from all harvest sites were positively correlated with water temperature. Shellfish from the Gulf Coast typically had higher densities of V. parahaemolyticus than did shellfish harvested from the North Atlantic or mid-Atlantic coast. Vibrio parahaemolyticus counts exceeded 1,000 CFU/g for only 5% of all samples. Pathogenic (tdh+) V. parahaemolyticus was detected in approximately 6% of all samples by both procedures, and 61.5% of populations in the positive samples from the direct plating procedure were at the lower limit of detection (10 CFU/g). The frequency of detection of pathogenic V. parahaemolyticus was significantly related to water temperature and to the density of total V. parahaemolyticus. The failure to detect pathogenic V. parahaemolyticus in shellfish more frequently was attributed to the low numbers and uneven distribution of the organism.

Animals↗

PubMed1991

Culture vs direct antigen assays for detection of microbial pathogens from lower respiratory tract specimens suspected of containing the respiratory syncytial virus.

Following the introduction of effective antiviral chemotherapy, rapid antigen assays have been utilized increasingly, instead of cell cultures, for detection of the respiratory syncytial virus from lower respiratory tract specimens. Because antigen assays, unlike cell culture, cannot amplify low levels of the virus to a detectable level, assay sensitivity is especially dependent on high-quality specimens. In addition, the assays are unable to detect other viruses or bacteria with which the patient may be infected. This review summarizes results from clinical studies of the performance of cell cultures and the more commonly used antigen assays, describes factors that may lead to false-positive or false-negative test results, and makes recommendations for the selection of procedures for the reliable detection of microbial pathogens from patients suspected of being infected with respiratory syncytial virus.

Antigens, Viral↗

PubMed1999

Evaluation of methodologies including immunofluorescent assay (IFA) and the polymerase chain reaction (PCR) for detection of human pathogenic microsporidia in water.

Microsporidia is a term used to describe a group of emerging protozoan pathogens whose environmental occurrence has only recently been documented due to lack of detection methodologies. This study evaluates and describes current methods for detection of microsporidia in water. Standard methods, for the collection and processing of large volumes of water to detect protozoa, showed only a 4.8% recovery, of microsporidia spores, from 100 l volumes of tap. Immunofluorescent assay (IFA) analysis was assessed using two different antibodies specific for human pathogenic microsporidia. Results indicated that the use of IFA for routine screening of water for microsporidia was not an acceptable approach. The antibodies tested for the IFA resulted in false positives and false negatives and did not react with Enterocytozoon bieneusi, which is an important human pathogenic microsporidia. Finally, the small sizes of the human pathogenic microsporidia prevent confirmation and species determination by light microscopic methods. Two methods for isolating microsporidia DNA from water for use in polymerase chain reaction (PCR) amplification of microsporidia target sequences were assessed. Both of these DNA isolation methods when combined with the PCR showed the ability to detect less than ten spores in purified water concentrates. Thus, this study represents the first documentation and evaluation of current methods for the detection of human pathogenic microsporidia in water.

Animals↗

PubMed1993

Polymerase chain reaction detection of nonviable bacterial pathogens.

Polymerase chain reaction (PCR) methodologies for detection of pathogens in environmental samples are currently available. However, positive amplification products for any set of primers only signal that the appropriate target nucleic acid sequences were present in the sample. The presence of the amplification products does not imply that the target organisms were viable. Here we show that PCR will detect nonviable cells, as long as intact target nucleic acid sequences are available. In an environmental water sample, nucleic acids degraded quickly and were not detectable by PCR after 3 weeks even when stored at 4 degrees C. However, these data show that there is a window of opportunity for PCR analyses to result in false positives with respect to viable cells. We further show that care must be taken in the way samples are stored for future PCR amplifications and that filter sterilization of media is not acceptable for long-term preservation of samples for PCR.

Bacteria↗