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P Fach

Publications and source records attributed to P Fach.

At least 19 recordsLinked to original sources
PubMed2005

Detection of Escherichia coli serogroup O103 by real-time polymerase chain reaction.

AIMS: The aims of the study were to identify the specific genes of O-antigen gene cluster from Shiga toxin-producing Escherichia coli (STEC) O103 and to provide the basis for a specific real-time PCR test for rapid detection of E. coli O103. METHODS AND RESULTS: The published primers complementary to JUMPstart and gnd gene, the conserved flanking sequences of O-antigen genes clusters in E. coli and related species, were used to amplify the 12-kbp O103 O-antigen biosynthesis locus of STEC O103. A DNA library representative of this cluster allowed two O103-specific probes to be identified in the flippase (wzx) and UDP-galactose-4-epimerase (galE) genes. Two specific O103 serotyping real-time PCR tests based on these two genes were successfully developed. CONCLUSIONS: These results confirm that the O-antigen gene cluster sequences of E. coli allow rapidly a specific O-antigen real-time PCR assay to be designed. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings increase the number of real-time PCR-assays available to replace the classical O-serotyping among E. coli O-antigen.

Blotting, Southern↗
PubMed2004

Validation of a PCR-based method for detection of food-borne thermotolerant campylobacters in a multicenter collaborative trial.

A PCR-based method for rapid detection of food-borne thermotolerant campylobacters was evaluated through a collaborative trial with 12 laboratories testing spiked carcass rinse samples. The method showed an interlaboratory diagnostic sensitivity of 96.7% and a diagnostic specificity of 100% for chicken samples, while these values were 94.2 and 83.3%, respectively, for pig samples.

Animals↗
PubMed2003

Comparison of a cultural method with ListerScreen plus Rapid'L.mono or PCR-ELISA methods for the enumeration of L. monocytogenes in naturally contaminated sewage sludge.

Cultural methods used to count Listeria monocytogenes in sewage sludge are laborious and time consuming, and alternative methods are needed to reduce analysis time and improve detection limits. In this study, a survey of L. monocytogenes in sewage sludge is presented with a comparative study between a cultural method and immunomagnetic separation using a ListerScreen test followed by identification of L. monocytogenes with Rapid'L.mono agar or PCR-ELISA. These two alternative methods improved the detection of L. monocytogenes in different types of sludge, irrespective of their physical and chemical characteristics. The ListerScreen method coupled with detection of L. monocytogenes on Rapid'L.mono offers the advantage of being less sophisticated than the molecular method and allows isolation of the organism, which may be useful in epidemiological studies. However, ListerScreen coupled with PCR-ELISA proved best for high-sensitivity detection of L. monocytogenes in sewage samples.

Colony Count, Microbial↗
PubMed2003

Development of a 5'-nuclease PCR assay for detecting Shiga toxin-producing Escherichia coli O145 based on the identification of an 'O-island 29' homologue.

AIMS: A DNA sequence, from Escherichia coli STEC O145, homologous to O-island 29 from STEC O157 is described, together with a real-time PCR assay for detecting it. METHODS AND RESULTS: PCR and sequencing were used to identify the 'O-island 29' homologous DNA sequence from STEC O145 (strain VTH34). The sequence divergence between the STEC O145 and O157 'O-island 29' allowed a STEC O145 5'-nuclease PCR assay to be developed. CONCLUSIONS: The characterization of a novel locus in STEC O145 has allowed a specific O145 serogroup 5'-nuclease PCR assay to be designed. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings increase the number of serogroup PCR assays available as alternatives to classical O-serotyping of E. coli.

Amino Acid Sequence↗
PubMed2003

Identification of a new putative enterotoxin SEU encoded by the egc cluster of Staphylococcus aureus.

AIMS: This paper reports on a new putative enterotoxin SEU encoded by the enterotoxin gene cluster egc from Staphylococcus aureus and on a real-time polymerase chain reaction (PCR) assay for detecting the seu gene. METHODS AND RESULTS: PCR and sequencing revealed a new putative enterotoxin SEU encoded by some egc clusters. The seu gene resulted from sequence divergence in the psient1 and psient2 pseudogenes previously described in the egc cluster (Jarraud et al. [2001] Journal of Immunology166, 669). The presence of the seu gene was investigated in a collection of S. aureus strains by conventional PCR and by a specific 5'-nuclease PCR assay. Among the 24 strains harbouring the egc cluster, four tested positive for the seu gene. CONCLUSIONS: The existence of the seu gene adds to the number of newly described se genes and underlines the need for a better understanding of their role in the pathogenesis of S. aureus. SIGNIFICANCE AND IMPACT OF THE STUDY: A thorough study of the seu gene should provide further insight into the phylogenetics of the staphylococcal enterotoxins.

Amino Acid Sequence↗
PubMed2003

Toward an international standard for PCR-based detection of food-borne thermotolerant Campylobacters: validation in a multicenter collaborative trial.

As part of a European research project, the performance of a PCR assay to detect food-borne thermotolerant campylobacters (Campylobacter jejuni, C. coli, and C. lari) was evaluated through an international collaborative trial involving 12 participating laboratories. DNA from 10 target and 8 nontarget strains was tested, and the results were reported as the presence of a positive signal after gel electrophoresis. The overall inclusivity (sensitivity) was 93.7%, and the exclusivity (specificity) was 100%. The results indicate that the assay can become an international standard and can be confidently applied in microbiological laboratories.

Arcobacter↗
PubMed2002

A PCR test for detecting Escherichia coli O157:H7 based on the identification of the small inserted locus (SILO157).

AIMS: This paper provides identification of a DNA sequence derived from Shiga toxin-producing Escherichia coli (STEC) O157:H7 and information on its utilization for detecting STEC O157 by PCR. METHODS AND RESULTS: Random Amplified Polymorphic DNA and DNA library were used to identify in STEC O157:H7 (strain EDL 933) a 2634-bp Small Inserted Locus, designated SILO157. Analysis of 211 bacterial strains showed that the PCR assays amplifying the SILO157 region could be used to detect STEC O157 with a good specificity. CONCLUSIONS: Characterization of a novel locus in STEC O157 is attractive since the serotype O157:H7 of STEC is still by far the most important serotype associated with more serious diseases. This island encodes putative proteins and especially one that is predicted to be an outer membrane protein designated IHP1. SIGNIFICANCE AND IMPACT OF THE STUDY: Further investigations could now be developed to appreciate the role of the SILO157 in pathogenicity.

Bacterial Outer Membrane Proteins↗
PubMed2002

Identification of the O-antigen biosynthesis genes of Escherichia coli O91 and development of a O91 PCR serotyping test.

AIMS: The aims of the study were to characterize the O91 O-antigen gene cluster from Shiga toxin-producing Escherichia coli (STEC) O91 and to provide the basis for a specific PCR test for rapid detection of E. coli O91. METHODS AND RESULTS: The published primers complementary to JUMPstart and gnd gene, the conserved flanking sequences of O-antigen genes clusters in E. coli and related species were used to amplify the 10-kbp O91 O-antigen biosynthesis locus of STEC O91. A DNA library representative of this cluster allowed two O91 specific probes to be identified, and two specific PCR O91 serotyping tests to be successfully developed. CONCLUSIONS: These results confirm that the O-antigen gene cluster sequences of E. coli allow rapidly a specific O-antigen PCR assay to be designed. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings increase the number of PCR-assays available to replace the classical O-serotyping among E. coli O-antigen.

Cloning, Molecular↗
PubMed2001

Comparison between a PCR-ELISA test and the vero cell assay for detecting Shiga toxin-producing Escherichia coli in dairy products and characterization of virulence traits of the isolated strains.

AIMS: This paper provides information on a PCR-ELISA method for detecting Shiga toxin-producing Escherichia coli (STEC), and on their prevalence in dairy products. METHODS AND RESULTS: The sensitivity and specificity of the test was evaluated using pure cultures, spiked and naturally-contaminated samples. A comparative study with vero cytotoxicity testing was conducted, and STEC isolated from naturally-contaminated samples were characterized. The PCR-ELISA test was highly specific and sensitive, and detected 14% more positive samples than the vero cell assay. The prevalence of STEC in raw milk and unpasteurized cheese was 21.5% and 30.5%, respectively, while samples from the 'dairy environment' and from pasteurized cheese were less contaminated. The 34 strains of STEC isolated from natural samples showed that some of them carried virulence genes. CONCLUSION: No conclusion can be drawn at the moment concerning the potential risk to consumers. SIGNIFICANCE AND IMPACT OF THE STUDY: These data show the necessity of valuable screening methods to appreciate the virulence of STEC.

Animals↗
PubMed2001

Screening for clostridium botulinum type A, B, and E in cooked chilled foods containing vegetables and raw material using polymerase chain reaction and molecular probes.

A molecular method was used for the detection of Clostridium botulinum spores of type A, B, and E in commercial cooked and pasteurized vegetable purées and in the raw materials (vegetables and other ingredients). The method allowed the detection of less than 8 spores/g of product for C. botulinum type A, less than 1 spore/g for proteolytic type B, less than 21 spores/g for nonproteolytic type B, and less than 0.1 spore/g for type E. Thirty-seven samples of raw vegetables and ingredients were tested for the presence of C. botulinum type A, B, and E; 88 and 90 samples of vegetable purées were tested, respectively, for the presence of C. botulinum type A and B and for the presence of C. botulinum type E. All samples were negative, suggesting that the prevalence of C. botulinum in these vegetable purées and the raw ingredients is probably low.

Clostridium botulinum↗
PubMed1999

Evaluation of a polymerase chain reaction-based test for detecting Salmonella spp. in food samples: Probelia Salmonella spp.

A commercially available polymerase chain reaction (PCR) kit was evaluated for the detection of Salmonella spp. in food samples. The test combines PCR amplification and sandwich hybridization of the amplified DNA in microtiter plates. The sensitivity and specificity was evaluated with 52 Salmonella strains and 51 non-Salmonella strains and showed that the test was entirely reliable. The threshold sensitivity was 10(2) CFU/ml. The limit of detection of dead cells that determines the minimum detection level of dead cells in food samples was superior to 10(6) CFU/25 g, a level rarely achieved in naturally contaminated samples. After an 18-h pre-enrichment step, the test could detect viable Salmonella in artificially contaminated food samples, even for the lower contamination level (3 CFU/25 g). There was complete agreement between the PCR test and the ISO 6579 bacteriological reference method with artificially contaminated samples. Regarding the accuracy of the results obtained from 253 naturally or noncontaminated foods and from 32 artificially contaminated foods, the agreement percentage was 99.6%. The fidelity of the technique was evaluated in a collaborative study with eight European laboratories and showed a correlation of 98.4%.

Colony Count, Microbial↗
PubMed1997

Salmonella DNA persistence in natural seawaters using PCR analysis.

The risks of false-positive responses were examined when using the polymerase chain reaction (PCR) method for the detection of Salmonella in the marine environment (water and shellfish). The degradation rates of DNA, both free and from dead Salmonella, were evaluated in natural seawaters maintained at 10 degrees and 20 degrees C, using PCR with Vir and invA primers. The DNA of dead Salmonella was detected up to 55 d in seawater collected in winter and stored at 10 degrees C. But in summer, the persistence was shorter: 10 d or even 2 d for a smaller inoculum (3 x 10(3) Salmonella ml-1). The role of the planktonic organisms present in spring and summer was pinpointed. For free DNA, the persistence times were shorter: from 2 to 4 d at 20 degrees C, and from 3 to 8 d at 10 degrees C showing that the nuclease activity of marine organisms is higher at warm temperatures. These data led us to recommend careful interpretations of direct PCR results, especially during cold periods and for samples collected close to terrestrial discharges of high concentrations of live, dead or lysed Salmonella. PCR is a rapid, specific and sensitive method, but should be applied with care to marine samples, in order to avoid false-positive responses.

Animals↗
PubMed1997

Detection of enterotoxigenic Clostridium perfringens in food and fecal samples with a duplex PCR and the slide latex agglutination test.

A duplex PCR procedure was evaluated for the detection of Clostridium perfringens in food and biological samples and for the identification of enterotoxigenic strains. This method uses two sets of primers which amplify in the same reaction two different DNA fragments simultaneously: the 283-bp C. perfringens phospholipase C gene fragment and the 426-bp enterotoxin gene fragment. Internal primers within the two primer sets confirmed the specificity of the method by DNA-DNA hybridization with the PCR products. No cross-reaction was observed with other Clostridium species or with other bacteria routinely found in food. The detection level was approximately 10(5) C. perfringens cells per g of stool or food sample. When overnight enrichment culture was used, 10 C. perfringens cells per g was detected in 57 artificially contaminated food samples. The duplex PCR is a rapid, sensitive, and reliable method for the detection and identification of enterotoxigenic C. perfringens strains in food samples. A slide latex agglutination test was also evaluated as a rapid, simple technique for the detection of C. perfringens enterotoxin in stool samples.

Clostridium perfringens↗
PubMed1996

Investigation of animal botulism outbreaks by PCR and standard methods.

A double PCR procedure is proposed for identification of Clostridium botulinum C and D. This method consists of a first PCR amplification with a degenerate primer pair able to amplify a 340 bp common DNA fragment from botulinum neurotoxin (BoNT) C1 and D genes, followed by two subsequent PCR amplifications with two primer pairs specific for BoNT/C1 and D respectively (198 bp DNA fragment). This method was found to be specific for C. botulinum C and D, amongst 81 strains of C. botulinum and 21 different species of other Clostridium and bacteria tested. The detection limit ranged from 10 to 10(3) bacteria in the reaction volume according to the C. botulinum C and D strains. In 160 naturally contaminated animal and food samples submitted to a 48 h enrichment culture, the double PCR showed an 89.4% correlation rate with the standard mouse bioassay. A clear distinction between botulism type C and D was obtained. The double PCR provides a reliable alternative for detection and identification of C. botulinum C and D in clinical and food samples.

Animals↗