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Biomedical subjects

Patrick Fach

Publications and source records attributed to Patrick Fach.

15 recordsLinked to original sources
PubMed2006

Screening food raw materials for the presence of the world's most frequent clinical cases of Shiga toxin-encoding Escherichia coli O26, O103, O111, O145 and O157.

This work aims to provide a strategy for rapidly screening food raw materials of bovine origin for the presence of the most frequent O-serogroups of Shiga toxin-encoding Escherichia coli (STEC) involved in food poisoning outbreaks. The prevalence of highly pathogenic serogroups of STEC was surveyed in 25 g portions of minced meat and raw milk using PCR-ELISA and multiplex real-time PCR assays. The prevalence of STEC in raw milk (n=205) and meat samples (n=300) was 21% and 15%, respectively. Contamination by the main pathogenic E. coli O-serogroups representing a major public health concern, including O26, O103, O111, O145, and O157, was potentially around 2.6% in minced meat and 4.8% in raw milk. The MPN values showed an overall contamination ranging from 1 to 2 MPN cells from highly pathogenic serogroups/kg. This survey would indicate that the human pathogenic potential of STEC present in these samples probably remains limited. No conclusion can be drawn at the moment concerning a potential risk for consumers. This rapid screening approach for evaluating the potential presence of highly pathogenic serogroups of STEC in food raw materials should help to improve risk assessment of food poisoning outbreaks.

Animals↗
PubMed2004

Prevalence of Clostridium botulinum in food raw materials used in REPFEDs manufactured in France.

Food raw materials used in refrigerated processed foods of extended durability (REPFEDs) manufactured in France were surveyed for Clostridium botulinum types A, B and E using PCR-Enzyme-linked Immunosorbent assay (PCR-ELISA) and mouse bioassay for detection respectively of cells and toxins in enrichment broth. Portions of 25 to 50 g of food were analysed. A total of 8 out of the 102 samples of fish and shellfish, 12 out of the 143 samples of meat and poultry, 1 out of the 62 samples of aroma, sauce and gravy, 4 out of the 25 samples of thickening agents, 3 out of the 26 samples of dehydrated dairy ingredients, and none of the 65 samples of spices, herbs and dehydrated mushroom were positive for C. botulinum in PCR-ELISA, i.e., 6.6% of all the samples tested. The 28 positive samples comprised 10 type A, 10 type B, 4 with both types A and B, and 4 undetermined by PCR typing. No sample positive for type E was detected. Of the 28 samples positive in PCR-ELISA, 15 were also positive in the mouse bioassay. The MPN count was between 1 and 3 C. botulinum/kg of food, which is similar to or in the lower range of values reported in the literature.

Clostridium botulinum↗
PubMed2004

Detection by 5'-nuclease PCR of Shiga-toxin producing Escherichia coli O26, O55, O91, O103, O111, O113, O145 and O157:H7, associated with the world's most frequent clinical cases.

This paper describes 5'-nuclease PCR assays for detecting eight O-serogroups, H7 flagellar antigen and stx genes from the Shiga toxin-producing Escherichia coli (STEC) associated with the world's most frequent clinical cases. A single set of primers was used to detect the genes stx1 and stx2 in the same reaction by 5'-nuclease PCR. Serotyping by 5'-nuclease PCR of STEC was based on the selection of primers and probes targeting the O-antigen gene clusters of E. coli O26, O55, O91, O111, O113, O157, the eae gene of E. coli O103, the O-island 29 of E. coli O145, and the flagellar H7 antigen gene. Results obtained on a collection of 190 strains indicate that the 5'-nuclease PCR assays used here could serve as a basis for rapid specific stx, O and H7 typing of these major pathogenic serogroups of E. coli. This work provides sensitive and specific tests for the rapid, reliable detection of the main pathogenic E. coli O-serogroups of major public health concern.

DNA Primers↗
PubMed2004

A LightCycler real-time PCR hybridization probe assay for detecting food-borne thermophilic Campylobacter.

In a previous study, we reported the performance of a PCR assay amplifying 287-bp of the 16S rRNA gene of thermo-tolerant Campylobacter (C. jejuni, C. lari, C. coli) through an international ring-trial involving 12 participating laboratories. Based on the validated set of primers, a LightCycler real-time PCR assay (LC-PCR), which used fluorescent hybridization probes was developed. The test incorporated an internal amplification control co-amplified with the 16S rRNA gene of Campylobacter to monitor potential PCR inhibitors and ensure successful amplifications. The specificity study involving 39 Campylobacter and nine strains of other species indicated that the LC-PCR test was highly specific, giving cross-reactivity with only one strain of C. upsaliensis (CCUG19559). The sensitivity of the LC-PCR assay, evaluated in 32 spiked poultry-rinse or pork carcass-swab samples, was determined at 10CFU/ml carcass-rinse. The prevalence of samples positive for thermo-tolerant Campylobacter was 58.8% in 68 naturally contaminated poultry rinse samples tested by LC-PCR and the data were in good concordance with those of bacteriological method. The Ct values of the three replicates obtained for each sample tested in three different runs demonstrate that the LC-PCR was highly reproducible and afford a powerful tool for rapid detection of the thermo-tolerant Campylobacter strains.

Animals↗
PubMed2004

Comparison of PCR-ELISA and LightCycler real-time PCR assays for detecting Salmonella spp. in milk and meat samples.

In a previous study, we reported the performance of a PCR assay amplifying 285-bp of the invA gene of Salmonella spp. through an international ring-trial involving four participating laboratories [Int. J. Food Microbiol. 89 (2003) 241]. Based on the validated set of primers and recent advancements in PCR technology, we have designed two specific PCR assays for detecting Salmonella spp. We have compared PCR-enzyme-linked immunosorbent assay (PCR-ELISA) and LightCycler real-time PCR assay (LC-PCR) with the standard ISO 6579 bacteriological reference method. The two PCR tests incorporated an internal amplification control (IAC) co-amplified with the invA gene of Salmonella to monitor potential PCR inhibitors and ensure successful amplification. The selectivity study involved 84 Salmonella and 44 non-Salmonella strains and the samples tested were represented by 60 artificially-contaminated samples of fish, minced beef and raw milk, and 92 naturally-contaminated milk and meat samples. When using either PCR-ELISA or LC-PCR assays, only Salmonella strains were detected. PCR-ELISA and LC-PCR assays gave with pure Salmonella cultures the same detection limit level of 10(3)CFU/ml, which corresponds respectively to 50 and 10 cells per PCR tube. Data on artificially contaminated samples indicated that both PCR methods were able to detect after enrichment less than five Salmonella cells in 25 g of food, giving 100% concordance with the ISO 6579 reference method. The results on naturally contaminated samples demonstrated that despite certain inhibition problems, LC-PCR and PCR-ELISA assays were highly specific and sensitive, and provide a powerful tool for detection of Salmonella in food samples.

Animals↗
PubMed2004

Diagnostic real-time PCR for detection of Salmonella in food.

A robust 5' nuclease (TaqMan) real-time PCR was developed and validated in-house for the specific detection of Salmonella in food. The assay used specifically designed primers and a probe target within the ttrRSBCA locus, which is located near the Salmonella pathogenicity island 2 at centisome 30.5. It is required for tetrathionate respiration in Salmonella. The assay correctly identified all 110 Salmonella strains and 87 non-Salmonella strains tested. An internal amplification control, which is coamplified with the same primers as the Salmonella DNA, was also included in the assay. The detection probabilities were 70% when a Salmonella cell suspension containing 10(3) CFU/ml was used as a template in the PCR (5 CFU per reaction) and 100% when a suspension of 10(4) CFU/ml was used. A pre-PCR sample preparation protocol including a preenrichment step in buffered peptone water followed by DNA extraction-purification was applied when 110 various food samples (chicken rinses, minced meat, fish, and raw milk) were investigated for Salmonella. The diagnostic accuracy was shown to be 100% compared to the traditional culture method. The overall analysis time of the PCR method was approximately 24 h, in contrast to 4 to 5 days of analysis time for the traditional culture method. This methodology can contribute to meeting the increasing demand of quality assurance laboratories for standard diagnostic methods. Studies are planned to assess the interlaboratory performance of this diagnostic PCR method.

Animals↗
PubMed2003

Interlaboratory diagnostic accuracy of a Salmonella specific PCR-based method.

A collaborative study involving four European laboratories was conducted to investigate the diagnostic accuracy of a Salmonella specific PCR-based method, which was evaluated within the European FOOD-PCR project (http://www.pcr.dk). Each laboratory analysed by the PCR a set of independent obtained presumably naturally contaminated samples and compared the results with the microbiological culture method. The PCR-based method comprised a preenrichment step in buffered peptone water followed by a thermal cell lysis using a closed tube resin-based method. Artificially contaminated minced beef and whole broiler carcass-rinse resulted in a detection limit of less than 5 cells per 25 g meat or 100 ml broiler rinse. A total of 435 samples from four countries, including pig carcass swabs (n = 285), whole broiler carcass-rinse (n = 25), various raw meat (n = 33), and environmental samples (n = 92) were investigated. The interlaboratory diagnostic accuracy, i.e. diagnostic specificity and sensitivity, was shown to be 97.5%. The co-amplification of an internal amplification control indicated possible inhibitory substances derived from the sample. This work can contribute to the quality assurance of PCR-based diagnostic methods and is currently proposed as international standard document.

Animals↗
PubMed2003

Evaluation of the performance of LNA and MGB probes in 5'-nuclease PCR assays.

The aim of this study was to evaluate the use of Locked Nucleic Acids (LNA) probes in 5'-nuclease PCR, by comparison with Minor Groove Binder (MGB) probes routinely practiced in laboratories on ABI Prism 7700. The comparison was made using a collection of Staphylococcus aureus strains that have already been characterized by MGB 5'-nuclease PCR assays in a previous study [Mol Cell Probes, submitted for publication]. The sensitivity and specificity of 5'-nuclease PCR assays targeting the Staphylococcal enterotoxin genes sea to see were compared and showed that the LNA and MGB methods were equivalent. In conclusion, the LNA 5'-nuclease PCR assays developed in this work provide a specific and sensitive alternative to the well-established MGB 5'-nuclease PCR assays used for the rapid detection of bacterial pathogens genes on ABI Prism 7700.

DNA Primers↗
PubMed2003

Comparison of different PCR tests for detecting Shiga toxin-producing Escherichia coli O157 and development of an ELISA-PCR assay for specific identification of the bacteria.

In an attempt to develop a standard for ELISA-PCR detection of Shiga toxin producing Escherichia coli (STEC) O157, six published PCR tests were tested in a comparative study on a panel of 277 bacterial strains isolated from foods, animals and humans. These tests were based on the detection of the genes rfbE [J. Clin. Microbiol. 36 (1998) 1801] and rfbB [Appl. Environ. Microbiol. 65 (1999) 2954], the 3' end of the eae gene [Epidemiol. Infect. 112 (1994) 449], the region immediately flanking the 5' end of the eae gene [Int. J. Food. Microbiol. 32 (1996) 103], the flicH7 gene [J. Clin. Microbiol. 35 (1997) 656], or a part of the recently described 2634-bp Small Inserted Locus (SILO(157) locus) of STEC O157 [J. Appl. Microbiol. 93 (2002) 250]. Unlike the other PCR assays, those amplifying the rfb sequences were unable to distinguish toxigenic from nontoxigenic O157. These assays were relatively specific to STEC O157, giving essentially a cross reaction with clonally related E. coli O55 and to a lesser extent with E. coli O145, O125, O126. They also detected the Shiga toxin (stx)-negative derivatives of STEC O157. Based on these results, an ELISA-PCR assay consisting of the solution hybridization of amplicons with two probes that ensured the specificity of the amplification was developed. The ELISA-PCR assay, which used an internal control (IC) of inhibition, was able to detect 1 to 10 copies of STEC O157 in the PCR tube. Adaptation of PCR into ELISA-PCR assay format facilitates specific and sensitive detection of PCR amplification products and constitutes a method of choice for screening STEC O157.

Adhesins, Bacterial↗
PubMed2003

Detection and genotyping by real-time PCR of the staphylococcal enterotoxin genes sea to sej.

This paper describes real-time fluorescence PCR assays for detecting and toxinotyping nine enterotoxin genes from Staphylococcus aureus. A universal set of primers allowed sea, seb, sec, sed, see, seg, seh, sei, sej enterotoxin genes from S. aureus to be detected in a single real-time PCR assay with the LightCycler (LC) instrument. Using the universal forward primer and a type-specific reverse primer, real-time PCR assays allowed the S. aureus enterotoxin genes to be specifically genotyped. A collection of S. aureus isolates (n=83) was detected and further characterised for sea, seb, sec, sed, see, seg, seh, sei, sej, using real-time PCR assays, and data were compared with those obtained by conventional block cycler PCR. Isolates were also tested for their ability to produce staphylococcal enterotoxins A, B, C and D by a commercial reversed passive latex agglutination (RPLA) test. Real-time PCR assays developed on the LightCycler system (LC-PCR) are a powerful tool for rapid detection and toxinotyping of the enterotoxin genes sea to sej from S. aureus. The work offers a very quick, reliable and specific alternative to conventional block cycler PCR assays to identify the enterotoxin profile of toxigenic S. aureus.

Bacterial Proteins↗
PubMed2003

A strategy based on 5' nuclease multiplex PCR to detect enterotoxin genes sea to sej of Staphylococcus aureus.

We describe the development of a strategy based on 5' nuclease multiplex PCR for the rapid detection of nine enterotoxin genes (sea, seb, sec, sed, see, seg, seh, sei, sej) of Staphylococcus aureus. The genotyping scheme consists in identifying these nine enterotoxin genes by three 5' nuclease Triplex-PCR assays. The strategy was evaluated using a collection of S. aureus reference strains previously examined with conventional PCR assays, and by testing previously characterized food S. aureus field strains. The 5' nuclease Triplex-PCR assays correctly detected the se genes in all the reference strains. In tests with field strains there was generally excellent agreement with the results obtained by conventional PCR, except for some strains harbouring variant se genes. The detection limits of the Triplex-PCR assays evaluated using fivefold dilution of recombinant plasmids for each se gene ranged from 16 to 2000 copies of target se genes in the PCR tube. The 5' nuclease Triplex-PCR assays developed are fast and specific, and provide a useful diagnostic tool for the detection and genotyping of se genes. The development of this method is an improvement that should facilitate epidemiological investigations of staphylococcal food poisoning outbreaks.

Base Sequence↗
PubMed2002

Detection by PCR-enzyme-linked immunosorbent assay of Clostridium botulinum in fish and environmental samples from a coastal area in northern France.

The prevalence of Clostridium botulinum types A, B, E, and F was determined in 214 fresh fish and environmental samples collected in Northern France. A newly developed PCR-enzyme-linked immunosorbent assay (ELISA) used in this survey detected more than 80% of samples inoculated with fewer than 10 C. botulinum spores per 25 g and 100% of samples inoculated with more than 30 C. botulinum spores per 25 g. The percent agreement between PCR-ELISA and mouse bioassay was 88.9%, and PCR-ELISA detected more positive samples than the mouse bioassay did. The prevalence of C. botulinum in seawater fish and sediment was 16.6 and 4%, respectively, corresponding to 3.5 to 7 and 1 to 2 C. botulinum most-probable-number counts, respectively, and is in the low range of C. botulinum contamination reported elsewhere. The toxin type identification of the 31 naturally contaminated samples was 71% type B, 22.5% type A, and 9.6% type E. Type F was not detected. The high prevalence of C. botulinum type B in fish samples is relatively unusual compared with the high prevalence of C. botulinum type E reported in many worldwide and northern European surveys. However, fish processing and fish preparation in France have not been identified as a significant hazard for human type B botulism.

Animals↗
PubMedDate not supplied

Multicenter validation of PCR-based method for detection of Salmonella in chicken and pig samples.

As part of a standardization project, an interlaboratory trial including 15 laboratories from 13 European countries was conducted to evaluate the performance of a noproprietary polymerase chain reaction (PCR)-based method for the detection of Salmonella on artificially contaminated chicken rinse and pig swab samples. The 3 levels were 1-10, 10-100, and 100-1000 colony-forming units (CFU)/100 mL. Sample preparations, including inoculation and pre-enrichment in buffered peptone water (BPW), were performed centrally in a German laboratory; the pre-PCR sample preparation (by a resin-based method) and PCR assay (gel electrophoresis detection) were performed by the receiving laboratories. Aliquots of BPW enrichment cultures were sent to the participants, who analyzed them using a thermal lysis procedure followed by a validated Salmonella-specific PCR assay. The results were reported as negative or positive. Outlier results caused, for example, by gross departures from the experimental protocol, were omitted from the analysis. For both the chicken rinse and the pig swab samples, the diagnostic sensitivity was 100%, with 100% accordance (repeatability) and concordance (reproducibility). The diagnostic specificity was 80.1% (with 85.7% accordance and 67.5% concordance) for chicken rinse, and 91.7% (with 100% accordance and 83.3% concordance) for pig swab. Thus, the interlaboratory variation due to personnel, reagents, thermal cyclers, etc., did not affect the performance of the method, which will be proposed as part of a developing international PCR standard.

Animals↗
PubMedDate not supplied

Detection of genetically modified corn (Bt176) in spiked cow blood samples by polymerase chain reaction and immunoassay methods.

The fate of DNA and protein transgenic sequences in products derived from animals fed transgenic crops has recently raised public interest. Sensitive molecular tests targeting the Bt176 genetic construct and the transgenic Cry1Ab protein were developed to determine whether plant sequences, especially transgenic sequences, are present in animal products. A protocol for total DNA extraction and purification from cow whole blood samples was first drawn up and assessed by spiking with known amounts of DNA from Bt176 maize. The limit of detection for transgenic sequences (35S promoter and Bt176-specific junction sequence) was determined by both the polymerase chain reaction-enzyme-linked immunosorbent assay (PCR-ELISA) and the 5'-nuclease PCR assay. Four additional PCR systems were built to substantiate the results. The first detects a mono-copy maize-specific sequence (ADH promoter). Two others target multi-copy sequences from plant nucleus (26S rRNA gene) and chloroplast (psaB gene). The last one, used as a positive control, targets a mono-copy animal sequence (alpha(s1)-casein gene). Both methods detected a minimum spiking at 25 copies of Bt176 maize/mL in 10 mL whole blood samples. The sandwich ELISA kit used detected down to 1 ng transgenic Cry1Ab protein/mL spiked whole blood.

Animals↗