Biomedical subjects

P Fach

Publications and source records attributed to P Fach.

24 records · Page 2Linked to original sources

PubMed1995

PCR and gene probe identification of botulinum neurotoxin A-, B-, E-, F-, and G-producing Clostridium spp. and evaluation in food samples.

A degenerate primer pair was selected to amplify specifically a 260-bp DNA fragment from Clostridium botulinum types A, B, E, F, and G, and five individual probes allowed identification of each toxinotype by hybridization of the PCR products. The 72 strains of different Clostridium species tested and 11 other bacterial species commonly found in food samples gave an amplification product. This assay was able to detect 1 C. botulinum type A or B and 10 C. botulinum type E strains per reaction. With 184 artificially contaminated food samples, after an 18-h enrichment step, the sensitivity was 10 bacteria per g of sample and the correlation with the mouse bioassay reached 95.6%.

Bacterial Typing Techniques↗

PubMed1994

Polymerase chain reaction for Salmonella virulence-associated plasmid genes detection: a new tool in Salmonella epidemiology.

The important role of plasmid genes in assessing virulence for BALB/c mice in salmonella, and the difficulty of using standard techniques to detect them, led us to develop a detection method by gene amplification. One hundred and forty-three strains (71 serovars) of salmonella and 35 strains of other species were tested using specific oligonucleotide primers. The amplification products were identified by a specific oligonucleotide probe. Forty-nine salmonella strains from ten serovars (S. abortus ovis, S. choleraesuis, S. dublin, S. enteritidis, S. gallinarum/pullorum, S. hessarek, S. typhimurium, S. IIIa 48:z4, z23, S. IV 43:z4, z23:-, S. V 28:a:-) produced a positive and specific response. Because of various origins of the strains possessing the gene sought and the diversity of the responses, both from one serovar to another and in the same serovar, this search has its place among the epidemiological markers in general use. This method appears well suited to the research and detection of plasmid genes associated with mouse virulence in salmonella.

Animals↗

PubMed1994

Evaluation of different DNA extraction procedures for the detection of Salmonella from chicken products by polymerase chain reaction.

Polymerase chain reaction (PCR) was used after a short pre-enrichment culture to detect Salmonella subspecies in chicken fillets. A direct PCR assay performed with chicken meat inoculated with Salmonella Typhimurium produced no PCR products. Six different DNA extraction protocols were tested to recover efficiently Salmonella DNA after a short incubation period. Three of them gave results that were reliable, rapid and sensitive. Successful protocols used Proteinase K and/or a centrifugation step to concentrate the samples. For reliable detection of Salmonella subspecies, a few thousand bacterial cells per ml must be present. To obtain this number of bacterial cells with an inoculation of about one cell in 25 g of ionized food products, it was necessary to incubate samples for at least 10 h before PCR. A larger inoculum of approximately 10 cells in 25 g of ionized food products, required 8 h in culture broth to give positive results by PCR-based assay.

Animals↗

PubMed1993

Polymerase chain reaction for the rapid identification of Clostridium botulinum type A strains and detection in food samples.

A polymerase chain reaction (PCR) was developed for the detection of Clostridium botulinum type A, a cause of human botulism. A two primer set and an oligonucleotide detection probe were used to specifically detect Cl. botulinum type A neurotoxin gene (BoNT/A). After 40 cycles of amplification, detection of a 798 bp amplified DNA fragment was carried out by agarose gel electrophoresis and Southern blot hybridization. This assay was able to detect 12.5 fg of purified target DNA or five bacteria per reaction. The sensitivity in artificially contaminated food samples after an 18 h enrichment step ranges from 10 to 10(3) bacteria per g according to the type of food samples. No cross-reactions were observed with the other Cl. botulinum toxinotypes and other bacteria found routinely in food. This PCR method may provide a suitable and rapid alternative to standard techniques for detection of Cl. botulinum type A in food samples.

Animals↗

PubMed1993

Detection by in vitro amplification of the alpha-toxin (phospholipase C) gene from Clostridium perfringens.

A polymerase chain reaction (PCR) with thermostable DNA polymerase from Thermus aquaticus is described for the specific amplification of the phospholipase C (alpha-toxin) gene of Clostridium perfringens. A set of primers selected for their high specificity could detect Cl. perfringens in stools with a detection limit of approximately 5 x 10(2) bacteria, after bi-amplification. A modified PCR without thermal steps was performed to rapidly amplify, with a yield of 60%, the DNA template. With this PCR method Cl. perfringens alpha-toxin gene could be detected within 2 h. The PCR method detected alpha-toxin positive Cl. perfringens but did not react with phospholipase C-producing Bacillus cereus, Pseudomonas aeruginosa, Cl. sordellii and Cl. bifermentans. The amplified PCR products were screened through ethidium bromide agarose gel electrophoresis or, in only 1 h, with the PhastSystem (Pharmacia). This PCR satisfies the criteria of specificity, sensitivity and rapidity required for a useful tool in epidemiology and for the diagnosis of the pathogen Cl. perfringens as it may be used directly on stool samples.

Bacterial Toxins↗

PubMed1991

Biotinylated probes to detect Leptospira interrogans on dot blot hybridization or by in situ hybridization.

Total genomic biotinylated probes which can identify leptospires by hybridization on filters or by in situ hybridization are described in this study. According to the weak G + C content of the strains studied (35-39%) and owing to the decreasing melting temperature (Tm) due to overbiotinylation, hybridization and wash temperatures were optimized at 33 degrees C and at 42 degrees C respectively. Fourteen serovars of Leptospira interrogans belonging to 11 different serogroups and three serovars of Leptospira biflexa were used in this study. Cross-hybridization results show that it is possible, by means of such probes, specifically to recognize pathogenic strains. These probes did not hybridize with the three saprophytic strains: L. buenos-aires, L. patoc and L. andamana. We also ran a total genomic probe, specific to the serovar buenos-aires which hybridizes only with homologous DNA.

Biotin↗