ABSTRACT
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.
Subject(s)
Campylobacter jejuni/isolation & purification , Dairy Products , Escherichia coli/isolation & purification , Listeria monocytogenes/isolation & purification , Milk , Animals , Campylobacter coli/isolation & purification , Electrophoresis, Agar Gel , Food Microbiology , In Vitro Techniques , Polymerase Chain Reaction/methodsABSTRACT
The polymerase chain reaction was used to obtain randomly-amplified polymorphic DNA (RAPD) profiles from Listeria spp. and enterobacteria. Eleven different oligonucleotides were evaluated. Only one, HR4 (19mer), generated reproducible and specific profiles for Listeria spp., while results for enterobacteria were controversial. A total of 57 different Listeria strains were subjected to the RAPD analysis and 27 different profiles were recognized. RAPD typing allowed strains of the same serotype to be distinguished but the same profile was obtained from different serotypes of L. monocytogenes in three cases and in one case two different serotypes of L. innocua yielded the same profile. RAPD-typing with HR4 allowed L. monocytogenes contamination in several food outlets to be traced back to a food processing plant. In additional experiments, the general utility of this RAPD system in typing Yersinia enterocolitica, verotoxigenic Escherichia coli and Salmonella enteritidis was evaluated.
Subject(s)
DNA, Bacterial/isolation & purification , Food Microbiology , Listeria monocytogenes/isolation & purification , Meat Products/microbiology , Polymerase Chain Reaction/methods , Bacterial Typing Techniques , Base Sequence , DNA, Bacterial/genetics , Enterobacteriaceae/classification , Enterobacteriaceae/genetics , Enterobacteriaceae/isolation & purification , Evaluation Studies as Topic , Food Contamination , Food Handling , Listeria monocytogenes/classification , Listeria monocytogenes/genetics , Molecular Sequence Data , Oligonucleotides , Polymorphism, GeneticABSTRACT
A rapid, sensitive and specific analysis of food samples determining wheat contamination was established using polymerase chain reaction (PCR) technology. First, primers specific for highly conserved eukaryote DNA sequences were used to prove isolated nucleic acid substrate accessibility to PCR amplification. Subsequently, a highly repetitive and specific genomic wheat DNA segment was amplified by PCR for wheat detection. This assay was tested with 35 different food samples ranging from bakery additives to heated and processed food samples. In addition, the PCR method was compared to an immunochemical assay that detected the wheat protein component gliadin. Combination of both assays allowed a detailed characterization of wheat contamination. Hence, wheat flour contamination could be distinguished from gliadin used as a carrier for spices as well as from wheat starch addition.
