Philipp Hübner

Polymerase chain reaction (PCR) in the quality and safety assurance of food : detection of soya in processed meat products

A new method for the specific and sensitive detection of soya (Glycine max) in processed meat products has been developed using polymerase chain reaction (PCR) technology. The presence of soya deoxyribonucleic acid (DNA) from several soya protein concentrates was determined with two pairs of specific oligonucleotides yielding a 414-bp (bp=base pair) fragment and an internal 118-bp fragment amplified from the soya lectinLe1 gene. The test detected DNA from textured soya protein concentrates in meat products at a level of 1% and was confirmed by a commercially available enzyme-linked immunosorbent assay (ELISA).

Quantitative competitive PCR for the detection of genetically modified organisms in food

Abstract Present polymerase chain reaction (PCR) detection methods only allow the qualitative detection of GMO in food without quantitation of the GMO content. Clearly, the availability of quantitative detection methods for GMO analysis is an important prerequisite for the introduction of threshold limits for GMOs in food. PCR is well known to be quantitative if internal DNA standards are co-amplified together with the target DNA. This quantitative competitive (QC) PCR was first described in the early nineties and is widely used nowadays. We have developed and evaluated QC–PCR systems for the quantitative detection of Roundup ReadyTM soybean (RRS) and Maximizer maize (MM) in food samples. Three DNA fragments differing from the GMO specific sequences by DNA insertions were constructed and used as internal standards in QC–PCR. These standards were calibrated by co-amplifying with mixtures containing defined amounts of RRS DNA and MM DNA, respectively. The calibrated QC–PCR systems were applied to several commercial food samples containing RRS and to three certified RRS flour mixtures (Fluka standards). Recently, quantitative methods for the detection of RRS were successfully tested in a collaborative study involving twelve European control laboratories. Thus, QC–PCR methods will allow to survey “de minimis thresholds” of GMOs in food.

Differentiation of sturgeon species by PCR-RFLP

Abstract A method for identification of sturgeon species in caviar has been developed based on the amplification of a region of the mitochondrial genome (tRNAGlu/cytochrome b) using the polymerase chain reaction (PCR). To distinguish between several types of sturgeon caviar the obtained 462bp long PCR-products were cut with different restriction endonucleases (RE) resulting in species-specific restriction fragment length polymorphisms (RFLP). The method is suitable to differentiate between 10 species of Acipenser and Huso originating from Europe and Asia.

Genotoxicity of ethyl carbamate (urethane) in Salmonella, yeast and human lymphoblastoid cells.

Ethyl carbamate is a known carcinogen occurring in fermented food and beverages and is therefore of interest for food safety assurance. We studied the genotoxicity of ethyl carbamate in Salmonella typhimurium, in Saccharomyces cerevisiae and in human lymphoblastoid TK6 cells. In absence of cytochrome P450 enzymes, no ethyl carbamate-mediated genotoxicity was observed in any of the three test systems in the non-cytotoxic range. In the presence of an activating system, ethyl carbamate was found to be mutagenic in Salmonella typhimurium strain TA100 but not in strains TA98 and TA102, indicating base-pair substitutions at G-C base pairs. In contrast, no significant mutagenicity of ethyl carbamate could be detected in human lymphoblastoid TK6 cells. However, applied in cytotoxic concentrations, ethyl carbamate was genotoxic for Saccharomyces cerevisiae in the absence of P450-mediated metabolic activation. Inhibitors of P450IIE1 (DMSO, ethanol and dithiodiethylcarbamate) diminished ethyl carbamate-mediated mutagenicity in Salmonella typhimurium strain TA100 in a dose dependent manner, suggesting that P450IIE1 is the activating enzyme.

Study of the presence of Campylobacter jejuni and C. coli in sand samples from four Swiss chicken farms

Chicken farms are frequently infected with Campylobacter jejuni and Campylobacter coli. The objective of the present study was to investigate environmental samples from chicken farms for the presence of C. jejuni and C. coli. Every week between July and November 1997, three sand samples from the runs of four chicken farms were analyzed by culture and directly by polymerase chain reaction (PCR). These two detection methods were compared to each other. A total of 231 samples were tested. Eleven samples (4%) were found to contain Campylobacter cells by culture, whereas 157 samples (68%) were positive by PCR. All samples which were positive by culture were also positive by PCR. All direct PCR products were further typed by restriction fragment length polymorphism (RFLP). Three different RFLP types and mixtures of these types were observed. Direct PCR products of one chicken farm were further typed by direct sequencing and two temporally separated sequence types could be distinguished. Campylobacter strains isolated by culture were also typed by RFLP and direct sequencing revealing close accordance with the corresponding direct PCR products.