The polymerase chain reaction (PCR) technique was applied to meat species identification in marinated and heat-treated or fermented products and to the differentiation of closely related species. DNA was isolated from meat samples by using a DNA-binding resin and was subjected to PCR analysis. Primers used were complementary to conserved areas of the vertebrate mitochondrial cytochrome b (cytb) gene and yielded a 359 base-pair (bp) fragment, including a variable 307 bp region. Restriction endonuclease analysis based on sequence data of those fragments was used for diffferentiation among species. Restriction fragment length polymorphisms (RFLPs) were detected when pig, cattle, wild boar, buffalo, sheep, goat, horse, chicken, and turkey amplicons were cut with Alul, Rsal, Taql, and Hinfl. Analysis of sausages indicates the applicability of this approach to food products containing meat from 3 different species. The PCR–RFLP analytical method detected pork in heated meat mixtures with beef at levels below 1%, and the method was confirmed with porcine- and bovine-specific PCR assays by amplifying fragments of their growth hormone genes. Inter- and intraspecific differences of more than 22 animal species with nearly unknown cytb DNA sequences, including hoofed mammals (ungulates), and poultry were determined with PCR–RFLP typing by using 20 different endonucleases. This typing method allowed the discrimination of game meats, including stag, roe deer, chamois, moose, reindeer, kangaroo, springbok, and other antelopes in marinated and heat-treated products.

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.

was used to analyze food for the presence of Listeria monocytogenes. Food samples were artificially contaminated to develop two procedures to detect the organism following enrichment steps. Procedure A was based on dilution of the enrichment broth followed by lysis of the bacteria and direct analysis of the lysate with PCR. With procedure A and artificially contaminated food samples, it was possible to detect fewer than 10 bacteria per 10 g of food. In procedure B, centrifugation was used to concentrate bacteria before lysis and PCR. With procedure A, 330 naturally contaminated food samples of several types were analyzed. Twenty samples were found to be positive for L. monocytogenes, which was in agreement with the classical culture technique. By using procedure B on a subset of 100 food samples, 14 were found to be positive by PCR whereas the classical culture method detected only 13. Analysis times, including enrichment steps, were 56 and 32 h with procedures A and B, respectively.

A major problem in the application of PCR is contamination with material amplified previously. Repeated PCRs result in the accumulation of intact and degraded amplicons and primer artifacts that can contaminate following amplification reactions. Post-PCR UV treatment and pre-PCR uracil DNA glycosylase (UDG) digestion have been recognized to efficiently inactivate or decompose intact amplification fragments. We show here that degraded amplification products and primer artifacts account for decreased sensitivity and may cause false-negative results. Our experiments indicate that partly degraded PCR products and primer artifacts containing sequences homologous to the primer oligonucleotides in the succeeding PCR reaction compete efficiently with sample DNA for the primers. The experiments done in this study may explain unexpectedly low PCR sensitivities reported in an increasing number of publications. In an attempt to solve this problem, we evaluated three post-PCR treatment methods to completely eliminate sequences competing for the amplification primers, namely, 8-methoxypsoralen (MOPS) or hydroxylamine treatment of amplified DNA and use of oligonucleotides containing $'-ChemiClamps. However, all three methods did not sufficiently inhibit artificially produced carryover contaminations. In conclusion, falsepositive results can be eliminated with UDG or UV treatment, but physical barriers are indispensable to avoid the occurrence of false-negative results. Production of Amplicons for Artificial Contamination Oligonucleotides were purchased from Anawa Trading SA

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