The strain A mouse has a high incidence of spontaneous lung tumors and is susceptible to lung tumor induction by chemical carcinogens. By utilizing transfection assay, Southern blot analysis, and DNA amplification techniques, we have detected an activated Ki-ras gene in the DNAs of both spontaneously occurring and chemically induced lung tumors of strain A mice. The point mutations in the spontaneous lung tumors were in both codon 12 (60%) and codon 61 (30%). In contrast, 100% of the mutations in the Ki-ras gene detected in methylnitrosourea-induced lung tumors and 93% of the mutations in the Ki-ras genes detected in benzo[ajpyreneinduced lung tumors were in codon 12, whereas 90% of the mutations in the Ki-ras genes detected in ethyl carbamateinduced lung tumors were in codon 61. The selectivity of mutations in the Ki-ras oncogene observed in chemically induced tumors, as compared to spontaneous tumors, suggests that these chemicals directly induce point mutations in the Ki-ras protooncogene. These data indicate that the strain A mouse lung tumor model is a very sensitive system to detect the ability of chemicals to activate the Ki-ras protooncogene in lung tissue.The strain A mouse lung tumor bioassay has been utilized for testing more than 300 compounds for carcinogenic activity (1-4). The strain A mouse also has a very high incidence of spontaneous lung tumors (nearly 100%o by 2 years of age), which is often a complicating factor in interpreting bioassay test results for carcinogenicity (1-4). The detection and characterization of activated oncogenes involved in the development of both spontaneously occurring and chemically induced lung tumors ofthe strain A mouse may offer one approach to define the mechanism(s) by which a chemical causes an increased incidence of lung tumors. Activated ras oncogenes have been detected in spontaneously occurring and chemically induced liver tumors of the B6C3F1 mouse (5-8) and the pattern of activating mutations in these tumors has been compared. More than 60% of the activated oncogenes detected in furan-and furfural-associated liver tumors of the B6C3F1 mouse were different from those seen in spontaneous B6C3F1 mouse liver tumors. Activating mutations in the Ha-ras gene and several activated Ki-ras genes were detected in chemically induced mouse liver tumors, indicating that furan and furfural caused an increase in mouse liver tumors at least in part by a genotoxic mechanism (7). Likewise, Wiseman et al. (8) MATERIALS AND METHODS Rodent Tumors. Spontaneous lung tumors were obtained from untreated, 24-to 27-month-old strain A/HeN mice (National Institute of Aging, Bethesda, MD). Strain A/J mice (6-8 weeks old, The Jackson Laboratories) were administered B[a]P by oral intubation at 3 mg per dose, once a week, for 4 weeks (9). EC and MNU were administered i.p. in a single injection with a dose of either 1000 mg/kg of body weight (for EC) or 150 mg/kg of body weight (for MNU) (10). At 12 months, individual lung tumors were collected and quick frozen in liquid nitrogen...

Recent outbreaks of listeriosis have emphasized the urgent need for rapid and reliable detection methods for Listeria spp., especially in food. Haemolysin production is a major factor in the pathogenesis of listeriosis and the polymerase chain reaction (PCR) was used to amplify two specific DNA fragments of the alpha- and the beta-haemolysin genes. The amplification system specifically recognized L. monocytogenes strains. The detection limit determined with pure cultures was 10 bacteria when estimated with alpha-haemolysin primers. In the analysis of 50 samples of cooked sausage products, bacterial colonies suspected to be Listeria spp. were isolated by conventional methods from six samples. PCR analysis identified three of six as L. monocytogenes. Subsequent serotyping showed perfect agreement with the PCR results. Since enrichment is the most time consuming step in conventional methods a PCR procedure which allows the direct detection of L. monocytogenes in milk was developed. Pasteurized milk was artificially contaminated with various levels of L. monocytogenes. The detection limit was determined to be 10 bacteria/10 ml milk and direct detection and identification of L. monocytogenes took less than two working days. These results show that this haemolysin gene amplification system is very rapid and reliable and therefore avoids cumbersome and lengthy cultivation steps.

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.

A polymerase chain reaction (PCR) method designed to sensitively detect and identify Campylobacterjejuni and Campylobacter coli without the need for isolating and culturing strains is described. The intergenic sequence between the flageilin genesfla& andflaB was amplified and characterized with a triple primer or seminested primer approach. A total of 50 bacterial strains, 27 of C. jejuni and C. coli and 23 of other species, were tested, giving no false-positive or false-negative results. The detection limit as determined by ethidium bromide staining of amplification products on agarose gels was 10 bacteria or less in artificially contaminated water, milk, and soft cheese samples with the seminested primer PCR assay. As an application of the PCR system, a set of 93 samples of milk and other dairy products was screened for the presence of C. jejuni and C. coli. We identified six positive samples (6.5%), while none were found with a conventional culture method.