Detection of wheat contamination in dietary non-wheat products by PCR.
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Celiac Disease↗
Biomedical subjects
U Candrian
Publications and source records attributed to U Candrian.
Evaluation of the ligase chain reaction (LCR) for the detection of point mutations.
The ligase chain reaction (LCR) was evaluated as an amplification method for an in vivo mutation assay. Specifically, the ligase was tested for its ability to selectively amplify a DNA sequence mutated at a single base, in the presence of an excess of wild-type DNA. As a model template a 370-bp DNA fragment of the mouse Ha-ras protooncogene containing an A to T mutation at the second position of codon 61 was used. With the commercially available ligase Ampligase (Epicenter), 250 molecules of mutant fragments could be detected by an enzyme-linked immunoassay with digoxigenin marker (giving a theoretical detection limit of 1 target gene per 10(4) copies of genome). In the analysis of mixtures with corresponding wild-type DNA fragments, a 1:1 mixture resulted in a clearly stronger signal than control samples lacking wild-type and mutant DNA. However, the signal obtained from a 100-fold dilution of the mutant DNA with wild-type DNA could not be distinguished from the background noise. In this particular form, LCR lacks sufficient selectivity to be applied to an in vivo situation, where the ratio of mutant to wild-type DNA sequences might be expected to lie around 1:10(6).
Animals↗
Polymerase chain reaction with additional primers allows identification of amplified DNA and recognition of specific alleles.
The use of additional primers in the standard two primer polymerase chain reaction (PCR) is described. This modification allows detection of a target gene in a single reaction, and identification of the amplification product obtained or recognition of a specific allele. The oligonucleotides used are internal to the original amplification primers and amplification-compatible with one of the original primers. Annealing of an additional primer to the target gene as well as to the primary amplification product will lead to the appearance of an additional smaller amplification fragment upon agarose gel electrophoresis of PCR products. Use of one or more allele-specific oligonucleotides as additional primers, in addition to two gene-specific primers, will allow recognition of different alleles of the target gene in a single PCR without further analysis except gel electrophoresis. The general applicability of the method was determined with several PCR assays for the detection of pathogenic bacteria.
Alleles↗
Use of polymerase chain reaction for detection of Listeria monocytogenes in food.
A previously described polymerase chain reaction (PCR) assay (B. Furrer, U. Candrian, C. Höfelein, and J. Lüthy, J. Appl. Bacteriol. 70:372-379, 1991) 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.
Animals↗
Activation of protooncogenes in spontaneously occurring non-liver tumors from C57BL/6 x C3H F1 mice.
The C57BL/6 x C3H F1 (hereafter called B6C3F1) mouse is an important animal model for long-term carcinogenesis studies. Maintained under normal laboratory conditions, these mice develop various types of spontaneous tumors during their lifetime. Activated Ha-ras genes have been detected in 66% of spontaneous hepatocellular tumors in the B6C3F1 mouse [Reynolds et al., Science (Washington DC), 237:1309, 1988]. In this study 49 spontaneous non-liver tumors were investigated for oncogene activation by DNA transfection techniques. Of the 49 tumor DNAs analyzed, only 5 yielded multiple foci in the NIH 3T3 focus assay: 2 of 10 pulmonary adenocarcinomas; 0 of 25 lymphomas; 2 of 2 Harderian gland adenomas; 0 of 1 adenocarcinoma of the small intestine; 1 of 6 malignant skin tumors; 0 of 4 hemangiosarcomas; and 0 of 1 lung metastasis of a hepatocellular carcinoma. DNA from six lymphomas which were negative in the NIH 3T3 focus assay were further analyzed for transforming genes by the nude mouse tumorigenicity assay. One of the five lymphomas tested positive with this assay. Southern blot analysis identified five activated ras genes: H-ras in two Harderian gland adenomas; K-ras in one pulmonary adenocarcinoma and in one s.c. adenocarcinoma; and N-ras in one lymphoma. The mutations involved were CG to AT and AT to TA in codon 61 of the Ha-ras genes, GC to AT or TA in codon 12 of the K-ras genes, and a GC to AT mutation in codon 12 of the N-ras gene. Transformant DNA from a pulmonary adenocarcinoma which yielded multiple foci in the transfection assay did not hybridize to DNA probes specific for the K-, H-, and N-ras, raf, neu, and met genes. Thirteen additional tumor DNAs yielded a single focus in the NIH 3T3 transfection assay. The transformant DNAs retransmitted in a second cycle transfection assay. Rearranged and/or amplified raf genes were detected in six of the transformant DNAs. At present we do not know whether these activated raf genes were present in the original tumor DNA. The other seven transformant DNAs did not hybridize with any of the above mentioned specific DNA probes utilized in Southern blot analysis. Unlike liver tumors, the activation of ras protooncogenes is not a frequent event in the development of spontaneous non-liver tumors of the B6C3F1 mouse. The results from this study should aid in understanding the neoplastic development associated with exposure to chemical carcinogens in the B6C3F1 mouse.
Adenocarcinoma↗
Detection of Escherichia coli and identification of enterotoxigenic strains by primer-directed enzymatic amplification of specific DNA sequences.
The polymerase chain reaction (PCR) was used to amplify DNA sequences from the malB operon of Escherichia coli. All E. coli strains tested yielded the specific DNA fragment. No amplification products were obtained with other Enterobacteriaceae. E. coli strains which produce enterotoxins were identified with additional primer pairs specific for the genes coding for the heat-labile toxin type I (LTI) and the heat-stable toxin type I (STI). Amplification products were identified by DNA-DNA hybridization. Alternatively, restriction endonuclease analysis was used for identification and to distinguish between different alleles of the enterotoxin genes. The detection limit was 10 bacteria. The PCR systems were validated by testing 27 E. coli of known enterotoxigenic properties. The PCR results were consistent with factual toxin production as determined by immunoassays. In addition, 58 E. coli strains isolated from soft cheese and mayonnaise were analyzed by PCR. One strain from a cheese sample was found to have the genetic information for STI production. This strain produced STI as determined by enzyme-linked immunosorbent assay.
Animals↗
Detection and identification of Listeria monocytogenes in cooked sausage products and in milk by in vitro amplification of haemolysin gene fragments.
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.
Animals↗
Use of inosine-containing oligonucleotide primers for enzymatic amplification of different alleles of the gene coding for heat-stable toxin type I of enterotoxigenic Escherichia coli.
A method which employs the polymerase chain reaction (PCR) to identify Escherichia coli strains containing the estA gene was developed. This gene codes for heat-stable enterotoxin type I. The use of an inosine-containing pair of amplification primers allowed the amplification of a specific 175-bp DNA fragment from several different estA alleles. The amplified fragments were identified and distinguished by allele-specific oligonucleotide hybridization and characterized by restriction endonuclease analysis. An extension of the classical two-primer PCR proved to be a very simple and rapid method to identify and characterize the estA alleles. Besides the inosine-containing pair of primers, which recognized all described alleles, additional oligonucleotides were used as primers. The sequence of each of these primers was allele specific, and each was amplification compatible with one of the inosine-containing primers. Thus, in one PCR the 175-bp fragment typical for all estA alleles and an allele-specific fragment of different size were produced. These fragments could be separated by agarose gel electrophoresis and were recognized by ethidium bromide staining. Twenty-seven E. coli strains were tested with this amplification system. The presence or lack of the genetic information for production of heat-stable enterotoxin type I was perfectly consistent with the ability of these strains to produce this enterotoxin, as determined by enzyme-linked immunosorbent assay.
Alleles↗
Improving PCR efficiency.
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Calcium-Binding Proteins↗
Detection and identification of E. coli producing heat-labile enterotoxin type I by enzymatic amplification of a specific DNA fragment.
The polymerase chain reaction (PCR) was used to identify strains of Escherichia coli which produce heat-labile toxin type I (LTI). Amplification primers were designed to detect E. coli strains of human as well as porcine origin. This assay was used to test the ATCC 37218 strain, which carries a recombinant plasmid with the genetic information for production of porcine LTI (pLTI). In addition, three clinical E. coli isolates of human and one of porcine origin were tested. All clinical isolates were reported to produce heat-labile enterotoxin (hLTI and pLTI, respectively) when tested by the Y1 adrenal cell method and/or by the CHO cell method. All strains yielded the expected 275 bp DNA fragment after enzymatic amplification. This fragment was further identified by allele specific oligonucleotide hybridization. Alternatively, the fragment was identified by a SmaI restriction enzyme site which is present in the genes of both the E. coli isolated from humans and pigs. The detection limit determined in water with the ATCC 37218 strain was 20 bacteria. The amplified sequence included a CfoI polymorphism which allowed to distinguish between the genes coding for pLTI and hLTI. All of the strains tested showed this polymorphism as expected. Depending on the identification method chosen, SmaI digestion or oligonucleotide hybridization, pure water can be analysed within 8 h or 12 h, respectively. This method may be adapted to environmental and food samples.
Animals↗
Activation of the Ki-ras protooncogene in spontaneously occurring and chemically induced lung tumors of the strain A mouse.
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[a]pyrene-induced lung tumors were in codon 12, whereas 90% of the mutations in the Ki-ras genes detected in ethyl carbamate-induced 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.
Animals↗
In vivo covalent binding of retronecine-labelled [3H]seneciphylline and [3H]senecionine to DNA of rat liver, lung and kidney.
Retronecine-labelled [3H]seneciphylline ([3H]SPH) and [3H]senecionine ([3H]SON) of high specific radioactivity (22 and 49 mCi/mmol, respectively) were prepared biosynthetically with seedlings of Senecio vulgaris L. using [2,3-3H]putrescine as precursor. [2,3-3H]Putrescine was synthesized by Gabriel synthesis of 1,4-diamino-2-butene from 1,4-dibromo-2-butene and catalytic hydrogenation of the product with tritium gas. Rats of both sexes were treated with the labelled pyrrolizidine alkaloids (PAs) (75-215 microCi SPH or 40-485 microCi SON/kg body wt.) and killed after 6 h or 4-5 days. SON-treated females excreted 83.4 +/- 0.2% of applied radioactivity in faeces and urine within 4 days whereas equally treated males excreted 90.9 +/- 3.2% in the same time. Excretion of 3H-activity from SPH-treated females was completed within 5 days (104.7 +/- 6.4%). Corresponding with these results, tissue levels were highest in SON-treated females. DNA and proteins were isolated from liver, lungs and kidneys and covalent binding of the alkaloids to DNA was determined. A Covalent Binding Index (CBI, mumol alkaloid bound per mol nucleotides/mmol alkaloid administered per kg body wt.) of 210 +/- 12 was found for the liver from SON-treated females whereas binding to liver DNA of males was lower by a factor of 4. The DNA damage determined six hours after treatment persisted during the following 4 days. Administration of [3H]SPH to female and male rats resulted in a CBI of 69 +/- 7 and 73/92, respectively, for the liver DNA. Furthermore we found binding of both alkaloids to DNA of lungs and kidneys in male and female rats. The in vivo formation of [3H]SON derived DNA adducts could be proved by HPLC analysis of hydrolyzed DNA.
Animals↗