Specific detection of buckwheat residues in processed foods by polymerase chain reaction

A specific and qualitative detection method for buckwheat in foods using the polymerase chain reaction (PCR) was developed. Trace amounts of buckwheat in commercial food products were qualitatively detected by this method. It should be reliable for detecting buckwheat residues in processed foods and practical for monitoring the labeling system for allergenic food materials.     

Specific detection of wheat residues in processed foods by polymerase chain reaction

A sensitive qualitative detection method for wheat in foods using polymerase chain reaction (PCR) was developed. Trace amounts of wheat in commercial food products could be qualitatively detected by this method. The sensitivity of the proposed PCR method appears to be similar to that of ELISA. The present method should be very useful for detecting wheat residues in processed foods.     

Specific detection of soybean residues in processed foods by the polymerase chain reaction

A sensitive qualitative detection method for soybeans in foods by using the polymerase chain reaction (PCR) was developed. For specific detection of soybeans with high specificity, the primer pair of Gym 81/Gym 82 was designed on the gene encoding the Glycine max repetitive sequence. The trace amount of soybeans in commercial food products could be qualitatively detected by this method.     

Specific detection by the polymerase chain reaction of potentially allergenic salmonid fish residues in processed foods

Salmonid fish is one of the allergenic items that are recommended to be labeled in the Japanese allergen-labeling system. This study develops a salmonid-specific polymerase chain reaction (PCR) method. A new primer pair, SKE-F/SKE-R, was designed to specifically detect the salmonid fish gene encoding mitochondrial DNA cytochrome b. Genomic DNAs extracted from 58 kinds of seafood and 11 kinds of processed food were individually subjected to PCR by using the primer pair, and a salmonid-specific fragment of 212 bp was only amplified in the salmonid samples and salmonid-containing processed foods. The detection limit of the PCR method was as low as 0.02 fg/μL of salmonid fish DNA (corresponding to 10 copies). There is no ELISA method for salmonid fish, making our PCR method the only reliable measure for detecting salmonid fish in processed foods.     

Detection of walnut residues in processed foods by polymerase chain reaction

A sensitive qualitative detection method for walnut (Juglans regia) using polymerase chain reaction (PCR) was developed. For detection of walnuts with high specificity, the primer pair WAL-F/WAL-R was designed based on walnut matK genes. Trace amounts of walnuts in commercial food products can be qualitatively detected using this method.     

The direct application of the polymerase chain reaction to DNA extracted from foods

Two methods for the successful extraction of DNA from foods are described. The rapid lysis method uses a proteinase K buffer system to lyse cells and solubilize food samples. DNA is then precipitated using isopropanol. The second method achieves cell lysis using toluene and mutanolysin, and solubilization using guanidium thiocyanate. Following protein removal with organic solvents DNA is precipitated with isopropanol. Both methods enabled the polymerase chain reaction to be applied directly to DNA extracted from samples of cheese, coleslaw and raw chicken and allowed the direct rapid, sensitive and specific detection of Yersinia enterocolitica, Aerococcus viridans and Listeria monocytogenes in these foods.     

Removal of contaminating DNA from polymerase chain reaction using ethidium monoazide

The presence of exogenous DNA in PCR reagents and DNA polymerase is a common occurrence. In particular, the amplification of 16S rRNA genes with universal primers for non-culture-based study is often hampered by the formation of false positives. Here, we describe the use of ethidium monoazide (EMA) to eliminate contaminating DNA in a polymerase chain reaction. The advantage of the proposed methodology is the retention of the highly sensitive nature of PCR with the ability to amplify template DNA at concentrations lower than those of contaminating DNA. The treatment of PCR master mix with EMA concentrations that exceeded those required to remove contaminating DNA can interfere with the amplification of low-template concentrations. The methodology presented is straightforward and can be accomplished within 10 min.     

DNA diagnosis of hydatidiform mole using the polymerase chain reaction

Summary We have used the polymerase chain reaction (PCR) technique for the diagnosis of hydatidiform mole, a trophoblastic disease. For this, we targeted the hypervariable 3 flanking region of the APOB gene (APOB/ VNTR) because of its high heterozygosity index (0.61) in the Japanese population. We examined seven clinical cases which were tentatively diagnosed as hydatidiform moles. Five of these revealed DNA segments unique to the paternal APOB allele, allowing us to diagnose a complete mole. The PCR technique for targeting the APOB/VNTR appears useful for early diagnosis of hydatidiform mole.     

A capillary polymerase chain reaction for Salmonella detection from poultry meat

AIMS: In this study, a capillary polymerase chain reaction (cPCR) was applied for Salmonella detection from poultry meat. METHODS AND RESULTS: Salmonella detection limits of the optimized cPCR were determined with DNA templates from the samples of tetrathionate broth (TTB), Rappaport Vassiliadis broth (RVB) and selenite cystine broth (SCB) artificially contaminated with 10-fold dilutions of 6 x 10(8) CFU ml(-1) of pure Salmonella enterica ssp. enterica serovar Enteritidis 64K stock culture. Detection limits of cPCR from TTB, RVB and SCB were found as 6, 6 x 10(1) and 6 x 10(4) CFU ml(-1), respectively. In addition, detection limits of bacteriology were also determined as 6 CFU ml(-1) with TTB and SCB, and 6 x 10(1) CFU ml(-1) with RVB. A total of 200 samples, consisting of 100 chicken and 100 turkey meat samples, were tested with optimized cPCR and bacteriology. Eight and six per cent of the chicken meat samples were found to harbour Salmonella by cPCR and standard bacteriology, respectively. Of six Salmonella isolates, four belonged to serogroup D, two to serogroup B. CONCLUSIONS: The TTB cultures of both artificially and naturally contaminated samples were found to be superior to those of RVB and SCB cultures in their cPCR results. This cPCR, utilizing template from 18-h TTB primary enrichment broth culture, takes approximately 40 min in the successful detection of Salmonella from poultry meat. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that cPCR from TTB enrichment culture of poultry meat would enable rapid detection of Salmonella in laboratories with low sample throughput and limited budget.     

Specific primer design for the polymerase chain reaction

The design of primers has a major impact on the success of PCR in relation to the specificity and yield of the amplified product. Here, we introduce the applications of PCR as well as the definition and characteristics for PCR primer design. Recent primer design tools based on Primer3, along with several computational intelligence-based primer design methods which have been applied in primer design, are also reviewed. In addition, characteristics of population-based methods used in primer design are discussed in detail.     

Human DNA quantitation using Alu element-based polymerase chain reaction

Human forensic casework requires sensitive quantitation of human nuclear DNA from complex sources. Widely used commercially available systems detect both nonhuman and human primate DNA, often require special equipment, and have a detection limit of approximately 0.1ng. Multicopy Alu elements include recently integrated subfamilies that are present in the human genome but are largely absent from nonhuman primates. Here, we present two Alu element-based alternative methods for the rapid identification and quantitation of human DNA, inter-Alu PCR and intra-Alu PCR. Using SYBR green-based detection, the effective minimum threshold level for human DNA quantitation was 0.01ng using inter-Alu- and 0.001ng using intra-Alu-based PCR. Background cross-amplification with nonhuman DNA templates was detected at low levels using inter-Alu-based PCR, but was negligible using intra-Alu-based PCR. These Alu-based methods have several advantages over currently available systems. First, the assays are PCR based and no additional unique equipment is required. Second, the high copy number of subfamily-specific Alu repeats in the human genome makes these assays human specific within a very sensitive linear range. The introduction of these assays to forensic laboratories will undoubtedly increase the sensitivity and specificity of human DNA detection and quantitation from complex sources.     

Hot start of the polymerase chain reaction using DNA helicase]

A novel method for the hot start of PCR using DNA helicases is developed. The addition of a DNA helicase prevents the random annealing of primers and synthesis of nonspecific products during the preparation of the reaction mixture and initial heating. The hot start of PCR occurs automatically after inactivation of the DNA helicase upon heating of the reaction mixture.     

Discrepancy between Penner serotyping and polymerase chain reaction fingerprinting of Campylobacter isolated from poultry and other animal sources

Thirty-four Campylobacter jejuni or coli strains, isolated from various livestock and darkling beetles from two Dutch poultry farms during different broiler production cycles, were subjected to Penner serotyping and polymerase chain reaction (PCR) fingerprint analysis. Ten different Penner serotypes were determined in the isolates. Visual scoring of the PCR fingerprints resulted in 14 clearly different profiles. Some strains with identical Penner serotypes exhibited different PCR fingerprints and conversely strains with different serotypes produced identical PCR fingerprints. Discrepancies between Penner serotyping and PCR fingerprinting were most obvious between isolates from different animal sources. Indications for the occurrence of genomic rearrangements were found. The inconsistency between serotyping and fingerprinting of Campylobacter strains suggests that conventional typing methods should be used in combination with fingerprinting if the epidemiological factors that contribute to Campylobacter colonization of live chickens are to be assessed reliably.     

Amplification of the phage lambda DNA sequence by polymerase chain reaction using thermostable DNA polymerase]

The efficiency of phage DNA amplification by the method of polymerase chain reaction (PCR) with Tth DNA-polymerase was studied for optimization of PCR conditions. The effect on amplification efficiency of medium ionic strength and pH, the presence of univalent cations, detergents, gelatin, ATP, pyrophosphate, SH-reagents and ratio of concentrations of Mg and dNTPs, primers and template was studied. It has been found that a pH optimum for PCR with Tth DNA-polymerase varies from 8.5 to 9.0. An ionic strength optimum for PCR is about 0.08. The influence of univalent cations on the activity of Tth DNA-polymerase can be expressed as NH4+ greater than Na+ greater than K+. 0.01% Tween-20 significantly increases the efficiency of PCR and 0.01% gelatin inhibits it. Addition of ATP, pyrophosphate, SH-reagents to the reaction mixture did not increase the yield of PCR product. It has been also shown that for the given PCR-system an optimum Mg/dNTPs molar ratio is within the range of 1.5-2.0. An optimum concentration of each of the pair of primers for this PCR-system is about 0.3 microM. The possibility of PCR-amplification of 500-8500 b.p. DNA fragments has been demonstrated.     

Specific detection of Neospora caninum oocysts in fecal samples from experimentally-infected dogs using the polymerase chain reaction

Neospora caninum oocysts, passed in the feces of a definitive host (dog), were isolated, and genomic DNA was extracted. A polymerase cahin reaction (PCR) targeting the N. caninum-specific Nc 5 genomic sequence was performed using the isolated DNA. A synthesized competitor molecule containing part of the Nc 5 sequence was included in the assay as a check against false-negative PCR results and to quantify N. caninum oocyst DNA in fecal samples. A standard curve of the ratio of fluorescence intensity of PCR-amplified competitor to that of oocyst DNA was constructed to compare oocyst equivalents from fecal samples containing unknown numbers of N. caninum oocysts and to assess the sensitivity of the assay. The specificity of the assay was determined using the Nc 5-specific primers in PCR assays against other parasites likely to be found in canine feces. Genomic DNA sequences from the canine coccidians Hammondia heydorni, Cryptosporidium parvum, Sarcocystis cruzi, S. tenella, and Isospora ohioensis and the canine helminth parasites Strongyloides stercoralis, Toxocara canis, Dipylidium caninum, and Ancylostoma caninum were not amplified. In addition, genomic DNA sequences from oocysts of coccidian parasites that might contaminate dog feces, such as Hammondia hammondi, Toxoplasma gondii, or Eimeria tenella, were not amplified in the PCR assay. The assay should be useful in epidemiological surveys of both domestic and wild canine hosts and in investigations of oocyst biology in experimental infections.     

Evaluation of different DNA extraction procedures for the detection of Salmonella from chicken products by polymerase chain reaction

Polymerase chain reaction (PCR) was used after a short pre-enrichment culture to detect Salmonella subspecies in chicken fillets. A direct PCR assay performed with chicken meat inoculated with Salmonella Typhimurium produced no PCR products. Six different DNA extraction protocols were tested to recover efficiently Salmonella DNA after a short incubation period. Three of them gave results that were reliable, rapid and sensitive. Successful protocols used Proteinase K and/or a centrifugation step to concentrate the samples. For reliable detection of Salmonella subspecies, a few thousand bacterial cells per ml must be present. To obtain this number of bacterial cells with an inoculation of about one cell in 25 g of ionized food products, it was necessary to incubate samples for at least 10 h before PCR. A larger inoculum of approximately 10 cells in 25 g of ionized food products, required 8 h in culture broth to give positive results by PCR-based assay.     

Mapping oxidative DNA damage using ligation-mediated polymerase chain reaction technology

Reactive oxygen species induce a pharmacopoeia of oxidized bases in DNA. DNA can be cleaved at most of the sites of these modified bases by digestion with a combination of two base excision repair glycosylases from Escherichia coli, Fpg glycosylase, and endonuclease III. The frequency of the resulting glycosylase-dependent 5'-phosphoryl ends can be mapped at nucleotide resolution along a sequencing gel autoradiogram by a genomic sequencing technique, ligation-mediated polymerase chain reaction (LMPCR). In cultured rat cells, the frequency of endogenous oxidized bases in mitochondrial DNA is sufficiently high, about one oxidized base per 100 kb, to be directly mapped from 0.1 microg of total cellular DNA preparations by LMPCR. Nuclear DNA has a lower frequency of endogenous oxidative base damage which cannot be mapped from 1-microg preparations of total cellular DNA. Preparative gel electrophoresis of the PGK1 and p53 genes from 300 microg of restriction endonuclease-digested genomic DNA showed a 25-fold enrichment for the genes and, after endonuclease digestion followed by LMPCR, gave sufficient signal to map the frequency of oxidized bases from human cells treated with 50 microM H2O2.     

Specific detection of Clostridium botulinum type B by using the polymerase chain reaction.

The polymerase chain reaction (PCR) and a radiolabeled oligonucleotide probe were used to specifically detect proteolytic and nonproteolytic Clostridium botulinum type B. Two synthetic primers deduced from the amino acid sequence data of type B neurotoxin were used to amplify a 1.5-kbp fragment corresponding to the light chain of the toxin. Although, nonspecific priming was observed when the PCR protocol was tested with other clostridial species, only the PCR product from C. botulinum type B isolates reacted with the radiolabeled internal probe. As little as 100 fg of DNA (approximately 35 clostridial cells) could be detected after only 25 amplification cycles.