Nuclease PCR Assay Research Articles

The TNF-A–857*T Polymorphism is Associated with Gastric Adenocarcinoma Risk in a Costa Rican Population

BackgroundCosta Rica is ranked as one of the countries with highest incidence of gastric cancer worldwide. Previous studies in Costa Rican populations have revealed associations between gastric cancer risk and several cytokine polymorphisms that seem to play a role in the regulation of the expression of these proteins. In this study, we assessed associations of the polymorphisms IL-6-174 G/C, IFNGR1-56 C/T, IL-8-251 T/A and TNF-A (-857 C/T, -308 A/G) with gastric pathologies in a high-risk population of Latin America. MethodsDNA samples of 47 patients with gastric adenocarcinoma, 53 with chronic gastritis, 56 with duodenal ulcer and 94 healthy controls, were genotyped for the five mentioned SNPs. All participants were ≥50-years-old. Genotyping was performed by PCR-RFLP and 5’-nuclease PCR assay. H. pylori infection, CagA status, pepsinogen (PG) I and II blood levels were determined by ELISA. Logistic regression analysis was used to determine possible associations of the polymorphisms with cancer, gastritis and duodenal ulcer, and linear regression analysis to determine associations with blood PG levels. ResultsA total of 86.6% of the population was positive for H. pylori; of them, 51.6% was CagA+. Patients with the TNF-A-857*T allele had an increased risk for gastritis (OR: 3.67, p = 0.015) and gastric adenocarcinoma (OR:6.15, p = 0.001). Associations between other polymorphisms and gastric diseases, or PG levels, were not found. ConclusionsOur results indicate that the TNF-A-857*T SNP is among the risk factors associated with the risk of gastric cancer in Costa Rica.

Genetic variants in TNFα, TGFB1, PTGS1 and PTGS2 genes are associated with diisocyanate-induced asthma

Diisocyanates are the most common cause of occupational asthma, but risk factors are not well defined. A case-control study was conducted to investigate whether genetic variants in inflammatory response genes (TNFα, IL1α, IL1β, IL1RN, IL10, TGFB1, ADAM33, ALOX-5, PTGS1, PTGS2 and NAG-1/GDF15) are associated with increased susceptibility to diisocyanate asthma (DA). These genes were selected based on their role in asthmatic inflammatory processes and previously reported associations with asthma phenotypes. The main study population consisted of 237 Caucasian French Canadians from among a larger sample of 280 diisocyanate-exposed workers in two groups: workers with specific inhalation challenge (SIC) confirmed DA (DA+, n = 95) and asymptomatic exposed workers (AW, n = 142). Genotyping was performed on genomic DNA, using a 5′ nuclease PCR assay. After adjusting for potentially confounding variables of age, smoking status and duration of exposure, the PTGS1 rs5788 and TGFB1 rs1800469 single nucleotide polymorphisms (SNP) showed a protective effect under a dominant model (OR = 0.38; 95% CI = 0.17, 0.89 and OR = 0.38; 95% CI = 0.18, 0.74, respectively) while the TNFα rs1800629 SNP was associated with an increased risk of DA (OR = 2.08; 95% CI = 1.03, 4.17). Additionally, the PTGS2 rs20417 variant showed an association with increased risk of DA in a recessive genetic model (OR = 6.40; 95% CI = 1.06, 38.75). These results suggest that genetic variations in TNFα, TGFB1, PTGS1 and PTGS2 genes contribute to DA susceptibility.

CTNNA3 (α-Catenin) Gene Variants Are Associated With Diisocyanate Asthma: A Replication Study in a Caucasian Worker Population

Recently, a genome-wide association study (GWAS) conducted in Korean subjects identified four CTNNA3 (alpha-T catenin) single nucleotide polymorphisms (SNPs) (rs10762058, rs7088181, rs1786929, and rs4378283) associated with diisocyanate-induced occupational asthma (DA). The CTNNA3 gene codes for a cadherin involved in formation of stretch-resistant cell-cell adhesions. We conducted a candidate gene association study to replicate these findings in Caucasian workers. Genotyping was performed on DNA using a 5' nuclease PCR assay collected from 410 diisocyanate-exposed and predominantly Canadian workers including 132 workers with DA confirmed by a specific inhalation challenge (DA+); 131 symptomatic workers in whom DA was excluded by a negative challenge (DA-); and 147 hexamethylene diisocyanate-exposed asymptomatic workers (AWs). As in the Korean study, highly linked CTNNA3 rs7088181 and rs10762058 SNPs (but not rs4378283 and rs1786929) were significantly associated with DA+ when compared with AWs but not in comparison with DA- workers (p ≤ 0.05). After adjusting for potentially confounding variables of age, smoking status, and duration of exposure, minor allele homozygotes of rs7088181 and rs10762058 SNPs were at increased risk for DA compared with AWs (OR = 9.05 [95% CI: 1.69, 48.54] and OR = 6.82 [95% CI: 1.65, 28.24], respectively). In conclusion, we replicated results from the only reported GWAS study of DA demonstrating an association between two closely linked CTNNA3 gene SNPs and DA. These findings lend further support to the clinical relevance of these genotypes in predicting susceptibility to DA and the potential importance of catenins in the disease process.

1273P - T790M Resistant Mutation in Plasma of Acquired Resistant EGFR Mutated Non Small Cell Lung Cancer (Nsclc) Patients - The Tarzo Trial (NCT00503971)

ABSTRACT Background EGFR-mutant NSCLC patients (pts) ultimately overcome resistant to tyrosine kinase inhibitors (TKIs). Among resistant mechanisms secondary EGFR T790M is the most frequent and account 50% of tumors. Previous data suggest that tumors with acquired T790M at post-progression biopsy specimen may have a more favorable prognosis and indolent progression. However, tumor re-biopsies at progression sites are scare in NSCLC patients and blood samples are a non-invasive method that may help to identify resistant mechanisms. Material and methods Pts with advanced NSCLC harboring EGFR mutations (Exon 19 and 21) and acquired resistance to EGFR tyrosine kinase inhibitors (TKIs) were eligible. All patients where included in a phase II trial (TARZO) and treated with erlotinib 150 mg PO daily plus oral vorinostat 400 mg QD on days 1–7 and 15–21 in a 28-day cycle. Blood samples were required at study entry. We aim to determine the feasibility and incidence of T790M resistant mutation from plasma DNA from patients by mutation from cell free circulating DNA from patients using a 5′nuclease PCR assay (TaqMan assay) with a FAM MGB-labeled probe for the wild-type and a VIC MGB-labeled probe for the mutant sequence in the presence of a protein nucleic acid (PNA) clamp, which was designed to inhibit the amplification of the wild-type allele (Pangaea Biotech SL patent). Results Twenty-five pts were included in the trial. From those, nineteen plasma specimens were obtained and used for DNA extraction. Overall T790M resistant mutation was detected from plasma DNA in 36.8% of the samples (7/19). There were no differences according patient characteristics (gender/ECOG-PS, smoking habits, number of previous treatments) or type of sensitizing mutation in tissue (Exon 19, L858R) and the presence of T790M. Median time to progression (TTP) and survival (OS) for the entire cohort was 2.4 months (IC 95% 1.2-3.6) and 13.2 months (IC 95% 2.23-26.9 months). Median TTP and OS were 2.4 vs 1.8 (p 0.076) and 13.7 vs 3.7 (p 0.095) in patients without and with T790M respectively. Conclusions T790M mutation is a common feature of resistant acquired EGFR mutated NSCLC patients and can be detected using plasma DNA. Disclosure All authors have declared no conflicts of interest.

Genetic Variants in Antioxidant Genes Are Associated With Diisocyanate-Induced Asthma

Diisocyanates are a common cause of occupational asthma, but risk factors are not well defined. A case-control study was conducted to investigate whether genetic variants of antioxidant defense genes, glutathione S-transferases (GSTM1, GSTT1, GSTM3, GSTP1), manganese superoxide dismutase (SOD2), and microsomal epoxide hydrolase (EPHX1) are associated with increased susceptibility to diisocyanate-induced asthma (DA). The main study population consisted of 353 Caucasian French-Canadians from among a larger sample of 410 diisocyanate-exposed workers in three groups: workers with specific inhalation challenge (SIC) confirmed DA (DA(+), n = 95); symptomatic diisocyanate workers with a negative SIC (DA(-), n = 116); and asymptomatic exposed workers (AW, n = 142). Genotyping was performed on genomic DNA, using a 5'-nuclease PCR assay. The SOD2 rs4880, GSTP1 rs1695, and EPHX1 rs2740171 variants were significantly associated with DA in both univariate and multivariate analyses. In the first logistic regression model comparing DA(+) and DA(-) groups, SOD2 rs4880, GSTM1 (null), GSTP1 rs762803, and EPHX1 rs2854450 variants were associated with DA (p = 0.004, p = 0.047, p = 0.021, p <0.001, respectively). Genotype combinations GSTT1*GSTP1 rs762803, GSTM1*EPHX1 rs2854450, EPHX1 rs2740168*EPHX1 rs1051741, and GSTP1 rs762803*EPHX1 rs2740168 were also associated with DA in this model (p = 0.027, p = 0.002, p = 0.045, p = 0.044, respectively). The GSTP1 rs1695 and EPHX1 rs1051741 and rs2740171 variants showed an association with DA in the second model comparing DA(+) and AW groups (p = 0.040, p = 0.019, p = 0.002, respectively). The GSTM3 rs110913*EPHX1 rs1051741 genotype combination was also associated with DA under this model (p = 0.042). The results suggest that variations in SOD2, GST, and EPHX1 genes and their interactions contribute to DA susceptibility.

Mutations of the catalytic subunit a of PI3K (PIK3CA) in erlotinib-treated non-small cell lung cancer (NSCLC) patients (p) with epidermal growth factor receptor (EGFR) mutations.

7588 Background: Stage IV NSCLC p with EGFR mutations treated with erlotinib had a median progression-free survival (PFS) of 14 months (m) and median survival (MS) of 27 m in our experience. However, EGFR mutations can only imperfectly predict outcome. PIK3CA mutations occur in several tumors and have prognostic and therapeutic implications. We examined the presence and potential influence of PIK3CA mutations on outcome to erlotinib in NSCLC p with EGFR mutations. Methods: PIK3CA mutation status was assessed in pretreatment paraffin-embedded tumor samples from 118 advanced NSCLC p with EGFR mutations treated with erlotinib. E542K, E545K and H1047R mutations were screened by 5’-nuclease PCR (Taqman) assay and confirmed by standard PCR followed by sequencing. Results: PIK3CA mutations were detected in 6 of 118 patients (5.1%). 84% of p with PIK3CA mutations had adenocarcinomas vs 50% of p with wild-type (wt) PIK3CA (P=0.01). Response rate was 50% for p with mutations vs 70% for p with wt PIK3CA (P=0.03). There were no other differences between p with and without PIK3CA mutations. 2 of the 6 p with PIK3CA mutations had the uncommon L861Q mutation in exon 21 of EGFR. 1 of the 6 p had complete response; 2 had partial response; 3 had progressive disease. There was a non-significant trend towards shorter PFS for the 6 p with PIK3CA mutations (9 vs 16 m; P=0.59). MS was not reached for the 6 p with PIK3CA mutations, while it was 29 m for p with wt PIK3CA (P=0.09). Conclusions: PIK3CA mutations can be present in some EGFR-mutant NSCLC p, with lower response and a trend towards shorter PFS. Treatment with EGFR tyrosine kinase inhibitors plus PIK3CA inhibitors should be tested in this small subset of p with double mutations.

Simplex and multiplex real-time PCR assays for the detection of flagellar (H-antigen) fliC alleles and intimin (eae) variants associated with enterohaemorrhagic Escherichia coli (EHEC) serotypes O26:H11, O103:H2, O111:H8, O145:H28 and O157:H7

To develop real-time PCR assays targeting genes encoding the flagellar antigens (fliC) and intimin subtypes (eae) associated with the five most clinically important serotypes of enterohaemorrhagic Escherichia coli (EHEC), i.e. O26:H11, O103:H2, O111:H8, O145:H28 and O157:H7. Primers and probes specific to fliC(H2) , fliC(H7) , fliC(H8) , fliC(H11) , fliC(H28) , eae-β1, eae-γ1, eae-ε and eae-θ were combined in simplex and multiplex 5'-nuclease PCR assays. The specificity of the assays was assessed on 201 bacterial strains and the sensitivity determined on serially diluted EHEC genomes. The developed PCR assays were found to be highly specific and detected as few as five EHEC genome equivalents per reaction. Furthermore, it was possible to detect the five major EHEC serotypes in cheese samples inoculated at concentration levels of ≤5CFU per 25g after overnight enrichment using the PCR assays. The PCR assays developed here were found to be sensitive and specific for the reliable detection of genes encoding the flagellar antigens and intimin variants belonging to the five most clinically relevant EHEC serotypes. Application of real-time PCR assays should improve the identification of foods contaminated by EHEC and facilitate the molecular typing of these organisms.

Rapid identification of chloroquine and atovaquone drug resistance in Plasmodium falciparum using high-resolution melt polymerase chain reaction

BackgroundDrug resistance determination for Plasmodium falciparum infections are important to determining the type of treatment to be given. Besides in vivo experiments, molecular methods, such as sequencing and PCR, are now increasingly being used. Here a cheaper alternative to sequencing or the use of multiplex 5'nuclease PCR assay for detection and differentiation of drug resistance haplotypes for chloroquine and atovaquone using polymerase chain reaction-high resolution melt (PCR-HRM) is reported.MethodsSeparate PCR-HRM assays were designed for the detection and differentiation of chloroquine and atovaquone drug resistance haplotypes in P. falciparum. PCR was conducted on a thermal cycler and melt curves generated using a LightScanner. These were tested against reference strains of P. falciparum from MR4 as well as 53 local isolates.ResultsThe PCR-HRM assays are able to detect and differentiate between the various haplotypes consistently. These assays can also be used to detect new variants.ConclusionsPCR-HRM is an inexpensive option for the determination of drug resistance profile in P. falciparum and will see increasing use as an alternative to sequencing and 5'nuclease PCR assays in reference laboratories or once PCR systems that are able to conduct HRM become commonplace.

Real-time PCR detection of environmental mycobacteria in house dust

Analysing the number and species of microbes in indoor dust is needed for assessment of human exposure to microbes in dwellings. Environmental mycobacteria are common heterotrophic bacteria in both natural and man-made environments and potential inducers of human immune system. Culture of mycobacteria from samples rich with other microbes is difficult due to the slow growth rate of mycobacteria and this has hampered the studies on their role in indoor human exposure. A quantitative, real-time 5'-nuclease (TaqMan) PCR assay was developed to detect environmental mycobacteria in indoor dust samples. The specificity of the primers and the probe targeting the 16S rDNA of mycobacteria, tested with 26 mycobacterial and 10 non-mycobacterial but related species, proved to be high. When tested on 20 indoor dust samples collected from five homes, the assay gave counts varying between 4.8 × 10 4 and 7.2 × 10 6 cell/g, being on average 1.1 × 10 3 times higher than culture. Seasonal variation in the dust counts of mycobacteria was observed by both culture and qPCR. Total of 140 isolates considered as mycobacteria by Ziehl–Neelsen staining and GLC-analyses were subjected to PCR analysis with the mycobacterial primers, and 39 isolates to partial 16S rDNA sequencing. All proved to be mycobacteria and revealed high diversity of mycobacterial species in the dust samples. Majority of the sequences were related to M. terrae and M. avium complexes.

T2009 Assessment of Novel Genetic Polymorphisms and Risk of Upper Gastrointestinal Carcinoma

Introduction Upper gastrointestinal malignancies comprising gastric and oesophageal cancers remain a major global health problem. Recently, genome wide association studies have been used to identify risk factors for several diseases. Four SNPs identified at chromosome regions 8q24 and 9p24 (rs6983267, rs10505477, rs1447295 and rs719725) have been shown to increase risk of various solid tumours including prostate and colorectal cancer, but have not been studied in the context of upper GI cancer. Methods Two case-control studies were used to assess SNP frequency: Study 1- a gastric cancer case-control study derived from a Caucasian population in Warsaw, Poland with DNA available from 312 non-cardia gastric adenocarcinoma patients and 419 controls, Study 2- a multicentre oesophageal and gastric cancer study from the US with DNA available from 307 subjects with gastric adenocarcinoma, 159 subjects with oesophageal cancer and 211 population controls. DNA was genotyped by 5′ nuclease PCR assay. Hardy-Weinberg equilibrium of alleles at individual loci was assessed using χ 2 test. OR with Cornfield 95% CIs were computed by logistic regression using Stata V.7.0 software. Results Genotyping and statistical analysis of the 4 SNPs was completed on 765 cases and 583 controls. Individuals carrying the C/C genotype of rs1447295 had an elevated risk for non-cardia gastric cancer (OR 1.57, 95% CI 1.03 to 2.45). Individuals carrying the A/A genotype of rs1447295 had an increased risk of squamous cell oesophageal cancer (OR 7.4, 95% CI 1.4 to 49). None of the other three SNPs tested in this study showed any statistically significant associations. Conclusion The association between the rs1447295 SNP and risks of gastric and oesophageal cancer are novel and offer an opportunity to explore the genetic basis of these malignancies. Our study has extended the genetic link between the 8q24 region and risk of yet another group of solid tumours. Extensive efforts should focus on evaluating the functional significance of this region in the context of the molecular pathogenesis of these malignancies.

Influence of cytokine gene variations on immunization to childhood vaccines

The magnitude of the immune response to vaccinations can be influenced by genetic variability. In the present study, we aimed to investigate whether cytokine or cytokine receptor gene polymorphisms were associated with variations in the immune response to childhood vaccination. The study group consisted of 141 healthy infants who had been immunized with hepatitis B vaccine (HBV), 7-valent pneumococcal conjugate (PCV7), and diphtheria, tetanus, acellular pertussis (DTaP) vaccines according to standard childhood immunization schedules. Genotype analysis was performed on genomic DNA using a 5′ nuclease PCR assay. Post vaccination total, isotypic, and antigen-specific serum antibody levels were measured using multiplex immunoassays. Significant associations were observed between SNPs in the TNFα, IL-12B, IL-4Rα, and IL-10 genes and vaccine-specific immune responses ( p < 0.05). In addition, SNPs in the IL-1β, TNFα, IL-2, IL-4, IL-10, IL-4Rα, and IL-12B genes were associated with variations in serum levels of immunoglobulins (IgG, IgA, IgM) and IgG isotypes (IgG1–IgG3) ( p < 0.05). These data suggest that genetic variations in cytokine genes can influence vaccine-induced immune responses in infants, which in turn may influence vaccine efficacy.

Development of a 5'-nuclease PCR assay for the identification of Escherichia coli strains expressing the flagellar antigen H21 and their detection in food after enrichment

To develop a real-time PCR assay targeting the Escherichia coli flagellar antigen H21 for identification and surveillance of clinically important Shiga toxin-producing E. coli (STEC) serotypes classified in seropathotype C. The fliC allele of STEC O91:H21 strain B2F1 was amplified and sequenced. The nucleotide sequence obtained was compared with fliC genes of E. coli O157:H21, O8:H21 and O113:H21 strains. A pair of oligonucleotide primers and a TaqMan minor groove binder probe specific for fliC-H21 were designed and used in a 5'-nuclease PCR assay. This method was evaluated using a panel of 138 diverse bacterial strains and was shown to be 100% specific for H21. PCR amplification of fliC-H21 from one cell per reaction mixture was possible, and an initial inoculum of 10 STEC H21 colony-forming units per 25 g of ground beef was detected after overnight enrichment. The PCR assay developed was found to be highly sensitive and specific for the identification and detection of E. coli H21 strains in ground beef. The real-time PCR assay targeting the H21 flagellar antigen described here offers a valuable method for the rapid detection and molecular typing of pathogenic STEC H21 strains in food.

Construction and evaluation of a microbiological positive process internal control for PCR-based examination of food samples for Listeria monocytogenes and Salmonella enterica

PCR assays for food-borne pathogens are widely available but have had more limited application to food testing compared with their use in clinical laboratories. When testing food samples, false negative PCR results can occur and may be due to interference with target-cell lysis necessary for nucleic acid extraction, nucleic acid degradation and/or direct inhibition of the PCR. Therefore, it is essential to include appropriate controls for the application of PCR to the detection of pathogens in food samples. The purpose of this study was to develop and evaluate a novel internal control (IC), capable of monitoring the complete detection procedure, from DNA extraction through to amplification and detection. A ‘positive process’ IC was developed for the reliable application of real-time PCR assays for the detection of Salmonella enterica or Listeria monocytogenes in enrichment broths. Two novel real-time 5′ nuclease PCR assays for the detection of a 77 bp fragment of the green fluorescent protein ( gfp) gene and a 91 bp fragment of the iroB gene of S. enterica were developed. These assays were specific and had detection limits of 5 ± 0.88 and 15 ± 1.03 CFU per PCR for the gfp and iroB genes respectively. The gfp PCR assay was combined with the iroB PCR assay, and also with a previously published real-time 5′ nuclease PCR assay for the detection of the hlyA gene of L. monocytogenes. Duplexed assays were optimised such that the target genes were simultaneously amplified at similar sensitivities to single reactions. The gfp gene was cloned into the chromosome of a non-pathogenic strain of Escherichia coli and the modified strain successfully encapsulated in LENTICULE discs. A single disc was added to 1 ml of standard enrichment broths immediately prior to DNA extraction, and used as an IC for the detection of L. monocytogenes and S. enterica by PCR. This method was evaluated using 393 naturally contaminated food or environmental samples, 267 for the detection of Salmonella spp. and 126 for Listeria spp. PCR inhibition was detected in 29 (8%) extracts, although neither pathogens were detected in these broths by culture. S. enterica was detected by PCR in 43 of 45 (96%) broths that were positive by conventional culture. The iroB gene was also detected in a further 2 broths by PCR alone. L. monocytogenes was detected in 6 broths by both PCR and conventional culture, plus an additional 7 by PCR only. Control strategies such as those described here are important tools for the interpretation of PCR assays by improving the reliability of detection of pathogens in food.

Detection of Pathogenic Yersinia enterocolitica in Meat using Real-Time PCR

Yersinia enterocolitica is an important zoonosis, which can cause disease in humans and animals. The culture methods available for detection of Y. enterocolitica in food samples are time-consuming and seldom successful. Using DNA-based methods, like PCR, this pathogen can be detected more rapidly and with greater sensitivity. The aim of this study was to establish a rapid and accurate real-time PCR method to detect pathogenic Y. enterocolitica in pork. The chromosomal ail−gene, which is only present in pathogenic Y. enterocolitica strains, was used as DNA target for the 5' nuclease PCR assay. The probe was labelled at the 5' end with the fluorescent reporter dye (FAM) and at the 3' end with the quencher dye (TAMRA). A 2-step protocol with 45 cycles was used in a multicolour real-time PCR detection system. A Ct value over 40 indicated a negative result. The DNA extraction procedure for the natural samples was rapid and simply. This qualitative real-time PCR method was shown to be specific and sensitive. Detection rate of ail-positive Y. enterocolitica in 200 pig tonsils was 88 % and 35 % with PCR and culture methods, respectively. When 100 raw pork samples were studied, 7 were positive with PCR and all were culture negative.

Association of genetic variations in inflammatory and fibrogenic genes with progressive massive fibrosis in coal miners (35.51)

Abstract Progressive massive fibrosis (PMF) is a chronic interstitial lung disease with a complex etiology. Cytokines, growth factors and cell-surface adhesion molecules play crucial roles in the pathogenesis of pulmonary fibrosis by mediating inflammation, fibroblast proliferation, angiogenesis and collagen synthesis. We examined the influence of functional single nucleotide polymorphisms (SNP) in interleukin-1 and 6, tumor necrosis factor-α, transforming growth factor-β, vascular endothelial growth factor, epithelial growth factor and intercellular adhesion molecule-1 genes on the development of PMF. A total of 648 ex-coal miners from the National Coal Workers Autopsy Study were examined in a case-control study. Genotype analysis was performed on genomic DNA using 5′ nuclease PCR assay. There were no significant differences in the frequencies of individual SNPs between PMF and control groups. However, gene-gene interactions were found between TNFα −308/−238, VEGF+405 / ICAM-1+241 and VEGF+405 / ICAM-1+241 / IL-6+174 (p&amp;lt;0.05) variants. These data suggest that genetic variant of cytokine and fibrogenic genes may play role in individual susceptibility to PMF initiated presumably by coal dust. Supported in part by the NIEHS-IAG (Y1-ES-0001). The findings have not been formally disseminated by the NIOSH and should not be construed to represent any agency determination or policy.&amp;lt;

Isolation and Characterization of Yersinia enterocolitica from Swine Feces Recovered during the National Animal Health Monitoring System Swine 2000 Study

A national study was conducted for the isolation of pathogenic Yersinia enterocolitica in pig feces in the United States as part of the National Animal Health Monitoring System's Swine 2000 study. Fecal samples collected from swine operations from September 2000 to March 2001 from 77 production sites in 15 of the top 17 swine-producing states were tested for the presence of pathogenic Y. enterocolitica. After enrichment of swine fecal samples in irgasan–ticarcillin–potassium chlorate broth, the enriched cultures were plated on cefsulodin-irgasan-novobiocin agar for isolation of presumptive Y. enterocolitica. The isolates were confirmed as pathogenic Y. enterocolitica by the fluorogenic 5′ nuclease PCR assay targeting the chromosomal attachment invasion ail gene. Of 2,793 fecal samples tested, 106 (3.80%) ail-positive strains of Y. enterocolitica were isolated. These 106 ail-positive isolates originated from 7 of the 15 participating states. The predominant serotype O:3 (n = 79 of 106) was distributed in five states (n = 5 of 7). Serotype O:5 (n = 27 of 106) was also found in five states (n = 5 of 7). All isolates contained the virulence plasmid and expressed virulence-associated phenotypic characteristics. These results indicate that swine in the United Stares harbor Y. enterocolitica that can potentially cause human illness.

Real-time PCR detection and quantification of vector trichodorid nematodes and Tobacco rattle virus

This report describes a novel diagnostic method for virus-vector trichodorid nematodes and associated Tobacco rattle virus (TRV) based on a real-time fluorogenic 5' nuclease PCR assay (TaqMan). Two independent primer/probe sets were designed targeting the 18S gene of the ribosomal cistron for the trichodorid species, Paratrichodorus pachydermus and Trichodorus similis. Assays using purified plasmid DNA containing clones of the 18S region and genomic DNA extracted from individuals from both nematode species displayed high specificity as no cros s-reaction was observed between the species or with two non-target trichodorid species Paratrichodorus anemones and Trichodorus primitivus. Relative quantification of target DNA present in unknown samples was performed by comparison of the fluorescence signals of the samples to those obtained from plasmid standard dilutions. Three primer/probe sets were also used to target TRV; one set for RNA1 and the two other sets for RNA2 of specific isolates (TRV-PpK20 and TRV-TpO1). Detection of both trichodorid species and TRV RNA1 and RNA2 from a single sample was achieved and field samples were used to demonstrate the potential of this assay to provide rapid, accurate and sensitive molecular information in relation to risk assessment in the field.

Prevalence of pathogenic Yersinia enterocolitica strains in pigs in the United States.

Yersinia enterocolitica is considered an important food-borne pathogen impacting the pork production and processing industry in the United States. Since this bacterium is a commensal of swine, the primary goal of this study was to determine the prevalence of pathogenic Y. enterocolitica in pigs in the United States using feces as the sample source. A total of 2,793 fecal samples were tested for its presence in swine. Fecal samples were collected from late finisher pigs from 77 production sites in the 15 eastern and midwestern pork-producing states over a period of 27 weeks (6 September 2000 to 20 March 2001). The prevalence of ail-positive Y. enterocolitica was determined in samples using both a fluorogenic 5' nuclease PCR assay and a culture method. The mean prevalence was 13.10% (366 of 2,793 fecal samples tested) when both PCR- and culture-positive results were combined. Forty-one of 77 premises (53.25%) contained at least one fecal sample positive for the ail sequence. The PCR assay indicated a contamination rate of 12.35% (345/2,793) compared to 4.08% (114/2,793) by the culture method. Of the 345 PCR-positive samples, 252 were culture negative, while of the 114 culture-positive samples, 21 were PCR negative. Among 77 premises, the PCR assay revealed a significantly (P < 0.05) higher percentage (46.75%, n = 36 sites) of samples positive for the pathogen (ail sequence) than the culture method (22.08%, n = 17 sites). Thus, higher sensitivity, with respect to number of samples and sites identified as positive for the PCR method compared with the culture method for detecting pathogenic Y. enterocolitica, was demonstrated in this study. The results support the hypothesis that swine are a reservoir for Y. enterocolitica strains potentially pathogenic for humans.

Detection and quantification of Shiga toxin-encoding genes in sheep faeces by real-time PCR

Sheep faeces may be an important source of Shiga toxin (Stx)-producing Escherichia coli. We have, therefore, established and evaluated a real-time 5'-nuclease PCR assay to quantify the stx(1) and stx(2) genes in sheep faeces. The detection limit of our assay for both stx(1) and stx(2) in spiked samples corresponded to 10(2)--10(3)CFU/g, which is lower than for other assays for measuring these genes in faecal samples. Quantification values for our assay ranged from 10(2) to 10(7)CFU/g faeces. The assay was evaluated on native, un-spiked faeces. All sheep tested (n=7) shed stx(1), and the quantitative results corresponded to the gene copies in 10(3)--10(4)CFU/g. The level of stx(2), however, was below the quantitative detection limit in all the samples analyzed. This quantitative stx(1) and stx(2) assay may be important in assessing whether sheep harbouring Shiga toxin-producing bacteria represent a potential hazard to human health.

THE EFFECTS OF INFANT FORMULAS CONTAINING EITHER PREBIOTICS OR PROBIOTICS ON THE FAECAL LACTOBACILLUS POPULATION

Aim: The intestinal microbiota of breast-fed infants is generally dominated by the genera Bifidobacterium and Lactobacillus. The intestinal microbiota composition is regarded as an important factor for the infants' health and well being, and several Lactobacillus species have been reported to inhibit pathogens or to stimulate the immune system. In this study the effects of infant formulas containing either prebiotics or probiotics on the percentage of faecal lactobacilli were studied. Methods: At birth, infants of whom the mother had decided not to breast- feed, were at random and double blindly allocated to one of the four formula groups. In total 36 infants received standard infant formula; 19 received a prebiotic formula containing a specific mixture of 0.6 g/100 ml GOS/FOS (ratio 9:1), 17 received a prebiotic formula containing 0.6 g/100 ml GOS and 19 received a probiotic formula containing 6.0 × 109 viable cells/100ml Bifidobacterium animalis Bb12 (ProF). A group of 101 breast-fed infants was included as a reference group. Faecal samples taken on age d5 and w12 were analysed, using a newly developed duplex 5′nuclease PCR assay to determine the percentage of lactobacilli. Results: In the breast-fed group, the percentages of lactobacilli increased significantly between d5 and w12 from 1.1 ± 0.4% to 6.4 ± 1.2% (mean ± SEM; p = 0.001). The GOS/FOS group also showed an increase of lactobacilli resulting in a significantly higher percentage of lactobacilli than in the standard formula group (6.1 ± 2.6% vs. 0.9 ± 0.4%; p = 0.007) at w12. No significant differences were found between GOS (1.1 ± 0.4%) or ProF (2.4 ± 1.7%) versus the standard formula group at w12. Conclusion: Addition of GOS/FOS to the standard infant formula stimulates the percentage of faecal lactobacilli similar to that found in breast-fed infants. In contrast no stimulation of lactobacilli was shown with standard infant formula or after the addition of 0.6g/100 ml GOS or viable Bifidobacterium animalis Bb12 to the standard formula.