Nuclease Polymerase Chain Reaction Assay Research Articles

Multi‐country outbreak of Shiga toxin‐producing Escherichia coli infection associated with haemolytic uraemic syndrome

EFSA Supporting PublicationsVolume 13, Issue 4 1017E Technical reportOpen Access Multi-country outbreak of Shiga toxin-producing Escherichia coli infection associated with haemolytic uraemic syndrome European Food Safety Authority and European Centre for Disease Prevention and Control, European Food Safety Authority and European Centre for Disease Prevention and ControlSearch for more papers by this author European Food Safety Authority and European Centre for Disease Prevention and Control, European Food Safety Authority and European Centre for Disease Prevention and ControlSearch for more papers by this author First published: 06 April 2016 https://doi.org/10.2903/sp.efsa.2016.EN-1017Citations: 3 Published date: 6 April 2016 Question number: EFSA-Q-2016-00232 AboutPDF ToolsExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL References Luna-Gierke RE, Wymore K, Sadlowski J, Clogher P, Gierke RW, Tobin-D'Angelo M, et al. Multiple-Aetiology Enteric Infections Involving Non-O157 Shiga Toxin-Producing Escherichia coli - FoodNet, 2001–2010. Zoonoses Public Hlth. 2014 Nov; 61(7): 492– 8. European Centre for Disease Prevention and Control. Escherichia coiifactsheet Stockholm: ECDC; [cited 2016 March 29]. Available from: http://ecdc.europa.eu/en/healthtopics/escherichia_coli/basic_facts/Pages/basic_facts.aspx. Fauci A, Braunwald E, Kasper D, Hauser S, Longo D, Jameson J, et al. Harrison's Principles of internal medicine. MH Medical, editor: McGraw Hill Medical; 2008. 1811– 5 p. EFSA (European Food Safety Authority) and ECDC (European Centre for Disease Prevention and Control). The European Union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in 2014. EFSA journal. 13(12): 4329,191. European Centre for Disease Prevention and Control. Surveillance Atlas of Infectious Diseases Stockholm: ECDC; 2016 [cited 2016 March 29]. Available from: http://atlas.ecdc.europa.eu/public/index.aspx. Espie E, Grimont F, Mariani-Kurkdjian P, Bouvet P, Haeghebaert S, Filliol I, et al. Surveillance of hemolytic uremic syndrome in children less than 15 years of age, a system to monitor O157 and non-O157 Shiga toxin-producing Escherichia coli infections in France, 1996–2006. Pediatr Infect Dis J. 2008 Jul; 27(7): 595– 601. Institut de Veille Sanitaire. Epidémie d'infections à E. coli producteurs de Shiga-toxines non 0157 liée à la consommation de camembert au lait cru, nord-ouest de la France, octobre-décembre 20052007 March 30. Available from: http://opac.invs.sante.fr/index.php2lvNnotice_display&id=9416. Ethelberg S, Smith B, Torpdahl M, Lisby M, Boel J, Jensen T, et al. Outbreak of non-O157 Shiga toxin-producing Escherichia coli infection from consumption of beef sausage. Clin Infect Dis. 2009 Apr 15; 48(8): e78– 81. Germinario C, Chironna M, CGallone M, Tafuri S, Desiante F, Calabrese G. Community-wide Outbreak of Haemolytic Uremic Syndrome Associated with Shiga Toxin 2-Producing Escherichia coli O26:H11 in Southern Italy, Summer 2013. Euro Surveill. 2016;(Forthcoming). International Organization for Standardization. ISO/TS 13136:2012 Microbiology of food and animal feed -Real-time polymerase chain reaction (PCR)-based method for the detection of food-borne pathogens -Horizontal method for the detection of Shiga toxin-producing Escherichia coli (STEC) and the determination of O157, O111, O26, O103 and O145 serogroups. Technical Specifications: ISO; 2012. p. 22. Peron E, Zaharia A, Zota L, Severi E, Mârdh O, Usein C, et al. Early findings in outbreak of haemolytic uraemic syndrome among young children caused by Shiga toxin-producing Escherichia coli, Romania, January to February 2016. Eurosurveillance. 2016; 21(11). Jenssen G, Hovland E, Bjerre A, Bangstad H-J, Nygard K, Vold L. Incidence and etiology of hemolytic-uremic syndrome in children in Norway, 1999–2008 - a retrospective study of hospital records to assess the sensitivity of surveillance. BMC Infectious Diseases. 2014; 14(1): 265. Nielsen EM, Andersen MT. Detection and characterization of verocytotoxin-producing Escherichia coli by automated 5' nuclease PCR assay. J Clin Microbiol. 2003 Jul; 41(7): 2884– 93. Citing Literature Volume13, Issue4April 20161017E ReferencesRelatedInformation

Association Between IL-1A Single Nucleotide Polymorphisms and Chronic Beryllium Disease and Beryllium Sensitization

To determine if single nucleotide polymorphisms (SNPs) in interleukin (IL) IL-1A, IL-1B, IL-1RN, IL-2, IL-9, and IL-9R were associated with chronic beryllium disease (CBD) and beryllium sensitization (BeS). Forty SNPs in six IL genes were evaluated in 85 individuals with CBD, 61 individuals with BeS, and 730 individuals without BeS or CBD (nonsensitized) using a 5' nuclease polymerase chain reaction assay. Logistic regression was used to evaluate the association between IL SNPs, CBD, and BeS, adjusting for plant-site and HLA-DPB1Glu69 in additive, dominant, and recessive inheritance models. IL-1A-1142, IL-1A-3769, and IL-1A-4697 were significantly associated with CBD in both the additive and dominant models compared to individuals with BeS or the nonsensitized. These results indicate that genetic variations in the IL-1A gene may play a role in the development of CBD but not BeS.

The relationship between Receptor Activator of Nuclear Factor-.KAPPA.B Ligand (RANKL) gene polymorphism and aortic calcification in Korean women

The aim of this study was to investigate the relationship between RANKL gene polymorphisms and aortic calcification in Korean women. In 237 healthy Korean women, aortic calcification in thoracic and abdominal aorta was examined in simple radiologic method and lumbar spine and femoral neck BMD were examined by dual energy X-ray absorptiometry. Serum OPG levels, bone turnover markers, such as ALP levels and urine deoxypyridinoline levels, and urine calcium excretion were measured. Genotyping of two RANKL gene polymorphisms, rs2277438 and rs9594782, was performed by allelic discrimination using the 5' nuclease polymerase chain reaction assay. The subjects with CT/CC genotypes of the rs9594782 polymorphism had a 3.9 times higher risk of aortic calcification compared with TT genotype. This significance was significant even after adjustment for age, BMI, blood pressure, fasting plasma glucose, serum high and low-density lipoprotein cholesterol levels. Mean levels of urine deoxypyridinoline were significantly higher in the subjects with AG/GG genotypes of the rs2277438 polymorphism compared with AA genotype, and this significance was persistent even after adjustment for age and BMI. There were no associations of mean values for age, BMI, serum OPG and ALP levels, urine calcium excretion, and BMD with RANKL gene polymorphisms. The RANKL gene rs9594782 polymorphism was associated with aortic calcification in Korean women. Rs2277438 polymorphism showed significant association with urine deoxypyridinoline levels, a bone resorption marker. These results suggest its role on vascular calcification and bone metabolism in humans.

Polymorphism in the promoter region of the klotho gene (G-395A) is associated with early dysfunction in vascular access in hemodialysis patients.

Background/AimsVascular access dysfunction is an important cause of morbidity and mortality in hemodialysis (HD) patients. Recent studies have shown that a klotho gene mutation is related to endothelial dysfunction, thrombosis, and arteriosclerosis, which are regarded as causes of vascular access dysfunction. We investigated the relationship between the klotho G-395A polymorphism and early dysfunction in vascular access in HD patients.MethodsPatients who underwent vascular access operations between 1999 and 2002 were enrolled (n=126). Genotyping was performed by allelic discrimination using a 5'-nuclease polymerase chain reaction assay. Clinical data that could be relevant to access dysfunction were obtained from medical records. Early dysfunction of vascular access was defined as the need for any angioplastic or surgical intervention to correct or replace a poorly or nonfunctioning vascular access within 1 year and at least 8 weeks after initial access placement.ResultsOf the 126 patients, the genotype frequency of G-395A was 72.2% for GG (n=91), 24.6% for GA (n=31), and 3.2% for AA (n=4), and the frequency of minor allele was 0.155. Clinical data were similar between the two groups, divided according to the status of the A allele. Early dysfunction occurred in 34 (27.0%) of patients, but it occurred at a significantly higher rate in A allele carriers (45.7%, 16/35) than in noncarriers (19.8%, 18/91; p=0.003).ConclusionsOur results suggest that the klotho G-395A polymorphism could be a risk factor for early dysfunction of vascular access in HD patients.

A Glu487Lys polymorphism in the gene for mitochondrial aldehyde dehydrogenase 2 is associated with myocardial infarction in elderly Korean men

Background A homozygous mutant ( ALDH2 ⁎ 2/ ⁎ 2) of the gene for mitochondrial aldehyde dehydrogenase 2 ( ALDH2) at codon 487 was reported to be associated with myocardial infarction (MI) among Japanese men. However, such an association has never been studied in a Korean population. Method The subjects consisted of 122 men (60–81 y) with MI recruited randomly from Yonsei University Medical Center, Korea. A total of 439 men (60–84 y) without MI were selected as controls from the Ansan Geriatric Study. ALDH2 genotypes were determined using the TaqMan fluorogenic 5′ nuclease polymerase chain reaction assay. Results Genotypes carrying the mutant ALDH2 allele ( ALDH2 ⁎ 1/ ⁎ 2 plus ALDH2 ⁎ 2/ ⁎ 2) were significantly more frequent in patients with MI than in the controls (42.6% vs. 30.5%, P = 0.0163). Multiple logistic regression analysis revealed that ALDH2 ⁎ 1/ ⁎ 2 plus ALDH2 ⁎ 2/ ⁎ 2, together with abnormal high density lipoprotein cholesterol and elevated body mass index, was an independent risk factor for MI in elderly Korean men (odds ratio = 1.976, 95% CI: 1.202–3.248). Conclusions ALDH2 polymorphisms may play an important role in the pathogenesis of MI in elderly Korean men.

A Functional Polymorphism of Toll-Like Receptor 4 Gene Increases Risk of Gastric Carcinoma and Its Precursors

TLR4 is a cell-surface signaling receptor involved in the recognition and host response to Helicobacter pylori. The TLR4+896A>G polymorphism linked with impaired reactivity to bacterial lipopolysaccharide may play a role in gastric carcinogenesis. We assessed associations with premalignant gastric changes in 149 relatives of gastric cancer patients, including 45 with hypochlorhydria and gastric atrophy. We also genotyped 2 independent Caucasian population-based case-control studies of upper gastrointestinal tract cancer, initially in 312 noncardia gastric carcinoma cases and 419 controls and then in 184 noncardia gastric carcinomas, 123 cardia carcinomas, 159 esophageal cancers, and 211 frequency-matched controls. Odds ratios were computed from logistic models and adjusted for potential confounding factors. TLR4+896G carriers had an 11-fold (95% confidence interval [CI], 2.5-48) increased odds ratio (OR) for hypochlorhydria; the polymorphism was unassociated with gastric acid output in the absence of H pylori infection. Carriers also had significantly more severe gastric atrophy and inflammation. Seventeen percent of gastric carcinoma patients in the initial study and 15% of the noncardia gastric carcinoma patients in the replication study had 1 or 2 TLR4 variant alleles vs 8% of both control populations (combined OR = 2.3; 95% CI = 1.6-3.4). In contrast, prevalence of TLR4+896G was not significantly increased in esophageal squamous cell (2%, OR = 0.2) or adenocarcinoma (9%, OR = 1.4) or gastric cardia carcinoma (11%, OR = 1.4). Our data suggest that the TLR4+896A>G polymorphism is a risk factor for noncardia gastric carcinoma and its precursors. The findings underscore the role of the host innate immune response in outcome of H pylori infection.

Relationship between polymorphisms G395A in promoter and C1818T in exon 4 of the KLOTHO gene with glucose metabolism and cardiovascular risk factors in Korean women

Recently, klotho has been proposed as a link between cardiovascular diseases and premature aging, but the relationship between KLOTHO genes and cardiovascular risk factors, especially glucose metabolism, in humans is unclear. We investigate the relationship between polymorphisms G395A in promoter and C1818T in exon 4 of the KLOTHO gene with glucose metabolism and cardiovascular risk factors in Korean women. In 251 women (mean age 51.3+/-6.9 yr), body mass index (BMI), waist circumference, blood pressure, fasting plasma glucose, insulin and lipid profiles were measured. The genotyping of polymorphisms G395A in promoter and C1818T in exon 4 of the KLOTHO gene was performed by allelic discrimination using a 5' nuclease polymerase chain reaction assay. Allele frequencies of G395A polymorphism was 0.829 for the G allele and 0.171 for the A allele and allele frequencies of C1818T polymorphism were 0.804 for the C allele and 0.196 for the T allele, both of which were in compliance with Hardy-Weinberg equilibrium and the two polymorphisms were in linkage disequilibrium (D'=0.43, p<0.01). Mean systolic blood pressure was significantly higher in A allele carriers of G395A polymorphism compared with non-carriers, and the significance was persistent even after adjustment for age and BMI. Mean fasting plasma glucose was significantly higher in T allele carriers of C1818T polymorphism compared with non-carriers, and the significance was persistent even after adjustment for age and BMI. Subjects without any minor allele from either single nucleotide polymorphisms (SNP) had significantly lower mean values for systolic, diastolic blood pressure and fasting plasma glucose levels compared with subjects with both minor allele from either SNP. We observed that KLOTHO G395A polymorphism was associated with blood pressure and KLOTHO C1818T polymorphism was associated with glucose metabolism in Korean women. Further studies are needed to clarify this relationship.

The relationship between four single nucleotide polymorphisms in the promoter region of the osteoprotegerin gene and aortic calcification or coronary artery disease in Koreans

Osteoprotegerin (OPG) is a recently identified cytokine that acts as a decoy receptor for the receptor activator of the NF-kappaB ligand (RANKL). OPG has been shown to be an important inhibitor of osteoclastogenesis and arterial calcification in animal models. OPG has been proposed as a link molecule between osteoporosis and arterial calcification, but the relationship between the OPG gene and the cardiovascular system in human populations is unclear. Thus, the aim of this study was to investigate the relationship between OPG gene polymorphisms and aortic calcification or coronary artery disease in Koreans. Genotyping of four polymorphisms, A163G, G209A, T245G and T950C, in the promoter region of the OPG gene was performed in 251 healthy Korean women (mean age 51.3 +/- 6.9 years) and in a second study population consisting of 100 patients who underwent coronary angiography (mean age 57.0 +/- 11.9 years), by allelic discrimination using the 5' nuclease polymerase chain reaction assay. Cardiovascular risk factors and serum OPG levels were measured and aortic calcification in thoracic and abdominal aorta was examined by simple radiological methods. In the first study population, the prevalence of aortic calcification increased significantly as the subjects grew older. The frequencies of mutant alleles were significantly higher in the subjects with aortic calcification compared with those without aortic calcification in G209A and T950C polymorphisms, although these significances were lost after adjustment for age. No significant relationship was found between OPG gene polymorphisms and serum OPG levels or cardiovascular risk factors. In the second study group, there were no associations between OPG promoter genotypes and aortic calcification, serum OPG levels, or coronary artery disease. We observed that the four polymorphisms in the promoter region of the OPG gene were not associated with aortic calcification or coronary artery disease in Koreans. Further studies are needed to clarify this relationship.

The Effects of Osteoprotegerin Polymorphism on Bone Mineral Metabolism in Korean Women with Perimenopause

Background: Osteoprotegerin (OPG) is a recently identified cytokine, which acts as a decoy receptor for the receptor activator of the NF-κB ligand (RANKL), and has also been shown to be an important inhibitor of osteoclastogenesis in animal models. However, the relationship between OPG gene polymorphism and female bone stati in human populations is unclear. In this study, the relationship between OPG gene polymorphisms and bone mineral metabolism in healthy Korean women was investigated. Methods: We observed 251 healthy women (mean age, 51.3 ± 6.9 yr). The serum OPG concentrations were determined using ELISA, and the biochemical markers of bone turnover and FSH measured using standard methods. The bone mineral densities at the lumbar spine and femoral neck were measured by dual energy χ-ray absorptiometry. The A163G, G209A, T245G and T950C polymorphisms of the OPG gene were analyzed by allelic discrimination using the 5 nuclease polymerase chain reaction assay. Results: The lumbar spine BMD of premenopausal women was marginally decreased in the variant allele group compared to the wild type group (A163G, 0.98 ± 0.14 g/cm 2 (GG +G A) vs. 1.05± 0.15 g/cm 2 (AA), P = 0.070; T245G, 0.97 ± 0.13 g/cm

The association between tumour necrosis factor-alpha gene polymorphism and the susceptibility to rugal hyperplastic gastritis and gastric carcinoma.

Some subjects infected by Helicobacter pylori have enlarged folds in the gastric body, the precise mechanism of which remains obscure. The aim of this study was to clarify the association of tumour necrosis factor-alpha (TNFA) gene polymorphism with susceptibility to hyper-rugosity. We also examined the association of TNFA polymorphism with gastric carcinoma. Four hundred and seventy-two subjects (male/female = 351/121, aged 26-81 years) without gastric carcinoma (control group), and 300 patients (male/female = 218/82, aged 32-91 years) with gastric carcinoma. Barium meal roentgenograms were performed in 396 subjects in the control group and fold width was measured at the greater curvature of the middle portion of the gastric body. Fasting plasma anti-H. pylori IgG titres, pepsinogens (PGs) I and II were analysed, and TNFA -857 promoter polymorphism was distinguished by the 5' nuclease polymerase chain reaction assay and polymerase chain reaction restriction fragment length polymorphism using HincII in both groups. Adjusted odds ratios of TNFA -857 T/T genotype and H. pylori seropositivity for hyper-rugosity (fold width = 6.0 mm) were 6.7 (95% confidence interval (CI) 1.5-28, P < 0.01) and 18.2 (95% CI 4.2-78, P < 0.0001), respectively. There were no significant differences in any genotype or allele frequencies between the control and total gastric carcinoma group. In a subgroup of gastric carcinoma patients who were negative for the PG assay, however, the odds ratio of the T allele was 1.4 (95% CI 1.0-2.0, P < 0.05). The TNFA -857 T/T genotype and H. pylori infection were strongly associated with rugal hyperplastic gastritis. The TNFA -857 T allele may promote gastric carcinoma without severe atrophy.

Optimization of a 3′-minor groove binder-DNA probe targeting the uidA gene for rapid identification of Escherichia coli O157:H7 using real-time PCR

Enterohemorrhagic Escherichia coli are harmful human pathogens capable of causing bloody diarrhea and vomiting. An important serotype commonly associated with human illness is the E. coli O157:H7 serotype. Unlike other real-time polymerase chain reaction (PCR) methods for identifying E. coli O157:H7, this study describes the development and optimization of a real-time PCR method targeting a conserved point mutation at +93 in the uidA ( gusA) gene that is unique to O157:H7, distinguishing it from non-O157:H7 serotypes. A TET-labeled Minor Groove Binder (MGB) DNA probe was designed for use in a 5′ nuclease PCR assay. Using a panel of two E. coli O157:H7 strains, three E. coli non-O157:H7 strains, and one non- E. coli species, the assay was optimized for the specific detection of the E. coli O157:H7 strains. Optimal conditions were identified at high anneal/extend temperatures, low magnesium concentrations, and low probe concentrations, resulting in correct identification of E. coli O157:H7 and non-O157:H7 strains. The improved specificity of MGB probes for single base pair mismatches such as the +93 uidA mutation provides a novel approach towards rapid identification of E. coli O157:H7.

Identification of a new putative enterotoxin SEU encoded by the egc cluster of Staphylococcus aureus.

This paper reports on a new putative enterotoxin SEU encoded by the enterotoxin gene cluster egc from Staphylococcus aureus and on a real-time polymerase chain reaction (PCR) assay for detecting the seu gene. PCR and sequencing revealed a new putative enterotoxin SEU encoded by some egc clusters. The seu gene resulted from sequence divergence in the psient1 and psient2 pseudogenes previously described in the egc cluster (Jarraud et al. [2001] Journal of Immunology166, 669). The presence of the seu gene was investigated in a collection of S. aureus strains by conventional PCR and by a specific 5'-nuclease PCR assay. Among the 24 strains harbouring the egc cluster, four tested positive for the seu gene. The existence of the seu gene adds to the number of newly described se genes and underlines the need for a better understanding of their role in the pathogenesis of S. aureus. A thorough study of the seu gene should provide further insight into the phylogenetics of the staphylococcal enterotoxins.

Nucleic Acid Molecular Biomarkers for Diagnostic Biodosimetry Applications: Use of the Fluorogenic 5′-Nuclease Polymerase Chain Reaction Assay

A reliable, relatively easy method for diagnostic assessment of radiation exposure is needed to support the triage of radiation casualties and medical treatment decisions in military defense operations. Our strategy is to identify radiation-responsive DNA mutations and gene expression targets that can be analyzed using polymerase chain reaction (PCR) assays and an existing fluorescence-based nucleic acid analysis system designed for forward-deployable laboratory applications. Using an in vitro model system of human peripheral blood lymphocytes, we identified a candidate nucleic acid biomarker (i.e., gene expression target) that is responsive to ionizing radiation. In this report, we describe our preliminary Haras gene expression findings. A dose-dependent elevation in Haras gene expression levels was demonstrated using Northern-blot analysis 17 hours after exposure to a 250-kVp dose of X-rays (25-100 cGy, 1 Gy/minute); c-Haras expression levels at 100 cGy were ninefold higher than those of controls. An alternative protocol to quantify the Haras cDNA target, using the rapid, real-time reverse transcriptase fluorogenic 5'-nuclease PCR assay, is described, along with a preliminary characterization of the dynamic range for detection. Our research shows that the analysis of multitarget nucleic acid biomarkers, using the multiplex fluorogenic 5'-nuclease PCR assay, has beneficial applications in radiation epidemiology, radiation therapy, and biodosimetry.

Quantitative plasmid mixture analysis using the fluorogenic 5'-nuclease polymerase chain reaction assay.

The fluorogenic 5'-nuclease polymerase chain reaction (PCR) assay has been shown to be useful for quantifying a given DNA target in a sample. Here we show how an existing PCR protocol can be amended for quantification by incorporating distinctive dual-labeled, sequence-specific oligonucleotide probes and resulting in a two- to threefold broader and more reliable dynamic range than that of conventional end-point analysis of PCR products. Moreover, we show a multiplex situation in which two targets, one normal and one mutated, can be amplified and quantified simultaneously and in the same reaction tube. Use of this novel approach for quantitative PCR applications eliminates the need for post-PCR processing and has clinical- and research-based diagnostic applications, particularly for measuring levels of mutations in a mixture.