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A bis(18-crown-6) Tröger's base receptor and 4-substituted hepta-1,7-diyl bisammonium salt ligands have been used as a model system to study the interactions between non-polar side chains of peptides and an aromatic cavity of a protein. NMR titrations and NOESY/ROESY NMR spectroscopy were used to analyze the discrimination of the ligands by the receptor based on the substituent of the ligand, both quantitatively (free binding energies) and qualitatively (conformations). The analysis showed that an all-anti conformation of the heptane chain was preferred for most of the ligands, both free and when bound to the receptor, and that for all of the receptor-ligand complexes, the substituent was located inside or partly inside of the aromatic cavity of the receptor. We estimated the free binding energy of a methyl- and a phenyl group to an aromatic cavity, via CH-π, and combined aromatic CH-π and π-π interactions to be -1.7 and -3.3â kJ mol-1 , respectively. The experimental results were used to assess the accuracy of different computational methods, including molecular mechanics (MM) and density functional theory (DFT) methods, showing that MM was superior.
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Simulación de Dinámica Molecular , Péptidos , Ligandos , Espectroscopía de Resonancia Magnética , Conformación MolecularRESUMEN
Clostridioides difficile (CD), previously known as Clostridium difficile, is an anaerobic Gram-positive rod-shaped bacterium that causes mild to severe diarrhea mainly in hospitalized patients. The bacteria are easily spread between patients and can persist in hospital wards due to its ability to form spores. An outbreak of CD causes great sufferings for patients and is in many aspects very expensive for the health care organization. Continuously monitoring circulating CD isolates in the hospital as well as being able to detect possible spread between patients at an early phase would be of great benefit. Recently a new method was published by Rizzardi et al. (2015) where CD can be typed to a High Molecular Weight (HMW)-profile using Matrix-Assisted Laser Desorption Ionization -Time of Flight Mass Spectrometry (MALDI-TOF MS). We analyzed 1000 isolates of toxin-positive CD with this method and compared the frequency of profiles within different hospitals as well as between two counties in the south-east part of Sweden. During the study period we could detect three outbreaks of CD in three different hospitals. One was an outbreak of CD with ribotype 027, resulting in severe consequences. The method was easily implemented at the clinical microbiology routine diagnostic laboratory and in collaboration with the hospitals Infection Control Units it is a very useful and cost-effective tool to detect outbreaks of CD at an early stage.
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Técnicas de Tipificación Bacteriana/métodos , Clostridioides difficile/aislamiento & purificación , Infecciones por Clostridium/microbiología , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Clostridioides difficile/química , Clostridioides difficile/genética , Infecciones por Clostridium/diagnóstico , Infecciones por Clostridium/epidemiología , Brotes de Enfermedades , Humanos , Peso Molecular , Suecia/epidemiologíaRESUMEN
Enhanced precision of epidemiological typing in clinically suspected nosocomial outbreaks is crucial. Our aim was to investigate whether single nucleotide polymorphism (SNP) analysis and core genome (cg) multilocus sequence typing (MLST) of whole genome sequencing (WGS) data would more reliably identify a nosocomial outbreak, compared to earlier molecular typing methods. Sixteen isolates from a nosocomial outbreak of ESBL E. coli ST-131 in southeastern Sweden and three control strains were subjected to WGS. Sequences were explored by SNP analysis and cgMLST. cgMLST clearly differentiated between the outbreak isolates and the control isolates (>1400 differences). All clinically identified outbreak isolates showed close clustering (≥2 allele differences), except for two isolates (>50 allele differences). These data confirmed that the isolates with >50 differing genes did not belong to the nosocomial outbreak. The number of SNPs within the outbreak was ≤7, whereas the two discrepant isolates had >700 SNPs. Two of the ESBL E. coli ST-131 isolates did not belong to the clinically identified outbreak. Our results illustrate the power of WGS in terms of resolution, which may avoid overestimation of patients belonging to outbreaks as judged from epidemiological data and previously employed molecular methods with lower discriminatory ability.
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Infección Hospitalaria/epidemiología , Brotes de Enfermedades , Infecciones por Escherichia coli/epidemiología , Tipificación de Secuencias Multilocus/métodos , Infección Hospitalaria/microbiología , Escherichia coli/enzimología , Escherichia coli/genética , Escherichia coli/aislamiento & purificación , Infecciones por Escherichia coli/microbiología , Genoma Bacteriano , Humanos , Epidemiología Molecular , Plásmidos/genética , Polimorfismo de Nucleótido Simple , Suecia/epidemiología , Secuenciación Completa del Genoma , Resistencia betalactámica , beta-Lactamasas/genéticaRESUMEN
A single-tube method, ligation-mediated real-time PCR high-resolution melt analysis (LMqPCR HRMA), was modified for the rapid typing of Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp. (ESKAPE) pathogens. A 97% agreement (60/62 isolates) was achieved in comparison to pulsed-field gel electrophoresis (PFGE) results, which indicates that LMqPCR HRMA is a rapid and accurate screening tool for monitoring nosocomial outbreaks.
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Técnicas de Tipificación Bacteriana/métodos , Bacterias Gramnegativas/clasificación , Infecciones por Bacterias Gramnegativas/microbiología , Bacterias Grampositivas/clasificación , Infecciones por Bacterias Grampositivas/microbiología , Epidemiología Molecular/métodos , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Infección Hospitalaria/epidemiología , Infección Hospitalaria/microbiología , ADN Bacteriano/química , ADN Bacteriano/genética , Bacterias Gramnegativas/genética , Bacterias Gramnegativas/aislamiento & purificación , Infecciones por Bacterias Gramnegativas/epidemiología , Bacterias Grampositivas/genética , Bacterias Grampositivas/aislamiento & purificación , Infecciones por Bacterias Grampositivas/epidemiología , Humanos , Factores de Tiempo , Temperatura de TransiciónRESUMEN
BACKGROUND: This prospective population based cohort study explores possible associations between host gene polymorphisms, blood group and life style factors on the one hand, and Helicobacter pylori infection, peptic ulcer, and the grade of inflammation, atrophy and intestinal metaplasia of the gastric mucosa, on the other hand. METHODS: The study population (472 volunteers) has previously undergone screening with gastroduodenoscopy, biopsy and blood sampling. The host gene polymorphisms of IL1B-31C/T, IFNGR1-56T/C, the IL1RN VNTR in exon 2 and the HLA-DRB1 gene alleles were analyzed using PCR and pyrosequencing. RESULTS: H. pylori infection was negatively related to HLA DRB1*03 (odds ratio (OR) 95% CI: 0.388 - 0.989) and was more frequent in individuals with blood group O than A (OR 95% CI: 1.121 - 2.677). There was a lower risk of moderate to severe inflammation in the antrum among individuals with IL1B-31 TC compared to CC carriers (OR 95% CI: 0.094 - 0.733). The IL1RN*L2 genotype was associated with higher risk of IM in the antrum than the *LL genotype (OR 95% CI: 1.570 - 15.878). There was a negative relation between the HLA DRB1 alleles *04 (OR 95% CI: 0.234 - 0.831) and *08 (OR 95% CI: 0.013 - 0.915), and IM in the antrum. CONCLUSION: The IL1RN VNTR and the IL1ß-31 alleles seem to be associated with intestinal metaplasia of the corpus mucosa and the grade of inflammation of the antrum, respectively. However, no unambiguous correlations could be identified between the host polymorphisms and the occurrence of H. pylori infection, peptic ulcer, and the grade of inflammation, atrophy and IM of the gastric mucosa.
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BACKGROUND: Chronic gastritis, peptic ulcer disease, and gastric cancer have been shown to be related to infection with Helicobacter pylori (H. pylori). Two major virulence factors of H. pylori, CagA and VacA, have been associated with these sequelae of the infection. In this study, total DNA was isolated from gastric biopsy specimens to assess the cagA and vacA genotypes. RESULTS: Variations in H. pylori cagA EPIYA motifs and the mosaic structure of vacA s/m/i/d regions were analysed in 155 H. pylori-positive gastric biopsies from 71 individuals using PCR and sequencing. Analysis of a possible association between cagA and vacA genotypes and gastroduodenal pathogenesis was made by logistic regression analysis. We found that H. pylori strains with variation in the number of cagA EPIYA motif variants present in the same biopsy correlated with peptic ulcer, while occurrence of two or more EPIYA-C motifs was associated with atrophy in the gastric mucosa. No statistically significant relation between vacA genotypes and gastroduodenal pathogenesis was observed. CONCLUSIONS: The results of this study indicate that cagA genotypes may be important determinants in the development of gastroduodenal sequelae of H. pylori infection. In contrast to other studies, vacA genotypes were not related to disease progression or outcome. In order to fully understand the relations between cagA, vacA and gastroduodenal pathogenesis, the mechanisms by which CagA and VacA act and interact need to be further investigated.
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Antígenos Bacterianos/genética , Proteínas Bacterianas/genética , Infecciones por Helicobacter/microbiología , Infecciones por Helicobacter/patología , Helicobacter pylori/patogenicidad , Biopsia , Mucosa Gástrica/microbiología , Mucosa Gástrica/patología , Genotipo , Helicobacter pylori/genética , Helicobacter pylori/aislamiento & purificación , Humanos , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADN , Suecia , Factores de Virulencia/genéticaRESUMEN
The mosaic structure of the cagA gene has been suggested to affect Helicobacter pylori CagA-associated pathogenesis. An improved polymerase chain reaction assay allowed for a rapid and detailed molecular analysis of the cagA gene 3'-region in a single amplification step, followed by amplicon sequencing using universal M13 and T7 sequencing primers.
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Antígenos Bacterianos/genética , Proteínas Bacterianas/genética , Helicobacter pylori/genética , Reacción en Cadena de la Polimerasa/métodos , Polimorfismo Genético , Cartilla de ADN/genética , Helicobacter pylori/aislamiento & purificación , Humanos , Análisis de Secuencia de ADNRESUMEN
The Helicobacter pylori cagA gene encodes a cytotoxin which is activated by phosphorylation after entering the host epithelial cell. Phosphorylation occurs on specific tyrosine residues within EPIYA motifs in the variable 3'-region. Four different cagA EPIYA motifs have been defined according to the surrounding amino acid sequence; EPIYA-A, -B, -C and -D. Commonly, EPIYA-A and -B are followed by one or more EPIYA-C or -D motif. Due to observed discrepancies in cagA genotypes in cultured H. pylori and the corresponding DNA extracts it has been suggested that genotyping assays preferentially should be performed directly on DNA isolated from biopsy specimens. Gastric biopsies randomly selected from a Swedish cohort were homogenised and used for both direct DNA isolation and for H. pylori specific culturing and subsequent DNA isolation. In 123 of 153 biopsy specimens, the cagA EPIYA genotypes were in agreement with the corresponding cultured H. pylori strains. A higher proportion of mixed cagA EPIYA genotypes were found in the remaining 30 biopsy specimens. Cloning and sequencing of selected cagA EPIYA amplicons revealed variations in number of cagA EPIYA-C motifs in the mixed amplicons. The study demonstrates that culturing of H. pylori introduces a bias in the number of EPIYA-C motif. Consistent with other H. pylori virulence genotyping studies, we suggest that cagA EPIYA analysis should be performed using total DNA isolated from biopsy specimens.
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Antígenos Bacterianos/genética , Proteínas Bacterianas/genética , Infecciones por Helicobacter/microbiología , Helicobacter pylori/genética , Helicobacter pylori/aislamiento & purificación , Secuencias de Aminoácidos/genética , Técnicas de Tipificación Bacteriana , Biopsia , Clonación Molecular , ADN Bacteriano/genética , Genotipo , Helicobacter pylori/patogenicidad , Humanos , Fosforilación , Estructura Terciaria de Proteína , Análisis de Secuencia de ADN , Manejo de Especímenes , VirulenciaRESUMEN
Methods for the confirmation of nosocomial outbreaks of bacterial pathogens are complex, expensive, and time-consuming. Recently, a method based on ligation-mediated PCR (LM/PCR) using a low denaturation temperature which produces specific melting-profile patterns of DNA products has been described. Our objective was to further develop this method for real-time PCR and high-resolution melting analysis (HRM) in a single-tube system optimized in order to achieve results within 1 day. Following the optimization of LM/PCR for real-time PCR and HRM (LM/HRM), the method was applied for a nosocomial outbreak of extended-spectrum-beta-lactamase (ESBL)-producing and ST131-associated Escherichia coli isolates (n = 15) and control isolates (n = 29), including four previous clusters. The results from LM/HRM were compared to results from pulsed-field gel electrophoresis (PFGE), which served as the gold standard. All isolates from the nosocomial outbreak clustered by LM/HRM, which was confirmed by gel electrophoresis of the LM/PCR products and PFGE. Control isolates that clustered by LM/PCR (n = 4) but not by PFGE were resolved by confirmatory gel electrophoresis. We conclude that LM/HRM is a rapid method for the detection of nosocomial outbreaks of bacterial infections caused by ESBL-producing E. coli strains. It allows the analysis of isolates in a single-tube system within a day, and the discriminatory power is comparable to that of PFGE.
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Brotes de Enfermedades , Infecciones por Escherichia coli/epidemiología , Escherichia coli/clasificación , Escherichia coli/genética , Tipificación Molecular/métodos , beta-Lactamasas/metabolismo , Anciano , Análisis por Conglomerados , Infección Hospitalaria/epidemiología , Infección Hospitalaria/microbiología , Escherichia coli/aislamiento & purificación , Infecciones por Escherichia coli/microbiología , Genotipo , Humanos , Epidemiología Molecular/métodos , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Temperatura de TransiciónRESUMEN
BACKGROUND: Two main types of receptors for gastrin and cholecystokinin (CCK) have been cloned and identified. CCK1 (CCK-A) receptors are expressed in the pancreas, the gallbladder, and parts of the brain, while CCK2 (CCK-B/gastrin) receptors (CCK2R) are expressed in gastric glands and in most of the brain. A splice variant of the CCK2R designated CCKRi4sv (CCK-C), which is constitutively expressed in human pancreatic cancer cells, has also been described. The purpose of the present investigation was to study CCK2R, CCK2i4svR, and gastrin mRNA expression in human pancreatic adenocarcinoma on the assumption that co-expression of CCK2R and gastrin or constitutive CCK2i4svR mRNA expression plays a pivotal role in the progression of pancreatic cancer. FINDINGS: PCR amplification using CCK2R specific primer-pairs, followed by ethidium-bromide stained agarose gel electrophoresis revealed the expression of wild-type CCK2R mRNA in 12 of 17 biopsy specimens. A CCK2R intron 4 specific nested PCR assay revealed that CCK2i4svR mRNA was expressed in only one of the biopsy specimen. The authenticity of PCR amplicons was confirmed by cloning of selected amplicons and DNA sequence analysis. Moreover, we found that hitherto undescribed multiple forms of 3'-end variant CCK2R mRNAs with various deletions in the retained intron 4 and exon 5, tentatively generating truncated proteins, were expressed in the pancreatic adenocarcinomas. CONCLUSION: Cloning and DNA sequencing of selected amplicons revealed that CCK2R and multiple CCK2i4svR-like mRNAs are expressed in human pancreatic adenocarcinoma. The originally described CCK2i4svR mRNA was only expressed in one of 17 tumours and appears to be rarely expressed in pancreatic adenocarcinoma. We report that CCK2R- and gastrin mRNA co-expression may play a role in a portion, but not in all of these tumours, and that aberrant splicing takes places in these tissues generating multiple forms of 3'-end variant CCK2R mRNAs.
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Molecular typing of Klebsiella species has become important for monitoring dissemination of ß-lactamase-producers in hospital environments. The present study was designed to evaluate poly-trinucleotide (GTG)(5)- and rDNA intergenic transcribed spacer (ITS)-PCR fingerprint analysis for typing of Klebsiella pneumoniae and Klebsiella oxytoca isolates. Multiple displacement amplified DNA derived from 19 K. pneumoniae (some with an ESBL-phenotype), 35 K. oxytoca isolates, five K. pneumoniae, two K. oxytoca, three Raoultella, and one Enterobacter aerogenes type and reference strains underwent (GTG)(5) and ITS-PCR analysis. Dendrograms were constructed using cosine coefficient and the Neighbour joining method. (GTG)(5) and ITS-PCR analysis revealed that K. pneumoniae and K. oxytoca isolates, reference and type strains formed distinct cluster groups, and tentative subclusters could be established. We conclude that (GTG)(5) and ITS-PCR analysis combined with automated capillary electrophoresis provides promising tools for molecular typing of Klebsiella isolates.
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Técnicas de Tipificación Bacteriana/métodos , Klebsiella/clasificación , Tipificación Molecular/métodos , Reacción en Cadena de la Polimerasa/métodos , Dermatoglifia del ADN/métodos , Cartilla de ADN/genética , ADN Bacteriano/genética , ADN Espaciador Ribosómico/genética , Genotipo , Humanos , Klebsiella/genética , Secuencias Repetitivas de Ácidos Nucleicos/genéticaRESUMEN
BACKGROUND: The presence of various EPIYA tyrosine phosphorylation motifs in the CagA protein of Helicobacter pylori has been suggested to contribute to pathogenesis in adults. In this study, a unique PCR assay and sequencing strategy was developed to establish the number and variation of cagA EPIYA motifs. FINDINGS: MDA-DNA derived from gastric biopsy specimens from eleven subjects with gastritis was used with M13- and T7-sequence-tagged primers for amplification of the cagA EPIYA motif region. Automated capillary electrophoresis using a high resolution kit and amplicon sequencing confirmed variations in the cagA EPIYA motif region. In nine cases, sequencing revealed the presence of AB, ABC, or ABCC (Western type) cagA EPIYA motif, respectively. In two cases, double cagA EPIYA motifs were detected (ABC/ABCC or ABC/AB), indicating the presence of two H. pylori strains in the same biopsy. CONCLUSION: Automated capillary electrophoresis and Amplicon sequencing using a single, M13- and T7-sequence-tagged primer pair in PCR amplification enabled a rapid molecular typing of cagA EPIYA motifs. Moreover, the techniques described allowed for a rapid detection of mixed H. pylori strains present in the same biopsy specimen.
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BACKGROUND: Homocysteine levels in circulation are determined by several factors and hyperhomocysteinemia is reportedly associated with cardiovascular diseases and dementia. The aim of this study is to determine the relation of chronic gastritis and other conditions to homocysteine levels and their relation to incident cardiovascular diseases and dementia. METHODS: An adult population-based cohort (N = 488) was screened for H. pylori infection, gastro-duodenitis (endoscopic biopsies), disease history, and lifestyle factors. Blood samples were analyzed for pepsinogen I and II (gastric function), vitamin B12, folate, homocysteine, and cystatin C (renal function). The methylenetetrahydrofolate reductase C677T polymorphism reportedly associated with hyperhomocysteinemia was analyzed by pyrosequencing. Incident cardiovascular diseases and dementia were monitored during a median follow-up interval of 10 years. RESULTS: At baseline, there was a positive relation of S-homocysteine to male gender, age, S-cystatin C, methylenetetrahydrofolate reductase 677TT genotype and atrophic gastritis. During follow-up, cardiovascular diseases occurred in 101/438 and dementia in 25/488 participants, respectively. Logistic regression analysis (adjusting for gender, age at baseline, follow-up interval, BMI, smoking, alcohol consumption, NSAID use, P-cholesterol, and P-triglycerides) showed an association of S-homocysteine higher than 14.5 mumol/l to cardiovascular diseases (OR 2.05 [95% c.i. 1.14-3.70]), but not to dementia overall. CONCLUSIONS: Gender, age, vitamin B12, folate, renal function, atrophic gastritis and the methylenetetrahydrofolate 677TT genotype were significant determinants of homocysteine levels, which were positively related to incident cardiovascular diseases.
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Enfermedades Cardiovasculares/epidemiología , Demencia/epidemiología , Gastritis/sangre , Homocisteína/sangre , Hiperhomocisteinemia/complicaciones , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores/sangre , Enfermedades Cardiovasculares/sangre , Enfermedades Cardiovasculares/etiología , Enfermedad Crónica , Demencia/sangre , Demencia/etiología , Ensayo de Inmunoadsorción Enzimática , Femenino , Estudios de Seguimiento , Gastritis/complicaciones , Humanos , Hiperhomocisteinemia/sangre , Hiperhomocisteinemia/epidemiología , Incidencia , Masculino , Persona de Mediana Edad , Factores de Riesgo , Suecia/epidemiologíaRESUMEN
Determination of an individual's aggregate dietary ingestion of pesticides entails analysis of a difficult sample matrix. Permethrin-specific molecularly imprinted polymer (MIP) solid-phase extraction cartridges were developed for use as a sample preparation technique for a composite food matrix. Vortexing with acetonitrile and centrifugation were found to provide optimal extraction of the permethrin isomers from the composite foods. The acetonitrile (with 1% acetic acid) was mostly evaporated and the analytes reconstituted in 90:10 water/acetonitrile in preparation for molecularly imprinted solid-phase extraction. Permethrin elution was accomplished with acetonitrile and sample extracts were analyzed by isotope dilution gas chromatography-ion trap mass spectrometry. Quantitation of product ions provided definitive identification of the pesticide isomers. The final method parameters were tested with fortified composite food samples of varying fat content (1%, 5%, and 10%) and recoveries ranged from 99.3% to 126%. Vegetable samples with incurred pesticide levels were also analyzed with the given method and recoveries were acceptable (81.0-95.7%). Method detection limits were demonstrated in the low ppb range. Finally, the applicability of the MIP stationary phase to extract other pyrethroids, specifically cyfluthrin and cypermethrin, was also investigated.
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Análisis de los Alimentos , Cromatografía de Gases y Espectrometría de Masas/métodos , Impresión Molecular , Permetrina/química , Residuos de Plaguicidas/química , Extracción en Fase Sólida/métodos , Isomerismo , Espectrometría de Masa por Ionización de Electrospray/métodosRESUMEN
BACKGROUND: Bacterial and cellular genotyping is becoming increasingly important in the diagnosis of infectious diseases. However, difficulties in obtaining sufficient amount of bacterial and cellular DNA extracted from the same human biopsy specimens is often a limiting factor. In this study, total DNA (host and bacterial DNA) was isolated from minute amounts of gastric biopsy specimens and amplified by means of whole genome amplification using the multiple displacement amplification (MDA) technique. Subsequently, MDA-DNA was used for concurrent Helicobacter pylori and human host cellular DNA genotyping analysis using PCR-based methods. RESULTS: Total DNA was isolated from gastric biopsy specimens of 12 subjects with gastritis and 16 control subjects having a normal mucosa. The DNA was amplified using a multiple displacement amplification (MDA) kit. Next, concurrent genotyping was performed using H. pylori-specific virulence gene PCR amplification assays, pyrosequencing of bacterial 16S rDNA and PCR characterisation of various host genes. This includes Interleukin 1-beta (IL1B) and Interferon-gamma receptor (IFNGR1) SNP analysis, and Interleukin-1 receptor antagonist (IL1RN) variable tandem repeats (VNTR) in intron 2. Finally, regions of the vacA-gene were PCR amplified using M13-sequence tagged primers which allowed for direct DNA sequencing, omitting cloning of PCR amplicons. H. pylori specific multiplex PCR assays revealed the presence of H. pylori cagA and vacA genotypic variations in 11 of 12 gastritis biopsy specimens. Using pyrosequencing, 16S rDNA variable V3 region signatures of H. pylori were found in 11 of 12 individuals with gastritis, but in none of the control subjects. Similarly, IL1B and IFNGR1-SNP and IL1RN-VNTR patterns could be established in all individuals. Furthermore, sequencing of M13-sequence tagged vacA-PCR amplicons revealed the presence of highly diverse H. pylori vacA-s/i/m regions. CONCLUSION: The PCR-based molecular typing methods applied, using MDA-amplified DNA derived from small amounts of gastric biopsy specimens, enabled a rapid and concurrent molecular analysis of bacterial and host genes in the same biopsy specimen. The principles and technologies used in this study could also be applied to any situation in which human host and microbial genes of interest in microbial-host interactions would need to be sequenced.
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Citocinas/genética , Infecciones por Helicobacter/genética , Helicobacter pylori/genética , Polimorfismo de Nucleótido Simple , Factores de Virulencia/genética , Adulto , Anciano , Técnicas de Tipificación Bacteriana/métodos , Biopsia , ADN Bacteriano/genética , ADN Ribosómico/genética , Femenino , Gastritis/genética , Gastritis/microbiología , Gastritis/patología , Genes Bacterianos , Genes de ARNr , Genoma Bacteriano , Genoma Humano , Genotipo , Infecciones por Helicobacter/microbiología , Infecciones por Helicobacter/patología , Helicobacter pylori/clasificación , Helicobacter pylori/patogenicidad , Humanos , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa/métodos , ARN Ribosómico 16S/genética , ARN Ribosómico 18S/genética , Alineación de Secuencia , Análisis de Secuencia de ADN , Estómago/microbiología , Estómago/patología , Virulencia/genéticaRESUMEN
BACKGROUND: DNA isolation from formalin-fixed paraffin-embedded tissue appears to be problematic due to degradation caused by fixative. Our aim was to investigate if the isolated genomic DNA from archival plasma/serum, combined with multiple strand displacement amplification (MDA) can be used for genotyping. METHODS: Nine archival plasma/serum samples and freshly frozen gastric biopsies from the same nine H. pylori-infected subjects were used for DNA isolation. Subsequently, MDA-DNA derived from the plasma/serum samples and DNA isolated from the antrum biopsies were analyzed by PCR amplification and pyrosequencing for the presence of interleukin-1beta gene (IL-1B) single nucleotide polymorphism (SNP). In addition, Southern blot and pyrosequencing analysis of H. pylori-specific PCR amplicons were performed. RESULTS: IL-1B SNP profiles obtained from the plasma/serum MDA-DNA and antrum biopsy DNA were identical. A C/C genotype was observed in 7 of 9 samples, and 2 of 9 revealed a C/T genotype for IL-1B -511. Similarly, 7 of 9 had a T/T, and 2 of 9 had a C/T genotype for IL-1B -31; 4 of 9 had a C/C, 4 of 9 had a C/T, and 1 of 9 had a T/T genotype, respectively, for IL-1B +3954. Moreover, pyrosequencing analysis revealed the presence of H. pylori 26695 and J99-like 16S rDNA variable V3 region sequence motifs in the antrum biopsies but not in the plasma/serum samples. CONCLUSIONS: We conclude that MDA combined with pyrosequencing enables a rapid and accurate molecular typing of cytokine single nucleotide polymorphisms from archival plasma/serum samples.