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Specific Polymerase Chain Reaction (specific + polymerase_chain_reaction)
Selected Abstractscag Pathogenicity Island of Helicobacter pylori in Korean ChildrenHELICOBACTER, Issue 4 2002Jae Sung Ko Abstract Background.cag pathogenicity island is reported to be a major virulence factor of Helicobacter pylori. The aim of this study was to investigate the status of cag pathogenicity island genes and gastric histology in Korean children with H. pylori gastritis. Methods.Helicobacter pylori DNA was extracted from antral biopsy specimens from 25 children with H. pylori gastritis. Specific polymerase chain reaction assays were used for four genes of cag pathogenicity island. The features of gastritis were scored in accordance with the updated Sydney System. Results.cagA was present in 23 (92%) of 25 children, and cagE in 24 (96%). Twenty-two (88%) children were cagT positive and 19 (76%) virD4 positive. All of the selected genes of the cag pathogenicity island were present in 17 (68%) children and completely deleted in one child. There were no differences in neutrophil activity and chronic inflammation between children infected with intact cag pathogenicity island strains and those with partially or totally deleted- cag pathogenicity island strains. Conclusion.cag pathogenicity island is not a uniform, conserved entity in Korea. Completeness of cag pathogenicity island may not be the major factor to determine the severity of H. pylori gastritis in children. [source] Infliximab and the risk of latent viruses reactivation in active Crohn's diseaseINFLAMMATORY BOWEL DISEASES, Issue 7 2007Alessandro Lavagna MD Abstract Background: Infliximab is used for refractory Crohn's disease but there are concerns regarding long-term safety. Recently, JC-polyomavirus (JCV) was studied after 3 cases of progressive multifocal leukoencephalopathy (PML) were found after treatment with natalizumab. The aim of this study was to investigate the short-term effect of infliximab on reactivation of several harmful latent viruses. Methods: Sixty consecutive patients scheduled for infliximab induction course were prospectively enrolled. Blood samples were taken before each infliximab infusion at 0, 2, 6, and 14 weeks. Specific polymerase chain reaction (PCR) analyses were performed to detect JCV, Epstein,Barr virus (EBV), human herpes virus-6, (HHV-6), -7, -8, and cytomegalovirus (CMV). Results: Indications to infliximab were luminal and fistulizing disease in 49 and 15 cases, respectively. Clinical improvement and remission were achieved in 54 (90%) and 39 (65%) of patients, respectively, at 6 weeks. No patient was JCV-positive at any timepoint. EBV serology was positive for 59/60 patients (98%); EBV-PCR tests were transiently positive (>40 copies/105 Peripheral blood mononuclear cells, PBMC) in 4 (7%) patients after infliximab, but in each case were negative at subsequent timepoints. All patients were negative for HHV-6, -7, and -8 at all timepoints. CMV serology was positive in 42 patients (70%), but no CMV-PCR-positive patient was observed. There was no association between concomitant treatments or clinical characteristics and viral status. Conclusions: Our results support the safety of short-term infliximab treatment with respect to latent virus reactivation. The long-term effects of infliximab, particularly for the issue of lymphoproliferative disorders, warrants further studies with larger populations, but so far data are reassuring. (Inflamm Bowel Dis 2007) [source] Detection of Enterohepatic and Gastric Helicobacter Species in Fecal Specimens of Children with Crohn's DiseaseHELICOBACTER, Issue 4 2008Si Ming Man Abstract Background: Although there is compelling evidence to support the role of bacteria in Crohn's disease (CD), there is currently no solid evidence to support the role of any one specific bacterial causative agent. Recent studies have suggested that members of the Helicobacteraceae may play a role in the development of CD. The aim of this study was to further investigate the presence of members of the Helicobacteraceae in children with and without CD. Materials and methods: Fecal specimens from 29 children with CD, 11 healthy, normal controls, and 26 symptomatic controls with non-inflammatory bowel disease (IBD) pathology were obtained for DNA extraction and subjected to Helicobacteraceae -specific polymerase chain reaction (PCR). All PCR-positive samples were sequenced. The association between the presence of members of the Helicobacteraceae and each study group was statistically analysed using the Fisher's exact test. Results: Based on Helicobacteraceae -specific PCR analysis, 59% (17 of 29) of the children with CD were positive, which was significantly higher than that in asymptomatic healthy children [9% (1 of 11); p = .01] and that in symptomatic children with non-IBD pathology [0% (0/26); p < .0001]. Sequencing of the 16S rRNA gene of positive samples revealed the presence of both enterohepatic Helicobacter species and Helicobacter pylori in fecal specimens. Conclusions: For the first time, enterohepatic and gastric Helicobacter species have been identified in fecal specimens from children diagnosed with CD using PCR. Our data suggest that Helicobacter species may have a pathogenic role in the development of CD in a considerable proportion of children. [source] Genetic association of vitamin D receptor polymorphisms with primary biliary cirrhosis and autoimmune hepatitisHEPATOLOGY, Issue 1 2002Arndt Vogel Autoimmune hepatitis (AIH) and primary biliary cirrhosis (PBC) are immune-mediated chronic inflammatory diseases of the liver of unknown etiology. Genetic factors appear to be involved in the pathogenesis of both diseases. 1,25-Dihydroxyvitamin D3 has been implicated as an immunomodulator, which acts through its own receptor (VDR). Polymorphisms of the VDR have been linked to a variety of autoimmune diseases. In this study VDR polymorphisms were analyzed in 123 patients with AIH, 74 patients with PBC, and 214 controls. VDR polymorphisms were assessed by BsmI, TaqI, ApaI, and Fok endonuclease digestion after specific polymerase chain reaction (PCR) amplification. We found a significant association between the BsmI polymorphisms in PBC patients in comparison with controls (,2 = 9.49, P = .009). Furthermore we detected a significant association of the Fok polymorphims in AIH patients in comparison to controls (,2 = 9.71, P = .008) indicating a genetic link of VDR polymorphisms to autoimmune liver diseases such as PBC and AIH in German patients. These findings contribute to the knowledge of the complex events determining immunologic tolerance in the liver. Further studies are needed to elucidate the mechanisms by which the vitamin D receptor contributes to the development of autoimmune diseases. [source] Integrated polymerase chain reaction-based procedures for the detection and identification of species and subspecies of the Gram-positive bacterial genus LactococcusJOURNAL OF APPLIED MICROBIOLOGY, Issue 2 2002Z.Y. Pu Aims:,Five species of the Gram-positive bacterial genus Lactococcus (Lactococcus lactis, L. garvieae, L. plantarum, L. piscium and L. raffinolactis) are currently recognized. The aim of this work was to develop a simple approach for the identification of these species, as well as to differentiate the industrially important dairy subspecies L. lactis subsp. lactis and L. lactis subsp. cremoris. Methods and Results:,Methods were devised based on specific polymerase chain reaction (PCR) amplifications that exploit differences in the sequences of the 16S ribosomal RNA genes of each species, followed by restriction enzyme cleavage of the PCR products. The techniques developed were used to characterize industrial cheese starter strains of L. lactis and the results were compared with biochemical phenotype and DNA sequence data. Conclusions:,The PCR primers designed can be used simultaneously, providing a simple scheme for screening unknown isolates. Strains of L. lactis show heterogeneity in the 16S ribosomal RNA gene sequence. Significance and Impact of the Study:,This work provides an integrated set of methods for differentiation and identification of lactococcal species associated with agricultural, veterinary, medical and processed food industries. [source] Malaysian Indians are genetically similar to Caucasians: CYP2C9 polymorphismJOURNAL OF CLINICAL PHARMACY & THERAPEUTICS, Issue 2 2006Z. Zainuddin MSc Summary Background:,CYP2C9 is one of the major drug metabolizing enzymes for many drugs including warfarin, NSAIDs and losartan. It is polymorphic in many populations. Data on the distribution of CYP2C9 and the implication of CYP2C9 polymorphism in the Malaysian population is lacking. Our objectives were therefore to investigate the prevalence of CYP2C9 variants among unrelated healthy volunteers of Malays, Chinese and Indians in Malaysia. Method:, Deoxyribonucleic acid was extracted using standard lysis methods. Allele specific polymerase chain reaction was performed for determination of CYP2C9*1, *2, *3, *4 and *5 variants according to Z. Zainuddin, L.K. Teh, A.W.M. Suhaimi, M.Z. Salleh, R. Ismail (2003, Clinica Chimica Acta, 336, 97). Result:, The Chinese had the highest frequency of CYP2C9*1 (321/330, 97·27%), followed by the Malays and the Indians (402 of 420, 95·71% and 291 of 330, 88·18%, respectively). CYP2C9*2 was not found in the Chinese. CYP2C9*3 were detected in all the three races with the Indians having the highest frequency of CYP2C9*3 (9·7%). The Indians had a frequency of CYP2C9*2 and *3 similar to Tamilians and Caucasians. Two of the Indians had *2/*3 and one had *3/*3 genotypes and are likely to be slow metabolizers. No subject with CYP2C9*4 and *5 were detected in our populations. Conclusion:,CYP2C9*2 and *3 were identified in our population. Indians are similar to Caucasians in terms of CYP2C9 genotypes and thus may respond to CYP2C9 substrates differently when compared with the Malays and Chinese in Malaysia. [source] Did the introduction of maize into Europe provide enemy-free space to Ostrinia nubilalis?JOURNAL OF EVOLUTIONARY BIOLOGY, Issue 2 2010Parasitism differences between two sibling species of the genus Ostrinia Abstract We examined whether maize offers enemy-free space (EFS) to its pest Ostrinia nubilalis, and may thereby have contributed to its divergence from the sibling species, Ostrinia scapulalis, feeding mainly on mugwort, when introduced into Europe five centuries ago. We collected Ostrinia larvae on maize (70 populations, 8425 individuals) and mugwort (10 populations, 1184 individuals) and recorded parasitism using both traditional (counting emerging parasitoids) and molecular methods (detection by specific polymerase chain reaction). The main parasitoid was Macrocentrus cingulum (Braconidae). On mugwort, parasitism was twice that on maize, and parasitoid-related mortality was 8 times higher. This suggests that maize affords substantial EFS to Ostrinia feeding on it. The lower Mortality:Infestation ratio in maize suggests that O. nubilalis' immune response might be stronger than that of O. scapulalis. If so, adapting to maize and diverging from O. scapulalis would decrease the impact of parasitism on O. nubilalis at both ecological and evolutionary levels. [source] Molecular cloning of Yersinia ruckeri aroA gene: a useful taxonomic toolJOURNAL OF FISH DISEASES, Issue 7 2001J Yugueros The aroA gene of Yersinia ruckeri, which encodes 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase was cloned by complementation of the aroA mutation in Escherichia coli AB2829 by using pUC18 plasmid as a vector. Nucleotide sequence of the aroA gene revealed an open reading frame of 427 amino acids showing a high degree of homology to other bacterial AroA proteins. A pair of primers with 23 and 20 nucleotides were selected from the 5, and 3, termini, respectively, and formed the basis of a specific polymerase chain reaction (PCR) assay. A 1165-bp deoxyribonucleic acid (DNA) fragment was amplified from all lysed Y. ruckeri strains. An identical size fragment was also amplified from lysed Y. pseudotuberculosis, Y. aldovae, Salmonella enteritidis and E. coli, but not from other enterobacteria. AluI restriction fragment length polymorphism (RFLP) of the PCR amplified products allowed for differentiation between Y. ruckeri and the other bacteria. Specificity and sensitivity make this PCR assay a useful method for rapid identification and diagnosis of Y. ruckeri infections. [source] Determination of the optimal cut-off value for the [13C]-urea breath test based on a Helicobacter pylori -specific polymerase chain reaction assayJOURNAL OF GASTROENTEROLOGY AND HEPATOLOGY, Issue 2 2000Haruhiko Yoshida Abstract Background: This study was conducted to determine the optimal cut-off value and breath sample collection time for the [13C]-urea breath test based on the assessment of Helicobacter pylori status with a gastric juice-based polymerase chain reaction (PCR) assay. Methods and Results: A total of 104 patients took 100 mg [13C]-urea orally and breath samples were collected at 5, 10, 20, 30 and 60 min. The increment of 13CO2: 12CO2 ratio from the baseline (,13C) was measured using a laser spectroanalyser. The PCR assay was positive in 63 and negative in 41 patients. The optimal cut-off value of ,13C was calculated for each sample collection time so that the distance from the geometric mean value among Helicobacter pylori -positive patients and that from the arithmetic mean value among negative patients were simultaneously maximized. The cut-off value of 2.7, at 20 min had the longest distance, being separated by 3.16 SD from the two mean values. Using this cut-off value, the urea breath test showed 100% specificity and 98% sensitivity for the diagnosis of Helicobacter pylori infection. [source] Mycoplasma genitalium: the aetiological agent of urethritis and other sexually transmitted diseasesJOURNAL OF THE EUROPEAN ACADEMY OF DERMATOLOGY & VENEREOLOGY, Issue 1 2004Jørgen Skov Jensen ABSTRACT Mycoplasma genitalium was first isolated in 1980 from two of 13 men with non-gonococcal urethritis (NGU). It shares several features with M. pneumoniae, a recognized respiratory tract pathogen. It is extremely difficult to isolate by culture. The development of sensitive and specific polymerase chain reaction (PCR) assays in the early 1990s made clinical studies possible and a significant number of publications have shown a strong association between M. genitalium and NGU, independent of Chlamydia trachomatis. The purpose of this review is to evaluate the currently available information on the associations between M. genitalium and urogenital tract infections in men and women and assess their fulfilment of the Henle,Koch postulates. It is concluded that there is very strong evidence that M. genitalium is a cause of NGU in men and cervicitis in women. Evidence for upper genital tract infections in women has begun to accrue, but further studies are needed. The optimal treatment of M. genitalium infections remains to be determined, but antibiotics of the macrolide group appear to be more active than tetracyclines. [source] Isolation and characterization of new microsatellite markers for rose bitterlings, Rhodeus ocellatusMOLECULAR ECOLOGY RESOURCES, Issue 3 2009Y. SHIRAI Abstract The Japanese rose bitterling (Rhodeus ocellatus kurumeus) is facing imminent extinction because of hybridization and competition from an invasive alien subspecies (Rhodeus ocellatus ocellatus). Eleven new microsatellite markers for the two subspecies were developed using dinucleotide repeat specific polymerase chain reaction. The number of alleles per locus and the heterozygosity in R. o. kurumeus were lower than those in R. o. ocellatus. Most of these microsatellite markers were successfully cross-amplified in three Acheilognathinae species. [source] Development of a PCR test to detect the downy mildew causal agent Plasmopara halstedii in sunflower seedsPLANT PATHOLOGY, Issue 2 2007R. Ioos Plasmopara halstedii, the causal agent of downy mildew of sunflower, is an obligate parasite but viable sporangia and oospores of the pathogen may be found in a quiescent state in seeds of sunflower and therefore may be transported with sunflower seeds in international commercial exchanges. In order to prevent the spread of this pathogen, especially the introduction of potentially new races, an efficient method to analyse sunflower seed samples is required. In this study, a P. halstedii -specific polymerase chain reaction (PCR) test was developed based on the ribosomal large sub unit (LSU) DNA. The forward (PHAL-F) and reverse (PHAL-R) PCR primers were designed from two polymorphic regions of LSU. After screening 22 isolates of P. halstedii corresponding to different races and countries and 32 other oomycete, deuteromycete and ascomycete isolates, the PHAL-F/R primers amplified only a single PCR band of c. 310 bp from P. halstedii. The PHAL-F/R PCR test could detect as little as 3 pg of P. halstedii genomic DNA per 20 µL reaction volume and enabled the direct detection of P. halstedii in 35 g sunflower seed samples without the need for any prior biological baiting step. An internal amplification control (IAC) was developed to help discriminate against false negative samples due to the potential presence of inhibitory compounds in DNA extracts. The test was successfully used on samples of naturally contaminated seeds. These new molecular tools should be of great interest for quarantine seed testing purposes. [source] Positive Correlation of Tissue Inhibitor of Metalloproteinase-3 and Death-Associated Protein Kinase Hypermethylation in Head and Neck Squamous Cell CarcinomaTHE LARYNGOSCOPE, Issue 8 2007Chetan S. Nayak MD Abstract Objectives/Hypothesis: Promoter hypermethylation of tumor suppressor genes is common in head and neck cancer as well as other primary cancers resulting in epigenetic gene silencing. Tissue inhibitor of metalloproteinase-3 (TIMP-3) has been shown to have promoter hypermethylation in several solid tumors, but has not been identified in head and neck squamous cell carcinoma (HNSCC). Our objective was to determine if TIMP-3 promoter was hypermethylated in HNSCC, if there was any correlation with death associated protein kinase (DAPK), a tumor suppressor whose promoter has been hypermethylated at high levels in HNSCC, and if any clinical factors influence hypermethylation of either of these genes. Study Design: Prospective study. Methods: Tumor samples from 124 patients with HNSCC were evaluated for promoter hypermethylation for TIMP-3 and DAPK using quantitative methylation specific polymerase chain reaction (qMSP). We compared both TIMP-3 and DAPK hypermethylation in HNSCC with each other as well as with other clinical variables. Results: We found that TIMP-3 was hypermethylated in approximately 71.8% of the tumor samples and DAPK was hypermethylated in 74.2%. The presence of TIMP-3 and DAPK promoter hypermethylation was significantly higher than in control specimens. More importantly, TIMP-3 and DAPK hypermethylations in these samples were highly correlated with a concordance of 78% (P < .001). DAPK was also correlated with current alcohol consumption (P < .028), but neither TIMP-3 nor DAPK hypermethylation was significantly correlated with other clinical variables or with survival. Conclusion: TIMP-3 promoter hypermethylation is elevated in HNSCC and is highly correlated with DAPK hypermethylation, implying a functional relationship between these genes. [source] Fumagillin for Treatment of Intestinal Microsporidiosis in Renal Transplant RecipientsAMERICAN JOURNAL OF TRANSPLANTATION, Issue 8 2010L. Champion We report 10 cases of intestinal microsporidiosis due to Enterocytozoon bieneusi in renal transplant (RT) recipients who were treated with fumagillin. All patients presented with afebrile subacute diarrhea (median of 2 weeks), associated with abdominal cramps (n = 5), and weight loss (n = 6), a mean of 68 months after RT. The diagnosis was made by the identification of microsporidial spores in stools with the use of appropriate staining and confirmed by a specific polymerase chain reaction assay for E. bieneusi in 7 patients. Median CD4 cell count was 292 cells/mm3. All patients received a median of 14 days of oral fumagillin (20 mg tid), and four patients also discontinued or tapered their immunosuppressive regimen (mycophenolate mofetil in 3, and azathioprine in 2). Clinical symptoms resolved rapidly with the clearance of microsporidial spores from stools in all patients. A severe but reversible thrombocytopenia was observed in one patient during fumagillin therapy, and another patient presented with abdominal cramps. Trough levels of tacrolimus measured in seven patients dropped below 5 ng/mL in six of them after 7,14 days of fumagillin. Intestinal microsporidiosis can cause subacute diarrhea in RT recipients. Fumagillin is an effective treatment with an acceptable safety profile, but monitoring of tacrolimus levels is warranted. [source] Methylation of the ASC gene promoter is associated with aggressive prostate cancerTHE PROSTATE, Issue 7 2006Rachael L. Collard Abstract Background The aim of this study was to investigate the methylation status of apoptosis-associated speck-like protein containing a CARD (ASC; TMS1; PYCARD) in prostate cancer cell lines and human tissues and to determine if those findings correlate with the clinicopathological features of prostate cancer. Methods Genomic DNA was isolated from prostate cell lines and microdissected tissues, bisulfite converted and analyzed by methylation specific polymerase chain reaction (MSP). Expression of ASC in prostate cancer cell lines treated with or without methylation inhibitors was determined by quantitative or qualitative RT-PCR. Results ASC gene expression was silenced or reduced in five prostate cancer cell lines and correlated with methylation status. Treatment of MDAPCa2b prostate cancer cells with the methylation inhibitors 5-aza-2-deoxycitidine and Zebularine reactivated expression of ASC. Of 58 prostate cancer specimens, methylation of the ASC promoter region was present in 65% of primary cancer tissue, 64% (7/11) of cancer-associated high grade-prostatic intraepithelial neoplasia (HG-PIN), and 28% of normal-appearing but adjacent to tumor prostate tissue. While ASC methylation was not related to Gleason score (P,=,0.46) or pathological stage (P,=,0.75), there was a significantly higher frequency of ASC methylation in the adjacent normal tissue for patients with biochemical recurrence (P,=,0.0383). Conclusions Methylation of the ASC gene promoter is both a frequent and early event in prostate cancer carcinogenesis. Surprisingly, methylation of the adjacent normal tissue occurs significantly more often in patients who later undergo biochemical recurrence, suggesting a role for inactivation of the ASC gene in the initial stages of aggressive disease. Prostate © 2006 Wiley-Liss, Inc. [source] Curing of four different plasmids in Yersinia pestis using plasmid incompatibilityLETTERS IN APPLIED MICROBIOLOGY, Issue 4 2008B. Ni Abstract Aims:, Plasmids are critical for the pathogenicity of Yersinia pestis. In order to carry out a systematic investigation of their role in pathogenesis, we cured plasmids from Y. pestis. Methods and Results:, Each plasmid's replicon of Y. pestis was cloned into plasmid pEX18Gm containing a counter-selectable sacB gene, and was then introduced into Y. pestis strain 201 by electroporation. Strains containing recombinant plasmids were cultivated under antibiotic selection. The resultant plasmid-curing colonies, identified by specific polymerase chain reactions, were then cured off pEX18Gm under sucrose pressure. This method was used to successfully cure all four plasmids of Y. pestis, singly or in different combinations. Conclusions:, Naturally evolving plasmids in Y. pestis are difficult to remove by conventional curing methods. We employed a method based on plasmid incompatibility to cure the plasmids from Y. pestis, which confirmed the efficacy of this method for curing plasmids with different types of replicons from one bacterium. Significance and Impact of the Study:, There have been no reports on the curing of multiple plasmids by using replication mechanisms from one bacterium with this technique. In the present study, we were able to successfully apply this methodology to cure four plasmids from Y. pestis, confirming its feasibility. [source] | |||||||||||||