Home About us Contact
|
Home About us Contact | ||
|
|
||
PCR Methods (pcr + methods)
Selected AbstractsCytochrome P450 2D6 and glutathione S-transferase M1 genotypes and migraineEUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 4 2000Mattsson Background Migraine is thought to be a disease of the brain and trigeminovascular system. Migraine patients often claim that stress, food, and beverages trigger their attacks. Chemical substances in these foodstuffs with the property of triggering migraine attacks have not yet been characterised. Cytochrome P450 2D6 (CYP2D6) and glutathione S-transferase M1 (GSTM1) are thought to be present in the brain. They metabolise numerous environmental compounds. The genes exhibit genetic polymorphism that is associated with altered enzyme activity. The aim of this study was to determine if the genotypes of these two enzymes are associated with migraine. Materials and methods The study included 100 female patients and 245 female controls from the general population. Genomic DNA was isolated from whole blood. Allele specific PCR methods were used to identify the normal CYP2D6*1 allele and the mutated CYP2D6*3 and CYP2D6*4 alleles. Initially all samples were genotyped only for GSTM1 plus (+) and GSTM1 null (,) variants. All samples positive for GSTM1 were further analysed for the presence of allelic variants GSTM1*A and GSTM1*B. Results None of the CYP2D6 and GSTM1 genotypes was associated with migraine. We observed an odds ratio (OR) for the poor metaboliser genotype of CYP2D6 of 1.4 (95% CI = 0.5,3.6) and for the GSTM1 null genotype of 1.0 (95% CI = 0.6,1.5). Conclusion The results of this study indicate that deficient metabolism because of mutated CYP2D6 alleles or GSTM1 allele variants is not important in the aetiology of migraine. [source] Comparison of three different PCR methods for detection of Brucella spp. in human blood samplesFEMS IMMUNOLOGY & MEDICAL MICROBIOLOGY, Issue 2 2002E Navarro In the diagnosis of human brucellosis, PCR could be a more sensitive technique than blood cultures and more specific than conventional serological tests. We compared three different PCR methods for the detection of Brucella spp. and we studied whether human genomic DNA affect the sensitivity of three primer pairs for the detection of Brucella DNA in a peripheral-blood PCR assay. These three pairs of primers amplified three different fragments included in: (i) a gene encoding a 31-kDa Brucella abortus antigen (primers B4/B5), (ii) a sequence 16S rRNA of B. abortus (primers F4/R2), and (iii) a gene encoding an outer membrane protein (omp -2) (primers JPF/JPR). The three primers assayed showed a difference in sensitivity for detecting purified Brucella DNA, ranging between 8 fg and 20 pg. However, the sensitivity of the primers F4/R2 and B4/B5 was affected by the presence of human DNA while the primers JPF/JPR were not. Therefore, although the sensitivity of PCR using primers F4/R2 is affected by human DNA, they are still the most sensitive and they could provide a useful tool for the diagnosis of human brucellosis. [source] Detection of drug-resistant HIV minorities in clinical specimens and therapy failureHIV MEDICINE, Issue 3 2008S Louvel Objective Particularly for therapy-experienced patients, resistance assessment by genotypic or phenotypic methods produces discordances. This study seeks proof that differences may arise from the fact that genotyping produces a single summary sequence whereas replicative phenotyping (rPhenotyping) functionally detects and assigns resistances in mixed HIV populations. Methods For validation, defined mixes of wild-type and M184V mutant were analysed by rPhenotyping or standard genotyping. Allele-specific and quantitative polymerase chain reaction (PCR) set detection and quantification limits for minor virus populations in vitro and in authentic clinical samples showing geno-/pheno-discrepant lamivudine resistance. Results Allele-specific and real-time PCR methods detected down to 0.3% of mutant M184V. The functional assessment was sensitive enough to reveal <1% of mutant M184V in mixed samples. Also in discordant samples from the diagnostic routine, in which rPhenotyping had identified drug resistance, real-time PCR confirmed minute amounts of mutant M184V. Conclusion By utilizing the replication dynamics of HIV under drug pressure, a rPhenotyping format potently reveals relevant therapy-resistant minority species, even of HIV known to possess reduced replicative fitness. With its rapid turnaround of 8 days and its high sensitivity, our rPhenotyping system may be a valuable diagnostic tool for detecting the early emergence of therapy-threatening HIV minorities or the persistence of residual resistant virus. [source] Prevalence, quantification and typing of adenoviruses detected in river and treated drinking water in South AfricaJOURNAL OF APPLIED MICROBIOLOGY, Issue 2 2005J. van Heerden Abstract Aims:, Human adenoviruses (HAds), of which there are 51 serotypes, are associated with gastrointestinal, respiratory, urinary tract and eye infections. The importance of water in the transmission of HAds and the potential health risks constituted by HAds in these environments are widely recognized. Adenoviruses have not previously been quantified in river and treated drinking water samples. In this study, HAds in river water and treated drinking water sources in South Africa were detected, quantified and typed. Methods and Results:, Adenoviruses were recovered from the water samples using a glass wool adsorption-elution method followed by polyethylene glycol/NaCl precipitation for secondary concentration. The sensitivity and specificity of two nested PCR methods were compared for detection of HAds in the water samples. Over a 1-year period (June 2002 to July 2003), HAds were detected in 5·32% (10/188) of the treated drinking water and 22·22% (10/45) of river water samples using the conventional nested PCR method. The HAds detected in the water samples were quantified using a real-time PCR method. The original treated drinking water and river water samples had an estimate of less than one copy per litre of HAd DNA present. The hexon-PCR products used for typing HAds were directly sequenced or cloned into plasmids before sequencing. In treated drinking water samples, species D HAds predominated. In addition, adenovirus serotypes 2, 40 and 41 were each detected in three different treated drinking water samples. Most (70%) of the HAds detected in river water samples analysed were enteric HAds (serotypes 40 and 41). One HAd serotype 2 and two species D HAds were detected in the river water. Conclusions:, Adenoviruses detected in river and treated drinking water samples were successfully quantified and typed. The detection of HAds in drinking water supplies treated and disinfected by internationally recommended methods, and which conform to quality limits for indicator bacteria, warrants an investigation of the risk of infection constituted by these viruses. The risk of infection may have implications for the management of drinking water quality. Significance and Impact of the Study:, This study is unique as it is the first report on the quantification and typing of HAds in treated drinking water and river water. This baseline data is necessary for the meaningful assessment of the potential risk of infection constituted by these viruses. [source] Simultaneous use of direct and indirect diagnostic techniques in atypical respiratory infections from Chlamydophila pneumoniae and Mycoplasma pneumoniaeJOURNAL OF CLINICAL LABORATORY ANALYSIS, Issue 4 2009S. Pignanelli Abstract In 2008, 50 samples (BAL), coming from hospital patients, with acute respiratory symptoms have been investigated using two real-time PCR methods: one assay for the single detection of Chlamydophila pneumoniae and Mycoplasma pneumoniae DNA and one commercially available real-time duplex PCR assay for the detection of C. pneumoniae and M. pneumoniae DNA. Both techniques used here showed compliant results, with 100% concordance for detection of C. pneumoniae and 98% for detection of M. pneumoniae. The positive results obtained agreed with the clinical suspicion of such infections in some cases and with the presence of IgM specific for C. pneumoniae and M. pneumoniae in all cases of acute infection. J. Clin. Lab. Anal. 23:206,209, 2009. © 2009 Wiley-Liss, Inc. [source] Diagnosis of Chlamydia trachomatis infectionJOURNAL OF CLINICAL LABORATORY ANALYSIS, Issue 1 2006Jayanti Mania-Pramanik Abstract Important progress in the diagnosis of Chlamydia trachomatis (C. trachomatis) includes the development of nucleic acid amplification techniques such as polymerase chain reaction (PCR) and ligase chain reaction (LCR). Commercial kits are available, but they are costly, sporadic in availability, must be imported, and are economically beyond the reach of common people. To overcome this limitation, most research laboratories have standardized their in-house-developed PCR methods for diagnosing this infection. However, each laboratory has to spend a great deal of time and money to accomplish this. Published reports do not always elaborate the steps involved in standardizing a test so that it can immediately be reproduced in another setting. In the present study we attempted to elaborate the steps involved in standardizing a sensitive and specific PCR technique followed by hybridization with specific C. trachomatis probe to diagnose this infection in cervical, introital, and urine specimens, and used it to determine the infection rate in a clinical population. J. Clin. Lab. Anal. 20:8,14, 2006. © 2006 Wiley-Liss, Inc. [source] Comparison of the efficiency and sensitivity of virus isolation and molecular methods for routine diagnosis of infectious haematopoietic necrosis virus and infectious pancreatic necrosis virusJOURNAL OF FISH DISEASES, Issue 2 2002-Maganja, D Barli Infectious haematopoietic necrosis virus (IHNV) and infectious pancreatic necrosis virus (IPNV) are widely distributed fish pathogens in Europe. A reverse transcriptase,polymerase chain reaction (RT,PCR) assay was developed for the detection of both viruses as an alternative method to virus assay in cell culture. Oligonucleotide primers corresponding to highly conserved regions of glycoprotein G-gene sequences were used for IHNV. For the detection of IPNV the VP2-coding region was selected for RT,PCR amplification. Products of the expected size were amplified from total ribonucleic acid (RNA) extracts of infected cells. The optimized RT,PCR methods successfully detected viral RNA from ovarian and seminal fluids and other organs. To enhance the sensitivity and specificity of RT,PCR, a semi-nested PCR assay was tested using additional specific inner primers for reamplification of products obtained by RT,PCR. Because of the possibility of template carry-over contamination, a closed one step RT,PCR method was tested. This technically simplified approach was then combined with the PCR,enzyme linked immunosorbent assay (ELISA) method for the detection of amplification products and verification using specific biotinylated probes. The test provides an additional tool for the detection of IHNV and IPNV which is rapidly and easily performed and is highly sensitive, especially for the detection of IHNV in fish samples coinfected with IPNV. The PCR,ELISA method for the detection of RT,PCR products enables the screening of large numbers of samples and offers the possibility for automatisation of diagnostic work. [source] Evaluation of the PCR method for identification of Bifidobacterium speciesLETTERS IN APPLIED MICROBIOLOGY, Issue 1 2008S.Y. Youn Abstract Aims:,Bifidobacterium species are known for their beneficial effects on health and their wide use as probiotics. Although various polymerase chain reaction (PCR) methods for the identification of Bifidobacterium species have been published, the reliability of these methods remains open to question. Methods and Results:, In this study, we evaluated 37 previously reported PCR primer sets designed to amplify 16S rDNA, 23S rDNA, intergenic spacer regions, or repetitive DNA sequences of various Bifidobacterium species. Conclusions:, Ten of 37 experimental primer sets showed specificity for B. adolescentis, B. angulatum, B. pseudocatenulatum, B. breve, B. bifidum, B. longum, B. longum biovar infantis and B. dentium. Significance and Impact of the Study:, The results suggest that published Bifidobacterium primer sets should be re-evaluated for both reproducibility and specificity for the identification of Bifidobacterium species using PCR. Improvement of existing PCR methods will be needed to facilitate identification of other Bifidobacterium strains, such as B. animalis, B. catenulatum, B. thermophilum and B. subtile. [source] Diagnosis of American foulbrood in honey bees: a synthesis and proposed analytical protocolsLETTERS IN APPLIED MICROBIOLOGY, Issue 6 2006D.C. De Graaf Summary Worldwide, American foulbrood (AFB) is the most devastating bacterial disease of the honey bee (Apis mellifera). Because the distinction between AFB and powdery scale disease is no longer considered valid, the pathogenic agent has recently been reclassified as one species Paenibacillus larvae, eliminating the subspecies designations Paenibacillus larvae subsp. larvae and Paenibacillus larvae subsp. pulvifaciens. The creamy or dark brown, glue-like larval remains of infected larvae continue to provide the most obvious clinical symptom of AFB, although it is not conclusive. Several sensitive and selective culture media are available for isolation of this spore-forming bacterium, with the type of samples that may be utilized for detection of the organism being further expanded. PCR methods for identification and genotyping of the pathogen have now been extensively developed. Nevertheless, biochemical profiling, bacteriophage sensitivity, immunotechniques and microscopy of suspect bacterial strains are entirely adequate for routine identification purposes. [source] Retinopathy of prematurity: Mutations in the Norrie disease gene and the risk of progression to advanced stagesPEDIATRICS INTERNATIONAL, Issue 2 2001Mohammad Z Haider AbstractBackground: Retinopathy of prematurity (ROP) is a retinal vascular disease that occurs in infants with short gestational age and low birth weight and may lead to retinal detachment and blindness. Missense mutations in the Norrie disease (ND) gene have been associated with the risk of progression to advanced stages in cases of ROP from the US and also in clinically similar ND and familial exudative vitreoretinopathy. Methods: We have screened two ND gene mutations, namely A105T and Val60Glu, by polymerase chain reaction,restriction fragment length polymorphism (PCR-RFLP) and allele-specific PCR methods, respectively, in 210 Kuwaiti premature newborns to replicate these findings in a different ethnic group. Results: In the Kuwaiti premature newborn cohort, 115 of 210 babies had no eye problems and served as controls, while 95 were cases of ROP. In 71 of 95 ROP cases, the disease regressed spontaneously on or before stage 3, while in 24 of 95 ROP cases the disease progressed to advanced stages 4 and 5. In case of missense mutation (A105T), the AA genotype was detected in 96% of controls compared with 87% of ROP cases (NS); similarly no significant difference was found between spontaneously regressed ROP cases and those who progressed to advanced stages. For the Val60Glu mutation, no significant association was detected between the genotype and progression of ROP to advanced stages. Conclusions: Unlike data from the US, our findings from a Kuwaiti cohort of ROP cases and controls suggest a lack of association between the two ND gene mutations (A105T and Val60Glu) and ROP and the risk of progression of the disease to advanced stages. [source] Genistein-induced neuroendocrine differentiation of prostate cancer cellsTHE PROSTATE, Issue 11 2006Jacek Pinski MD Abstract BACKGROUND Neuroendocrine (NE) cells are present in normal prostate and their number appears to be increased in advanced prostate cancer (PCA). In this study, we studied the effect of the phytoestrogen, genistein, on NE differentiation of LNCaP cells in vitro. METHODS Neuroendocrine marker expression of LNCaP cells exposed to genistein was measured by immunohistochemistry, Western blot, and real-time PCR methods. Western blot analysis was used to study cell cycle and signaling pathways induced by genistein treatment. RESULTS Six days after continuous genistein treatment, the majority of genistein-surviving cancer cells underwent transdifferentiation into a NE-like phenotype overexpressing the NE markers chromogranin A, synaptophysin, serotonin, and beta-III tubulin. This NE differentiation process was associated with upregulation of the cell cycle modulators p21, p27, and p53, and activation of the MAPK and STAT3 pathways. CONCLUSION Our data indicate that genistein evokes not only apoptosis but also NE transdifferentiation of PCA cells. Prostate © 2006 Wiley-Liss, Inc. [source] Lymphoid tumours of the ocular adnexa: a morphologic and genotypic study of 15 casesACTA OPHTHALMOLOGICA, Issue 3 2003Margret Sigurdardottir Abstract. Purpose:, To examine all lymphoproliferative lesions of the ocular adnexa diagnosed in Iceland during 1983,2000 and to determine whether polymerase chain reaction (PCR) methods to determine clonality are helpful in characterizing these lesions. Methods:, All patients diagnosed with lymphoproliferative lesions in the ocular adnexa in the years 1983,2000 were included in the study. Polymerase chain reaction studies for clonality were performed on these lesions. Results:, Fifteen cases were identified. Seven were classified as inflammatory pseudotumour, one as lymphoid hyperplasia, four as atypical lymphoid hyperplasia and three as lymphoma. Of 12 cases examined by PCR, three were monoclonal for B-cells (one lymphoma, one inflammatory pseudotumour and one atypical lymphoid hyperplasia) while the remaining lesions (including two lymphomas) appeared polyclonal. Conclusion:, The results of this study suggest that analysis of clonality by PCR methods may be of limited use in classifying lymphoproliferative lesions of the ocular adnexa as benign or malignant. These results underscore the importance of using several techniques when determining clonality. [source] Is quantitative PCR for the pneumolysin (ply) gene useful for detection of pneumococcal lower respiratory tract infection?CLINICAL MICROBIOLOGY AND INFECTION, Issue 6 2009G. Abdeldaim Abstract The pneumolysin (ply) gene is widely used as a target in PCR assays for Streptococcus pneumoniae in respiratory secretions. However, false-positive results with conventional ply -based PCR have been reported. The aim here was to study the performance of a quantitative ply -based PCR for the identification of pneumococcal lower respiratory tract infection (LRTI). In a prospective study, fibreoptic bronchoscopy was performed in 156 hospitalized adult patients with LRTI and 31 controls who underwent bronchoscopy because of suspicion of malignancy. Among the LRTI patients and controls, the quantitative ply -based PCR applied to bronchoalveolar lavage (BAL) fluid was positive at ,103 genome copies/mL in 61% and 71% of the subjects, at ,105 genome copies/mL in 40% and 58% of the subjects, and at ,107 genome copies/mL in 15% and 3.2% of the subjects, respectively. Using BAL fluid culture, blood culture, and/or a urinary antigen test, S. pneumoniae was identified in 19 LRTI patients. As compared with these diagnostic methods used in combination, quantitative ply -based PCR showed sensitivities and specificities of 89% and 43% at a cut-off of 103 genome copies/mL, of 84% and 66% at a cut-off of 105 genome copies/mL, and of 53% and 90% at a cut-off of 107 genome copies/mL, respectively. In conclusion, a high cut-off with the quantitative ply -based PCR was required to reach acceptable specificity. However, as a high cut-off resulted in low sensitivity, quantitative ply -based PCR does not appear to be clinically useful. Quantitative PCR methods for S. pneumoniae using alternative gene targets should be evaluated. [source] | |||||||||||||