摘要:

BackgroundIn recent years PCR has proven to be a highly sensitive and specific method for the diagnosis of infections caused byNeisseria meningitidis.Study designWe developed and evaluated aN. meningitidisLightCycler real-time duplex PCR (NM-LCdPCR) capable of simultaneously detecting and distinguishing between two separate genes on theN. meningitidisgenome.MethodsThe NM-LCdPCR was developed on the LightCycler platform (Roche Diagnostics, Castle Hill, NSW, Australia) and comprised two primer pairs and two hybridization probe sets, enabling the detection of both theporAandctrAgenes within the same reaction mix. To distinguish between the fluorescence emitted by each hybridization probe set, each downstream probe was labeled with a different fluorophore (either LC-Red640 or LC-Red705). The results obtained by the NM-LCdPCR were then compared with the results obtained by a mono-specific LightCycler assay targeting theporAgene only (porA-LCPCR).PatientsOne-hundred and forty-eight clinical samples from patients with suspected meningococcal infection were evaluated.ResultsThe results of the NM-LCdPCR and porA-LCPCR gave 100% agreement;N. meningitidisDNA was detected in 25 samples whereas 123 samples were negative by both assays. The breakdown of the NM-LCdPCR results show that both genes were detected in 26 of the 28 positive samples.DiscussionBy targeting two separateN. meningitidisgenes, the NM-LCdPCR has the potential to prevent the false-positive results which may arise from sequence variation. In addition, the ability to detect and discriminate between the two differentN. meningitidisgenes within the same reaction mix offers a rapid means for confirming the presence ofN. meningitidisDNA in clinical samples, thereby reducing the need for subsequent confirmatory assays to be performed.ConclusionsThe sensitivity and specificity of the NM-LCdPCR assay, combined with its ability to detect and discriminate both theN. meningitidis porAandctrAgenes, make it suitable for the diagnosis ofN. meningitidisinfections in the routine clinical laboratory.

ISSN:1084-8592    

出版商:ADIS    

年代:2003

数据来源: ADIS

摘要:

BackgroundThe implementation of cost-effective intervention strategies for zoonotic protozoa relies on the development of sensitive and accurate diagnostic methods. We carried out a study to evaluate the accuracy of a PCR method for the detection ofCryptosporidiumspp. oocysts in fecal samples from cattle.MethodsFecal samples were spiked with different numbers of oocysts and the limit of detection of the method was determined. Two methods of DNA extraction were assessed: glass beads and freeze-thawing using liquid nitrogen. A nested PCR approach was developed targeting theCryptosporidiumSSUrRNAandTRAP-C2genes. Agreement between the diagnosis ofCryptosporidiumspp. at the SSU rRNA and TRAP-C2 loci was quantified using the κ-coefficient.ResultsCompared with the freeze-thawing method, the glass beads method was found to be a better way of extracting DNA fromCryptosporidiumoocysts (sensitivities were 83 and 100%, respectively). The limits of detection for glass beads and freeze-thaw were low, 1 and 10 oocyst/g fecal samples, respectively. Forty-six percent of the field samples previously classified as negative forCryptosporidium parvumby the floatation-concentration and enzyme-linked immunosorbent assay methods showed DNA with the PCR protocol.ConclusionPrimers for SSU rRNA are more successful in producing an amplification than primers for theTRAP-C2gene which makes the former PCR protocol the approach of choice for detectingCryptosporidium parvumoocysts in field samples.

ISSN:1084-8592    

出版商:ADIS    

年代:2003

数据来源: ADIS

摘要:

BackgroundLyme disease is a multisystem, multistage infection caused by three genospecies of theBorrelia burgdorferisensu lato species. The diagnosis of Lyme disease is based on a history of tick-bite, physical examination, and serological tests. In the seronegative patients with Lyme borreliosis symptoms, additional testing should be introduced.MethodsThe study group was composed of 240 hospitalized patients presented with various clinical symptoms suggesting Lyme borreliosis: 221 of the patients with neurological abnormalities and 19 with oligoarticular arthritis. Citrated blood and serum samples were collected from the patients for culture and serological examination, respectively. Moreover, 173 cerebrospinal and 6 synovial fluid samples were tested. New oligonucleotide primers based onB. burgdorferisensu lato 16SrRNA gene sequences were designed for the detection of the bacteria in blood, cerebrospinal, and synovial fluid specimens with PCR. Levels of specific antibodies were measured in serum, cerebrospinal fluid and synovial fluid samples using ELISA and Western blot.B. burgdorferispirochetes from blood, cerebrospinal fluid, and synovial fluid samples were cultured in cell line. Extracted and purifiedB. burgdorferiDNA was identified by PCR with new oligonucleotide primers. Then three genospecies were identified by PCR amplification with other primer sets specific for 16S rDNA and/or by the restriction fragment length polymorphism of 23S(rrl)–5S(rrf).ResultsBacterial DNA were found in samples from 32 patients, including 28 patients with neuroborreliosis and 4 with Lyme arthritis.B. burgdorferi-specific IgM and/or IgG serum antibodies were detected in 14 of these patients. Fourteen strains ofBorrelia garini, 4 strains ofBorrelia afzeliiand 1 strain ofB. burgdorferisensu stricto were identified by PCR. Genospecies were not recognized in 13 specimens.ConclusionsThe procedure can be a rapid and sensitive diagnostic method for the detection of etiological agents in clinical materials derived from patients with the clinical symptoms of Lyme borreliosis. It can be utilized for both basic research as well as routine laboratory diagnosis.

ISSN:1084-8592    

出版商:ADIS    

年代:2003

数据来源: ADIS

摘要:

BackgroundOur understanding of fragile X syndrome can be improved by reversing the expression of the silenced fragile X mental retardation 1 (FMR1) gene in immortalized cells from these patients. Epstein-Barr virus (EBV) infection has been extensively used to transform B cells into a permanent lymphoblastoid cell line.MethodsWe immortalized B lymphocytes from three different fragile X patients and one normal male. We analyzed the CGG triplet repeats and methylation status of the FMR1 and interferon (IFN)-γ promoter. We also assayed FMR1 mRNA levels by real-time PCR and FMR1 protein (FMRP) by Western blot.ResultsWe observed that EBV transformation may induce the instability of CGG repeats and DNA demethylation that can lead to the modification of mRNA expression.ConclusionsEBV transformation may induce variable changes in the genome that can lead to the misinterpretations of experimental data obtained from these cells. Thus, periodic testing of DNA from immortalized cells should be routinely undertaken to detect undesired effects.

ISSN:1084-8592    

出版商:ADIS    

年代:2003

数据来源: ADIS

摘要:

AimDNA testing can provide a definitive diagnosis of familial hypercholesterolemia (FH), even in the absence of the clinical characteristics of this inherited cardiovascular disease (CVD) subtype. Our aim was to design a rapid diagnostic assay capable of simultaneously analyzing seven point mutations in the low-density lipoprotein receptor (LDLR) gene, which occur at high frequency in South African FH patients.MethodsThe test is based on multiplex DNA amplification and hybridization to membrane strips presenting a parallel array of immobilized allele-specific oligonucleotide probes.ResultsA reverse-hybridization assay for genotyping LDLR point mutations D154N, D200G, D206E, C356Y, G361V, V408M, and P664L was set-up and validated using pretyped human DNA samples, as well as recombinant plasmid clones containing mutant alleles. The procedure is rapid (6 hours) and may be automated to a large extent.ConclusionsThe new FH strip-assay forms an important part of the comprehensive cardiovascular genetic screen offered routinely to high-risk population groups in South Africa. A genetic approach based on FH testing in conjunction with other ‘genetic’ CVD risk factors is feasible and justified, since the spectrum of disease-related mutations have been defined to a large extent in the genetically distinct population groups of South Africa. Knowledge of a significantly increased CVD risk due to the presence of gene variations, which can be targeted for risk reduction by the avoidance of relevant environmental risk factors and the appropriate treatment, provides a powerful message to motivate people into implementing preventative measures based on their genetic profile.

ISSN:1084-8592    

出版商:ADIS    

年代:2003

数据来源: ADIS

摘要:

BackgroundAnalyzing the molecular variants of immunological system genes helps to develop our understanding of the pathogenesis of several diseases. The tumor necrosis factor (TNF) is an important cytokine of cellular response and inflammation. TheTNFgene is located within the MHC region on chromosome 6p21.3. Single nucleotide polymorphisms in theTNFgene, such as the one at a position −308, probably have a direct influence on TNF production. Myeloperoxidase, a heme enzyme, participates in micro-organism killing. The myeloperoxidase (MPO) gene is located on chromosome 17. In the promoter region, at position −463, G to A transition has been found, which causes decreased gene expression.AimThe aim of our study was to analyze the genetic polymorphisms of theTNFandMPOgenes in patients with chronic renal failure.MethodsThe study included 95 patients with chronic renal failure and 115 healthy individuals. All participants were genotyped forTNF−308 and MPO promoter region polymorphisms by PCR, followed by digestion and gel electrophoresis. Genotype distribution was compared between patients and controls. For statistical analysis the Statistica PL 6.0 program was used. The Kruskal-Wallis and median test were employed; to evaluate relationship between quantitative data chi-square test was used.ResultsThere were no significant differences in genotype distribution ofTNForMPOpolymorphisms between patients and controls. Some differences may be associated with gender because theTNF1/TNF1 genotype was significantly more common in healthy women in comparison with women with chronic renal failure (p < 0.05). In men, no such differences were found. ForMPOpolymorphism, in men with renal failure the GG genotype was significantly more frequent than in healthy men (p < 0.05). Comparing theMPOgenotype distribution in diabetic nephropathy patients and nondiabetic patients, we found a statistically significant difference: GG and AA genotypes were more frequent in diabetic nephropathy than in other renal diseases (73 versus 60% and 10.8 versus 1.7%, respectively; p < 0.05). The genotype distribution in patients with other renal diseases was similar to the control group. There was a correlation between theTNFgenotype and the age of onset of glomerulonephritis. For myeloperoxidase, there was a significant association between genotype and the age of onset of renal disease. There was no relationship between theTNFandMPOgenotypes and time to end-stage renal disease.ConclusionOur studies show that theTNFandMPOgenes may play a role in chronic renal failure. The relationship observed between polymorphisms of theTNFandMPOgenes and chronic renal failure may depend on the pathophysiological changes in different diseases underlying renal failure.

ISSN:1084-8592    

出版商:ADIS    

年代:2003

数据来源: ADIS

摘要:

BackgroundMethylenetetrahydrofolate reductase (MTHFR) plays a critical role in folate metabolism and displays common genetic polymorphisms affecting the enzyme activity. TheMTHFRgenetic polymorphisms have been associated with a decrease in the risk of developing the lymphoid but not myeloid form of pediatric and adult leukemias.AimIn this study we describe the genotyping of theMTHFR C677Tpolymorphism by melting curve analysis with the LightCycler®in a case-controlled study of patients with acute lymphocytic leukemia (ALL), myelogenous leukemia (AML), and chronic myelogenous leukemia (CML), and assess the effect of this common polymorphism on the leukemia risk in adult patients in Turkey.MethodsDNA from peripheral blood lymphocytes was used for genotyping in the LightCycler®PCR by melting curve analysis. The risk of leukemia associated with theMTHFRpolymorphism was evaluated by comparing the genotype frequencies between the control and patient groups.ResultsThe frequency of the homozygote variant genotype (677TT) was lower than that in healthy individuals in all three leukemia groups. The677TTgenotype did not appear to have a protective effect in patients with ALL (Odds ratio [OR] = 0.78 with a 95% confidence interval [CI] = 0.24–2.59), compared with healthy controls. The difference was higher (4.3-fold) in patients with AML, but still non-significant (OR = 0.23 with a 95% CI = 0.03–1.83). In patients with CML, the frequencies of both heterozygous (677CT) and homozygote variant genotypes were lower (OR = 0.72 and 0.66, respectively).ConclusionsOur results suggest that theMTHFR C677Tpolymorphism displays a similar distribution pattern in lymphoid and myeloid leukemias and that the frequency of the homozygote variant genotype (677TT) is lower in all leukemia types.

ISSN:1084-8592    

出版商:ADIS    

年代:2003

数据来源: ADIS

摘要:

BackgroundNuclear factor-kappa B (NF-κB) is an important transcription factor involved in the regulation of immune responses as well as in cell proliferation and survival. An abnormal and constitutive activation of NF-κB is observed in many pathological states as diverse as inflammation, neurological diseases, and cancer.Methods and resultsTermination of NF-κB transcription is mediated through the NF-κB-dependent synthesis of the IκB-α inhibitory subunit. To quantify NF-κB activation we measured by real-time PCR the expression of IκB-α mRNA. The PCR data perfectly matched the results obtained by Northern blot or gene reporter analysis when Jurkat leukemic T cells or HeLa carcinoma cells were stimulated with various activators of NF-κB, such as the cytokine tumor necrosis factor (TNF)-α or the phorbol ester PMA. Constitutive NF-κB activation in Hodgkin’s lymphoma cell line could also be evaluated by this approach. Kinetic experiments in HeLa cells show that TNF stimulation first induced NF-κB DNA binding within 30 minutes, followed by IκB-α gene transcription 30 minutes later. Removal of TNF after stimulation resulted in a faster decrease in both NF-κB DNA binding activity and IκB-α mRNA levels. No accumulation or stabilization of IκB-α mRNA was detected that could bias interpretation of the results. The sensitivity of the method allowed the detection of NF-κB activation in stimulated normal peripheral blood lymphocytes.ConclusionThe real-time PCR measure of IκB-α mRNA levels is a rapid, sensitive, and powerful method to quantify the transcriptional power of NF-κB. It can be easily used for clinical evaluation of NF-κB status.

ISSN:1084-8592    

出版商:ADIS    

年代:2003

数据来源: ADIS

摘要:

BackgroundThe Liverpool epidemic strain (LES) ofPseudomonas aeruginosais widespread among patients with cystic fibrosis (CF) in specialist centers around Liverpool and elsewhere in the UK. This study evaluates a new diagnostic PCR assay based on a unique DNA sequence (PS21) of LES, for its identification of colonies directly from sputum.MethodsOne hundred and fifty-eight sputum samples from 92 patients were cultured andP. aeruginosaisolates were typed by PS21 PCR and pulsed-field gel electrophoresis (PFGE). Subsequently, PS21 PCR was performed directly on sputum and the results were compared with culture, PFGE, and PS21 PCR typing.ResultsEighty patients were colonized withP. aeruginosa, 63 by LES (79%). There was 100% concordance between PS21 PCR on colonies and PFGE typing. The sensitivity and specificity of PS21 PCR directly on sputum was 98.2% and 93.6%, respectively.ConclusionsThis study shows that PS21 PCR can be used for simple and rapid screening of LES colonization in CF patients.

ISSN:1084-8592    

出版商:ADIS    

年代:2003

数据来源: ADIS

摘要:

BackgroundGene promoter methylation is a mechanism for tumor suppressor gene silencing and inactivation. The development of highly sensitive methods for revealing aberrant cancer-associated DNA methylation allows the identification of tumor markers not only in tumor samples, but also in body fluid, an approach that can be useful in the early detection of neoplasms.MethodsWe analyzed the methylation status at 16 loci in tumor samples of the gastrointestinal tract and in early or pre-neoplastic lesions of the colon.ResultsTumor samples revealed that methylation at the transmembrane protein containing epidermal growth factor and follistatin domains (TPEF) locus had the best ratio of discrimination between tumor samples versus normal tissues (83 versus 0%). Its combination with hypermethylated in cancer 1 (HIC1), death-associated protein kinase (DAPK) andO-6-methylguanine DNA methyltransferase (MGMT), allowed the detection of aberrant methylation in 98% of colorectal carcinomas and 100% of gastric carcinomas. The same alterations were also detected in colon adenomas and tissues surrounding the adenomas, indicating that hypermethylation at these loci occurred early in tumor progression. Analysis of DNA from peripheral blood revealed thatTPEFmethylation was detectable in colorectal tumor patients and patients with early or pre-neoplastic lesions, but not in healthy volunteers.ConclusionsOur results identifyTPEFas a tumor marker that could be useful in the follow-up of gastrointestinal cancer patients or the screening of individuals at risk of developing gastrointestinal neoplasms.

ISSN:1084-8592    

出版商:ADIS    

年代:2003

数据来源: ADIS