Molecular diagnosis : a journal devoted to the understanding of human disease through the clinical application of molecular biology最新文献

Background: The 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 of Cryptosporidium spp. oocysts in fecal samples from cattle.

Methods: Fecal 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 the Cryptosporidium SSU rRNA and TRAP-C2 genes. Agreement between the diagnosis of Cryptosporidium spp. at the SSU rRNA and TRAP-C2 loci was quantified using the kappa-coefficient.

Results: Compared with the freeze-thawing method, the glass beads method was found to be a better way of extracting DNA from Cryptosporidium oocysts (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 for Cryptosporidium parvum by the flotation-concentration and enzyme-linked immunosorbent assay methods showed DNA with the PCR protocol.

Conclusion: Primers for SSU rRNA are more successful in producing an amplification than primers for the TRAP-C2 gene which makes the former PCR protocol the approach of choice for detecting Cryptosporidium parvum oocysts in field samples.

Background: The Liverpool epidemic strain (LES) of Pseudomonas aeruginosa is 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.

Methods: One hundred and fifty-eight sputum samples from 92 patients were cultured and P. aeruginosa isolates 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.

Results: Eighty patients were colonized with P. 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.

Conclusions: This study shows that PS21 PCR can be used for simple and rapid screening of LES colonization in CF patients.

Background: It is widely known that the efficiency of fluorescence in situ hybridization (FISH) probes applied to formalin-fixed, paraffin-embedded tissues is affected by the conditions under which the tissues are fixed and embedded. However, relatively few studies address exactly how tissue archiving conditions affect the performance of FISH probes. We report our experience based on use of an ALK FISH probe, during the validation of its diagnostic utility.

Methods: We applied the probe to 77 formalin-fixed, paraffin-embedded tissue blocks archived from 1991 through to 2000, and studied the interrelationship between the archival age (which ranged up to 10 years), type and condition of tissue, duration required for optimum hydrolysis, and obtainability of hybridization signals.

Results: We found that as archival age and tissue collagen content increased, not only did hydrolysis times have to be prolonged in order to yield interpretable hybridization signals, but also the likelihood of blocks becoming non-signaling increased. The most striking positive correlations were seen between the archival age of signaling lymphoid blocks and their requisite hydrolysis times.

Conclusions: The difficulty in applying FISH on archival tissue increases with its archival age and collagen content, and may necessitate changes in laboratory protocol accordingly.

Background: Analyzing 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. The TNF gene is located within the MHC region on chromosome 6p21.3. Single nucleotide polymorphisms in the TNF gene, 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.

Aim: The aim of our study was to analyze the genetic polymorphisms of the TNF and MPO genes in patients with chronic renal failure.

Methods: The study included 95 patients with chronic renal failure and 115 healthy individuals. All participants were genotyped for TNF-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.

Results: There were no significant differences in genotype distribution of TNF or MPO polymorphisms between patients and controls. Some differences may be associated with gender because the TNF1/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. For MPO polymorphism, in men with renal failure the GG genotype was significantly more frequent than in healthy men (p < 0.05). Comparing the MPO genotype 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 the TNF genotype 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 the TNF and MPO genotypes and time to end-stage renal disease.

Conclusion: Our studies show that the TNF and MPO genes may play a role in chronic renal failure. The relationship observed between polymorphisms of the TNF and MPO genes and chronic renal failure may depend on the pathophysiological changes in different diseases underlying renal failure.

Introduction: Mutations at the codon 6 of the beta-globin gene (hemoglobin [Hb] S and HbC) can be routinely identified by various methods and prenatal diagnosis has been available to affected families for several years. However, the presence of maternal cells in fetal samples constitutes a serious potential source of prenatal misdiagnosis and most methods currently used to detect maternal contamination are based on the analysis of highly polymorphic loci. In addition, these methods are labor intensive and time consuming and risk carry-over contamination.

Method: We describe here a one-step method for mutation detection that uses fluorescent hybridization probes with melting curve analysis for both simultaneously prenatal diagnosis of sickle cell disease and potential maternal contamination.

Results: Retrospective and prospective prenatal diagnosis studies (conducted in 20 and 50 cases, respectively), using both the regular procedure and real-time PCR assay show perfect concordant results. We show in addition, that as little as 5% maternal contamination can be detected and that genotype determinations are unambiguous.

Background: Oral lichen planus (OLP) is a chronic relapsing cell-mediated condition of unknown etiology. The purpose of this study was to ascertain if the human herpesviruses (HHVs) or human papillomaviruses (HPVs) act as possible factors or co-factors in the pathogenesis of OLP.

Methods: Thirty-eight histologically confirmed OLP and 20 normal control buccal mucosa tissue samples were analyzed. Polymerase chain reaction analysis was employed to detect members of the HHV and HPV families.

Results: The Epstein-Barr virus and HHV-7 were detected in a small percentage of tissue samples. However, HPV-16 was detected in 26.3% of OLP samples and 0% of the normal control tissues. The epidermodysplasia verruciformis-related HPV types were detected in 42% of OLP samples and 45% of normal control samples.

Conclusion: The results of this study do not suggest a causative role for members of the HHV family in the pathology of OLP. However, a statistical association was found between HPV-16 presence and OLP.

Background: Among the well characterized X-linked conditions causing mental retardation, mutations in the methyl-CpG-binding protein 2 gene (MECP2) in Xq28 have been found in up to 85% of patients with Rett syndrome, a neurologic disorder which, in addition to other symptoms, severely affects higher cognitive functions in females. Mutations in the MECP2 gene are involved in a broad spectrum of phenotypes from classical Rett syndrome to mild intellectual difficulties in females and neonatal encephalopathy in males. Recently, mutations in the MECP2 gene were reported in males with non-specific mental retardation suggesting that defects in MECP2 could be responsible for up to 2% of X-linked mental retardation.

Methods: We screened by denaturing high-pressure liquid chromatography the entire coding region and flanking intronic sequences of the MECP2 gene in a cohort of 354 mentally retarded males found negative for an expansion across the FRAXA CGG repeat and in a family in which a boy and his sister were mentally retarded.

Results: We identified mainly silent polymorphisms within the MECP2 gene, together with four sequence alterations of unknown significance, i.e. three missense mutations (T197M, T228S, and P376S) and one substitution at position -19 in intron 3 (378-19delT). Further familial investigations allowed us to ruled out a pathogenic effect for the intronic variant, the T228S and the P376S missense mutations.

Conclusions: These results confirm that MECP2 mutations in males are far more rare than initially thought and call for a careful evaluation of the pathogenicity of the MECP2 missense mutations identified in mentally retarded males before genetic counseling is proposed to the relatives.

Background: Interferon-beta (IFNbeta) has proven to be an important advance in the therapy of multiple sclerosis (MS), but optimal markers for bioactivity have not been identified. To accurately measure bioactivity in MS patients treated with IFNbeta, we developed and tested a real-time reverse transcriptase (RT)-PCR assay for gene expression of MxA, an IFNbeta-induced gene in the peripheral blood of patients treated with IFNbeta.

Methods: We compared IFNbeta-treated patients with MS to controls in expression of MxA relative to the house-keeping gene, GAPDH. 2'-5'oligoadenylate synthetase (OAS) gene expression was also tested by real-time RT-PCR on RNA from the same patient specimens. Anti-IFNbeta antibody was measured by ELISA and a cytopathic effect assay.

Results: Seven of 54 patients were found to have complete loss of bioactivity. MxA expression correlated well with OAS expression. All patients with lost bioactivity had high levels of binding antibodies or neutralizing antibodies.

Conclusions: This is the first demonstration that a real-time RT-PCR assay can be used to monitor therapy with interferons. These data identify MxA mRNA as an excellent biomarker for INFbeta action on the IFN receptor, and clarify the relationship between anti-IFNbeta antibodies and bioactivity in patients with MS treated with IFNbeta.