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Results for “Nested Genes”

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PubMed1996

[Detection of bcl-2/JH fusion gene in patients with non-Hodgkin's lymphoma by semi-nested polymerase chain reaction].

To study the incidence and clinical significance of bcl-2/JH fusion in 45 Chinese patients with non-Hodgkin's lymphoma, semi-nested polymerase chain reaction (PCR) was used to detect the fusion gene of non-Hodgkin's lymphoma. A total of 12 patients gave positive results, they include 9 of the 15 patients with follicular lymphoma and 3 of the 30 patients with diffuse B-cell lymphoma. The results were negative in 5 patients with reactive hyperplasia. The breakpoint was located within the major breakpoint region in 11 of the 12 patients, and the remaining patient had bcl-2 translocation in the minor cluster region. The results of the study show that bcl-2/JH fusion gene in non-Hodgkin's lymphomas is an important molecular biological marker and has a significant role in differentiating benign or malignant hyperplasia, determination of clonality, predicting prognosis and in the discovery of minor residual disease.

Diagnosis, Differential↗

PubMed2006

Polymorphisms of prostaglandin-endoperoxide synthase 2 gene, and prostaglandin-E receptor 2 gene, C-reactive protein concentrations and risk of atherothrombosis: a nested case-control approach.

OBJECTIVE: Recent data have shown an association between polymorphisms of prostaglandin-endoperoxide synthase-2 gene (PTGS2; alias COX-2), and prostaglandin-E receptor-2 gene (PTGER2) and risk of atherothrombotic disorders. METHODS: We evaluated two PTGS2 (rs20417, rs689470), and three PTGER2 (rs708494/uS5, rs708495/uS7, and chr14: 50 764 013/uS10) gene polymorphisms among 600 Caucasian male participants of the Physicians' Health Study with incident myocardial infarction (MI) or ischemic stroke and 600 age- and smoking-matched controls who remained free of all reported cardiovascular disease. RESULTS: Genotype distributions were in Hardy-Weinberg equilibrium in the control groups. Genotype and allele distribution were similar between cases and controls. The polymorphisms tested were in linkage disequilibrium. Results from the adjusted haplotype-based conditional logistic regression analysis showed a modest association of the PTGER2 2-1-1 haplotype with reduced risk of MI (odds ratio = 0.50, 95% CI; CI = 0.26-0.97, P = 0.04), and the 2-2-1 haplotype with reduced risk of ischemic stroke (odds ratio = 0.68, 95% CI = 0.47-0.99, P = 0.048). In contrast to prior data, we found no evidence for an association of the PTGS2 polymorphisms/haplotypes tested with risk of incident MI nor with ischemic stroke. However, we found suggestive evidence for an association of specific PTGER2 haplotypes with reduced risk of these outcomes. CONCLUSION: Although these prospective data implicate the potential involvement of prostaglandin-E receptor-2 gene variation in atherothrombosis, external validation of our findings is needed.

Adult↗

PubMed2005

Inhibition effect of Chinese herbal medicine on transcription of hepatitis C virus structural gene in vitro.

AIM: To investigate the inhibitory effect of Chinese herbal medicine on the transcription of hepatitis C virus (HCV) structural gene in Hela D cells. METHODS: Hela cell line was transfected with recombinant pBK-CMV-HCV containing HCV structural gene by Lipofectamine. RT-nested-PCR and Western blot assay were used to testify the HCV gene expression in Hela cells. The Hela cells expressing HCV structural protein were named Hela D cells. Prescriptions of Xiao chaihu Decoction (XCHD), Fufang Huangqi (FFHQ) and Bingganling (BGL) were respectively added to Hela D cells in various concentrations. Semi-quantitative RT-nested-PCR product analysis was performed according to the fluorescent density between HCV DNA band and GAPDH DNA band in gel electrophoresis after screened. RESULTS: Recombinant pBK-CMV-HCV could correctly express the HCV structural gene in Hela D cells. After co-culture of Hela D cells with three prescriptional different concentrations for 48 h respectively, the transcription of HCV gene decreased with increasing of the concentration of each prescription. The lightness ratio of HCV product bands to GAPDH product bands was 0.24, 0.10 and 0.12 in Hela D cells incubated with 0.1 g/mL of XCHD, FFHQ and BGL respectively and the lightness ratio HCV product bands to GAPDH product bands was 0.75, 0.67 and 0.61 respectively in the control cells. CONCLUSION: The prescriptions of XCHD, FFHQ and BGL partly inhibit the transcription of HCV structural gene in Hela D cells.

Base Sequence↗

PubMed1998

Localization of hepatitis B virus DNA in hepatocellular carcinoma by polymerase chain reaction in situ hybridization.

The polymerase chain reaction in situ hybridization (PCR-ISH) is a new technique that combines the sensitivity of PCR with the localizing ability of ISH. To investigate the expression pattern of hepatitis B virus (HBV) in the tissue of hepatocellular carcinoma (HCC), we detected HBV-DNA with PCR-ISH in paraffin-embedded tumor and corresponding non-tumor tissues from 11 HCC patients. HBV-DNA was detected in 4 of 11 tumor tissues and in 7 of 10 non-tumor tissues. In tumor tissues, positive signals were scattered in the tissue with occasional clustering, and were found mainly in the cytoplasm of HCC cells rather than in the nucleus. In non-tumor tissues, the number of positive signals was higher than in tumor tissues and they were found in regenerating nodules with differing patterns and intensities. When we compared the detection rate of PCR-ISH with nested PCR among 10 tissue samples, HBV-DNA was detected in 5 tissue samples by PCR-ISH, but the S gene was detected in 10, precore gene in 9 and X gene in 8 by nested PCR. The findings suggest that PCR-ISH is a sensitive technique for localizing HBV in tissue sections and that the low level of HBV replication persists in HCC cells.

Carcinoma, Hepatocellular↗

PubMed2006

Comparison between two real-time PCR assays and a nested-PCR for the detection of Toxoplasma gondii.

BACKGROUND AND AIM OF THE WORK: In recent years, the diagnosis of toxoplasmosis has been improved by Real-time PCR assays. In this study we compared the performances of two Real-time PCRs (FRET and TaqMan protocols) already described in the literature, and one nested-PCR, currently used in our laboratory for the molecular diagnosis of toxoplasmosis. METHODS: We evaluated the sensitivity and the specificity of a FRET- and a TaqMan-based Real-time PCRs targeting a 529 bp repeat region and the 18S RNA gene, respectively, and a nested-PCR, targeting the B1-gene of Toxoplasma gondii. We also tested, through nested-PCR, 46 biological samples obtained during a period of 29 months from pregnant women or immunocompromised patients with suspected T. gondii infection. RESULTS: The analytical sensitivity of nested and TaqMan PCRs was approximately 10(3) tachyzoites/ml. FRET assay showed a sensitivity of 102 tachyzoites/ml. Three out of 46 biological samples were nested-PCR-positive and these results were also confirmed by both Real-time PCRs. CONCLUSIONS: Nested- and real-time PCRs evaluated in this study resulted very sensitive and specific; in particular FRET PCR resulted more sensitive than the other assays, probably because of the greater copy number of the target sequence. Real-time PCR assays are easy-to-use, producing results faster than conventional PCR systems and reducing contamination risks.

Adult↗

PubMed2002

Fate of coliphage in waste water treatment process and detection of phages carrying the Shiga toxin type 2 gene.

Abundances of phages specific to Escherichia coli in the wastewater treatment process were analyzed. Relatively abundant coliphages were detected in sewage influent. Phages in the influent were found both suspended in liquid phase and attached on the solid particles. Phage concentration was not reduced in the settling tank without chemical agglutination. Anaerobic followed by aerobic treatment of the sewage reduced concentration of suspended phages. Almost no phage was detected as a suspended form in the aerobic tank. Most of the phages were detected as attaching form and were excluded by aggregation with sludge. Using an experimental approach based on the detection of Shiga toxin 2 (Stx 2) gene by a phage enrichment culture followed by nested PCR, bacteriophages carrying Stx 2 gene were detected in the influent, settling tank, and anaerobic tank. It was revealed that the presence of phages carrying Stx 2 gene is common in sewage and these phages are effectively eliminated through sewage treatment process.

Coliphages↗

PubMed1994

Competitive nested polymerase chain reaction for quantification of human MDR1 gene expression.

Tumor cell resistance to cytotoxic drugs is considered one of the major obstacles to successful chemotherapy. Multidrug resistance (MDR) describes the simultaneous expression of cellular resistance to a wide range of structurally and functionally unrelated drugs. The development of the multidrug resistance phenotype is accompanied by multiple morphological and biochemical changes: (a) increased glutathione levels in the cytoplasm, (b) modified levels of enzymes in the nucleus, particularly topoisomerase II, (c) increased DNA repair capacity and (d) overexpression of the (human) MDR1 gene encoding a transmembrane efflux pump (P-glycoprotein, gp-170), which leads to decreased intracellular accumulation and therefore to resistance to a variety of cytotoxic drugs. In this report we describe a competitive polymerase chain reaction (PCR) assay for the absolute quantification of MDR1 mRNA. This assay uses a transcript generated in vitro as an internal standard which is later coamplified together with the MDR1 cDNA. Both cDNAs exhibit the same MDR1 primer sites but differ in the length of the amplicon. For a second round of amplification we applied nested MDR1 primers and were successful in improving the sensitivity of this competitive PCR system. This test for characterizing the MDR1 expression offers high sensitivity and specificity and is therefore of great clinical relevance. It should be useful in improving monitoring and design of chemotherapy.

Base Sequence↗

PubMed2000

Detection of tumor cells in blood using CD45 magnetic cell separation followed by nested mutant allele-specific amplification of p53 and K-ras genes in patients with colorectal cancer.

A new method for detecting circulating tumor cells that is based on magnetic-activated cell separation (MACS) and nested mutant allele-specific amplification (nested MASA) was evaluated in patients with colorectal cancer using the p53 and K-ras genes as genetic markers. By negative selection with anti-CD45 monoclonal antibody-conjugated supermagnetic microbeads, the proportion of tumor cells was enriched 9-fold. By the combination of MACS and nested MASA, 10 tumor cells in 10(7) normal peripheral blood mononuclear cells could be detected without false-positives. Using this method, we examined blood taken from the tumor drainage veins of 23 patients with colorectal cancer. Eighty-seven percent (20/23) of primary tumor tissues showed p53 and/or K-ras gene mutations. Forty-five percent (9/20) of patients with p53 and/or K-ras mutations in the primary tumor showed the same mutated genes in the blood samples. There was a significant association between the presence of p53 and K-ras gene mutation in the blood and tumor size, depth of invasion, and venous invasion. Blood gene mutation was detected in 80% (4/5) of samples from patients with synchronous liver metastases. Sixty percent (3/5) of patients with mutant genes in the blood developed asynchronous liver metastases after surgery. The overall survival of patients with p53 and/or K-ras gene mutation-positive findings in blood was significantly shorter than that of patients testing negative on Kaplan-Meier analysis. Our results suggest that the method may be useful for reliable detection of tumor cells circulating in the blood and may help to identify patients at high risk for relapse.

Adenocarcinoma↗

PubMed2000

Evaluation of a specific nested PCR targeting domain III of the 23S rRNA gene of "Tropheryma whippelii" and proposal of a classification system for its molecular variants.

"Tropheryma whippelii"-associated infections are usually confirmed histopathologically by using light microscopy. PCR assays targeting the 16S rRNA gene (16S rDNA) of "T. whippelii" are increasingly being applied for this purpose. Compared to microscopic analysis, PCR seems to be more sensitive, as indicated by the fact that several cases of Whipple's disease with negative histopathological findings but positive PCR results have been reported. Considering the lack of pathognomonic clinical features for this disease and the fact that "T. whippelii" DNA has repeatedly been found in patients without clinical Whipple's disease, such PCR results should be confirmed by additional tests. We have, therefore, evaluated a "T. whippelii"-specific nested PCR targeting domain III of the 23S rDNA with 41 clinical specimens known to contain "T. whippelii" 16S rDNA. All of these specimens were also positive for "T. whippelii" 23S rDNA. The specificity of the test was shown by sequencing of the amplicons and by the absence of amplicons in 38 negative controls. We consider this PCR test to be a suitable tool for confirming the presence of "T. whippelii" DNA in specimens with inconclusive histopathological findings. The information derived from sequencing of the partial "T. whippelii" 23S rDNA was then combined with our recent data of the 16S-23S rDNA spacer region of this organism. Overall, four different rDNA types are recognized in our proposed classification system for molecular variants of "T. whippelii." This preliminary scheme may provide a basis for further epidemiological and clinical studies with "T. whippelii" and associated diseases.

Actinobacteria↗

PubMed2001

Characterization of cDNA encoding molt-inhibiting hormone of the crab, Cancer pagurus; expression of MIH in non-X-organ tissues.

Synthesis of ecdysteroids (molting hormones) by crustacean Y-organs is regulated by a neuropeptide, molt-inhibiting hormone (MIH), produced in eyestalk neural ganglia. We report here the molecular cloning of a cDNA encoding MIH of the edible crab, Cancer pagurus. Full-length MIH cDNA was obtained by using reverse transcription-polymerase chain reaction (RT-PCR) with degenerate oligonucleotides based upon the amino acid sequence of MIH, in conjunction with 5'- and 3'-RACE. Full-length clones of MIH cDNA were obtained that encoded a 35 amino acid putative signal peptide and the mature 78 amino acid peptide. Of various tissues examined by Northern blot analysis, the X-organ was the sole major site of expression of the MIH gene. However, a nested-PCR approach using non-degenerate MIH-specific primers indicated the presence of MIH transcripts in other tissues. Southern blot analysis indicated a simple gene arrangement with at least two copies of the MIH gene in the genome of C. pagurus. Additional Southern blotting experiments detected MIH-hybridizing bands in another Cancer species, Cancer antennarius and another crab species, Carcinus maenas.

Amino Acid Sequence↗

PubMed1990

Characterization of a potato leafroll luteovirus subgenomic RNA: differential expression by internal translation initiation and UAG suppression.

Northern blot analysis of Solanum tuberosum infected with potato leafroll luteovirus revealed the 6 kb genomic RNA and a major 2.3 kb subgenomic RNA. The 5' end of the subgenomic RNA was located at nucleotide 3653 in an intergenic region located at the centre of the viral genome upstream of three open reading frames (ORFs). Transient expression in tobacco and potato protoplasts of the beta-glucuronidase reporter gene fused to various putative regulatory sequences present in the subgenomic RNA was used to study their influence on expression levels. We observed a suppression of the amber stop codon separating the coat protein (CP) gene from a downstream ORF (56K protein), to a level of 0.9% to 1.3%. Translation initiation at the AUG of an ORF (17K protein) which is nested within the CP gene, exceeds translation of the CP gene itself by a factor of 7.

Base Sequence↗

PubMed2004

Detection of tet(M) gene from raw milk by rapid DNA extraction followed by a two-step PCR with nested primers.

The likelihood that milk and milk products may act as a vehicle for antibiotic-resistant bacterial genes has become a concern to the food industry and a public health issue, and the demand for rapid tests has increased. The purity of DNA extracted from food samples is a key issue in the sensitivity and usefulness of biological analyses, such as PCR for pathogens and nonpathogens. A rapid, phenol-chloroform free method based on a modification of a sodium iodide DNA extraction, followed by a two-step PCR was developed for direct detection of the tet(M) gene in milk samples within a single working day. This study compares the proposed method with a traditional phenol solvent extraction method and with a commercial kit (QIAamp DNA blood mini kit, Qiagen). The three DNA extraction methods were used to ensure access to the tet(M) gene from 1 ml of raw milk, inoculated with a strain of Enterococcus faecalis, which carries the tet(M) gene. The proposed method, followed by a two-step PCR with nested primers specific for the tet(M) gene, was able to reach a detection limit below 10 CFU/ml in less than 4 h, including the two amplification cycles, thus outperforming in sensitivity and rapidity both the traditional and the commercial method.

Animals↗

PubMed2004

Pathogens and symbionts in ticks: prevalence of Anaplasma phagocytophilum (Ehrlichia sp.), Wolbachia sp., Rickettsia sp., and Babesia sp. in Southern Germany.

Tick-transmitted diseases like tick-borne encephalitis and Lyme borreliosis have been well known in Germany for decades. Ongoing research now gives an additional focus to a broad range of other bacteria and parasites in ticks like Anaplasma phagocytophilum, former Ehrlichia sp., Rickettsia sp. and Babesia sp. Knowledge about the prevalence of these infectious agents in ticks is an important prerequisite for risk assessment of human diseases. Therefore nymphs and adult Ixodes ricinus ticks were collected and examined for Anaplasma phagocytophilum (n = 5424 ticks), Rickettsia sp. (n = 1187), and Babesia sp. (n = 3113). For the detection of Anaplasma phagocytophilum, DNA from the 16S rDNA gene was amplified by nested PCR and hybridized with a DIG-labeled oligonucleotide probe. The examination of Rickettsia sp. was performed by single PCR. A partial sequence of the citrate synthase gene was amplified. As a target for the detection of Babesia sp., DNA from the 18S rDNA gene was amplified, also by single PCR. All positive PCR products were sequenced to control specificity. Anaplasma phagocytophilum was detected by PCR in n = 103 (1.9%) out of 5,424 examined ticks from 11 investigation areas. However, not all positive PCR products hybridized using DIG-labeled oligonucleotide probe. Thus, the result of sequencing indicated that only 1.0% (n = 54) belonged to Anaplasma phagocytophilum and nearly half of these PCR products (0.9%) were identified as Wolbachia sp. Rickettsia sp. in Ixodes ricinus ticks from 3 areas were found in n = 105 (8.9%) out of 1,187 ticks examined (range from 13.3% to 5.6%). Sequencing showed Rickettsia helvetica exclusively. In about 2.6% of Rickettsia-positive ticks, double infection with Anaplasma phagocytophilum was found. Babesia sp. was detected in n= 31 (1.0%) out of 3,113 ticks examined, which originated from 4 different areas. By sequencing, n = 28 (90.0%) were identified as Babesia divergens. Three of all Babesia-positive ticks were identified as harboring Babesia microti. The detection of Anaplasma phagocytophilum, Rickettsia sp. and Babesia sp. demonstrates their possible role as a source of human infection in Germany.

Anaplasma phagocytophilum↗

PubMed2005

Coccidioidomycosis and blastomycosis: advances in molecular diagnosis.

Clinical isolates of Coccidioides spp. and Blastomyces dermatitidis can be identified by chemiluminescent DNA probes and PCR assays targeting multicopy genes. In fixed tissue samples, cells of the two fungi are specified by in situ hybridization and PCR assays targeting 18S rDNA but sequencing of the products is mandatory. Nested PCR assays targeting genes encoding species- or genus-specific proteins like proline rich antigen of Coccidioides spp. and B. dermatitidis adhesin facilitate amplification of specific DNA from fixed tissue samples. The value of DNA amplification from native specimens of suspected cases of coccidioidomycosis or blastomycosis still needs to be determined.

Animals↗

PubMed2000

Low in vivo levels of human anx7 (annexin vii) gene expression are due to endogenous inhibitory promoter sequences.

ANX7 is a Ca(2+)/GTP-dependent membrane fusion protein, which is involved in regulating exocytotic secretion. While the nullizygous ANX7 (-/-) knockout mutation in the mouse is lethal, ANX7 is nonetheless the least expressed member of the annexin gene family in mammalian and lower organisms. To investigate the basis for low ANX7 expression level we have studied the human ANX7 promoter. In our early cloning studies we learned that the human ANX7 promoter lacks TATA and CCAAT boxes, but is GC rich and contains several SP1 binding sites. To study how expression of the ANX7 gene is controlled, we fused nested deletion mutants of the 5'-flanking region to a luciferase reporter gene. We find that the maximal level of luciferase activity is obtained by a construct comprising the domain -889/+34. However, a repressor effect observed in the region between -240 and +34 is dramatically eliminated either by the inclusion of 177 bp upstream sequences, or by the presence of an upstream SV40 enhancer sequences. ANX7 gene expression is also cell type specific and is subject to different transcriptional controls in different cellular environments. The fact that the SV40 enhancer sequences not only eliminates the suppression of luciferase activity in the inhibitory region of the ANX7 promoter, but also reverses the inhibitory effect of phorbol ester, suggests that endogenous cis-acting elements or transacting factors may be responsible for the physiologically low level of ANX7 protein expression.

Animals↗

PubMed2005

Detection of cultured and uncultured Burkholderia cepacia complex bacteria naturally occurring in the maize rhizosphere.

The species composition of a Burkholderia cepacia complex population naturally occurring in the maize rhizosphere was investigated by using both culture-dependent and culture-independent methods. B. cepacia complex isolates were recovered from maize root slurry on the two selective media Pseudomonas cepacia azelaic acid tryptamine (PCAT) and trypan blue tetracycline (TB-T) and subjected to identification by a combination of restriction fragment length polymorphism (RFLP) analysis and species-specific polymerase chain reaction (PCR) tests of the recA gene. DNA extracted directly from root slurry was examined by means of nested PCR to amplify recA gene with species-specific B. cepacia complex primers and to obtain a library of PCR amplified recA genes. Using the culture-dependent method the species Burkholderia cepacia, Burkholderia cenocepacia, Burkholderia ambifaria and Burkholderia pyrrocinia were identified, whereas using the culture-independent method also the species Burkholderia vietnamiensis was detected. The latter method also allowed us to highlight a higher diversity within the B. cenocepacia species. In fact, by using the culture-independent method the species B. cenocepacia recA lineages IIIA and IIID besides B. cenocepacia recA lineage IIIB were detected. Moreover, higher heterogeneity of recA RFLP patterns was observed among clones assigned to the species B. cenocepacia than among B. cenocepacia isolates from selective media.

Base Sequence↗

PubMed2005

Fetal gender determination and BclI polymorphism using nucleated erythrocytes in maternal blood.

This study demonstrated determination of fetal gender from nucleated red blood cells (NRBCs) in maternal blood and attempted to apply prenatal diagnosis of hemophilia A using BclI DNA polymorphism. Venous blood was drawn from 20 pregnant women, and NRBCs were recovered by magnetic activated cell sorting and anti-GPA (glycophorin A) immunostaining. After microdissector isolation of the NRBCs, primer extension preamplification (PEP) and nested PCR of the amelogenin gene were performed to determine fetal gender. We also performed PEP and nested PCR of BclI polymorphism to verify the validity of prenatal diagnosis of hemophilia A. DNA amplification was achieved in 107 cells (51.9%) and fetal gender determined with 65.0% accuracy. Unfortunately, we could not verify the validity within the scope of this study. However, in a larger number of cases that are informative in BclI polymorphism, we will be able to identify patients affected by hemophilia A using fetal NRBCs in maternal blood.

Amelogenin↗

PubMed1998

[Rhesus factor determination of the genome of stereomicroscopy selected amniocytes].

This study intended to develop a molecular biological method for the determination of the rhesus blood type through the genome of amniocytes. Using micropipettes amniocytes were isolated under a stereomicroscope from amniotic fluid samples. Single or several (> or = 10) amniocytes, respectively, were pipetted into 0.2 ml vials each, and were lysated at 65 degrees C for 15 min using an alkaline solution making the DNA accessible to polymerase chain reaction. Thereafter, the rhesus D gene as well as the CE gene were amplified with nested primers in two subsequent PCR. Finally, the amplification product was electrophoretically analyzed using agarose gel. When single aminocytes were analysed the amplification rate was 64% for the rhesus D gene, and 80% for the rhesus CE gene; "allele specific non amplification" was observed in 24% of cases. With 10 or more amniocytes per vial the amplification rate was 100%. This method permits to reliably predict the fetal rh-type at low costs within about 4 hours.

Amniocentesis↗