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Specific Primers (specific + primer)
Terms modified by Specific Primers Selected AbstractsTracing the route of Sphaerospora truttae from the entry locus to the target organ of the host, Salmo salar L., using an optimized and specific in situ hybridization techniqueJOURNAL OF FISH DISEASES, Issue 11-12 2003A S Holzer Abstract Sphaerospora truttae is an important pathogen of Atlantic salmon parr in Scottish aquaculture. To trace the early development of S. truttae and to overcome the common problem of detecting low numbers of cryptic, early myxosporean stages, a DNA-based approach was applied in this study. Specific primers were designed for S. truttae from Atlantic salmon, based on 18S rDNA sequences, obtained from isolated myxosporean spores. These were 5, biotin-labelled and used in an optimized and rapid in situ hybridization (ISH) protocol, which provided a strong and specific signal of the parasite within host tissue sections and, at the same time, minimized structural damage to tissues due to processing. This methodology provided a reliable tool enabling the detection of S. truttae stages down to single cell level. Using ISH the epithelium of the gills was identified as the predominant entry locus of the parasite. By 3 days after infection S. truttae had penetrated the vascular epithelia and thereafter proliferated in the blood for at least 10 days before exiting the vascular system through capillary walls. From day 12 post-infection onwards, the kidney, as well as the spleen and the liver, were invaded. Numbers of S. truttae invading the kidney (37.3%) differed little from numbers found in the spleen (35.3%) and the liver (27.4%). The latter organs represented a dead end in the development of S. truttae as all stages in these organs degenerated and sporogony was found to take place exclusively inside the renal tubules. Early sporogonic stages were found from day 25 post-infection but mature spores only developed after at least 15 days of proliferation within the tubules. [source] Development of a reverse transcription-polymerase chain reaction (RT-PCR) assay for the detection of Amasya cherry diseasePLANT PATHOLOGY, Issue 6 2007Z. Kozlakidis A reverse transcription-polymerase chain reaction (RT-PCR) procedure was developed to detect Amasya cherry disease (ACD) in naturally infected sweet cherry (Prunus avium) leaves sampled from Turkey. The procedure was based on detection of the presence of a mycoviral-like double-stranded RNA (dsRNA) of 5·3 kbp always found in association with ACD, which is probably caused by a fungus. Specific primers were designed to amplify a fragment of the diagnostic dsRNA. The method will improve routine diagnosis of ACD in Prunus spp. [source] A case of mucosal leishmaniasis: beneficial usage of polymerase chain reaction for diagnosisINTERNATIONAL JOURNAL OF DERMATOLOGY, Issue 12 2001Hironori Onuma MD A 36-year-old woman, who had emigrated from Japan to Paraguay as a 4-year-old child before returning to Japan in 1991, visited our clinic on November 10, 1997. She had suffered from a persistent ulcer on her forearm as a 6-year-old child and received intravenous injections for a few months, although she did not remember the details of therapy. Since May 1997, she had been aware of redness and swelling on her nose and had been treated with topical corticosteroid, but no improvement had been noted. Physical examination revealed erythematous plaque with crust from the left internal naris to nasolabial region (Fig. 1a). The atrophic plaque that had resulted from prolonged ulceration was found on the right forearm (Fig. 1b). In a biopsy specimen from the erythematous plaque on the nasolabial region, mononuclear dermal infiltrate, consisting of lymphocytes and histiocytes, was seen (Fig. 2a). The histiocytes were filled with Leishman-Donovan (L-D) bodies on a Giemsa staining sample (Fig. 2b). Fiberscopic examination revealed white plaque in the pharynx. The biopsy from the affected mucosa showed the same histopathological finding as with the skin. Figure 1. (a) Erythematous plaque with crust from the left internal naris to nasolabial region. (b) Atrophic plaque on the right forearm Figure 2. (a) In the biopsy specimen from the erythematous plaque on the nasolabial region, a mononuclear dermal infiltrate consisting of lymphocytes and histiocytes was seen. (Hematoxylin-Eosin stain, × 100) (b) The histiocytes were filled with Leishman-Donovan bodies. (Giemsa staining, × 400) Total DNA was purified from the skin biopsy specimen for polymerase chain reaction (PCR) analysis using a specific primer for L (V) braziliensis.1,2 A 70-bp product was amplified (Fig. 3a); furthermore, the specificity of the PCR product was confirmed by Southern hybridization with the probe for L (V) braziliensis (Fig. 3b) and DNA sequence analysis (data not shown). From December 2, 1997, the patient received 20 mg/kg/day sodium stibogluconate (PentostamTM) intravenously for 20 days. After 5 days of treatment, the redness and swelling of the skin lesion was improved, and faint erythema remained at the end of 20 days' treatment. After a 2-week interval, since the erythema remained, another 20-day treatment was performed. All of the skin lesion became scar tissue and L-D bodies could not be found in a skin biopsy specimen. However, L-D bodies were still found in a biopsy from the pharyngeal mucosa that had a normal appearance. Though another additional treatment was planned, the patient refused it. Figure 3. (a) The results of PCR. 70-bps bands appear in lanes 2 and 6. Lane 1, a size marker (pUC19/HapII); lane 2, DNA extracted from the formalin-fixed patient's sample; lane 3, DNA extracted from a formalin-fixed control sample; lane 4, DNA (,); lane 5, DNA extracted from L (V) tropica; lane 6, DNA extracted from L (V) braziliensis. (b) Results of Southern blotting using the PCR products. The PCR products were transferred from agarose gel as shown in Fig. 3 (a). Specific probes were hybridized with 70-bps bands on lanes 2 and 6 [source] Detection of chimerism following vascularized bone allotransplantation by polymerase chain reaction using a Y-chromosome specific primerJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 6 2003Keiichi Muramatsu Abstract Chimerism following allogeneic organ transplantation is a phenomenon known to occur and be associated with development of immunologic tolerance in allotransplantation. However, little is known about graft cell migration following vascularized bone allografting. In this study, chimerism was assessed following vascularized tibia transplantation from male DA or PVG donors to female PVG rat recipients using a semi-quantitative polymerase chain reaction for the Y-chromosome. FK-506 (Tacrolimus) was administered after transplantation for immunosuppression. All immunosuppresssed PVG rat recipients of PVG bone grafts showed a high level of chimerism (1%) in the thymus, spleen, liver and cervical lymph nodes at 18 weeks post-transplant. Donor cells were also detected in the contralateral tibia and humerus. In non-immunosuppressed PVG rat recipients of DA bone grafts, donor cells were detected in the spleen in three of five rats within 2 weeks post-transplant. In these animals the bone grafts were severely rejected. In immunosuppressed PVG rat recipients of DA bone grafts, two of five, four of eight and eight of 10 rats showed low level chimerism (0.1%) in peripheral blood at 1, 12, and 18 weeks post-transplant. Six rats showed a high level of chimerism in the spleen and thymus. Histological studies revealed no rejection findings through 18 weeks post-transplant. Our results indicate that chimerism, or the presence of graft cells in host tissue, may occur in the face of acute rejection and be demonstrable following vascularized isograft and allograft living bone transplantation when chronic immunosuppression is maintained. Graft vascular patency during the short-term likely allows cellular migration, even in the face of acute rejection. Long-term survival and proliferation of graft marrow elements in host tissue may be possible with adequate immunosuppression. © 2003 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved. [source] Use of polymerase chain reaction to detect the soft rot pathogen, Pythium myriotylum, in infected ginger rhizomesLETTERS IN APPLIED MICROBIOLOGY, Issue 2 2003P.H. Wang Abstract Aims: The aims are to establish a polymerase chain reaction (PCR)-based method for detecting Pythium myriotylum in the rhizome of ginger and diagnosing ginger soft rot and screening health seed ginger. Methods and Results: A booster PCR method was established for detection of P. myriotylum using a specific primer selected from rDNA ITS1 region coupled with universal primer ITS2. It successfully applied to the detection of P. myriotylum in naturally infected ginger rhizomes but not from DNA of ginger rhizomes collected from field without target fungus. Conclusions: A specific method for detecting P. myriotylum was achieved. Significance and Impact of the Study: The new PCR method has allowed us to monitor ginger for the presence of P. myriotylum as a way of disease diagnosis or healthy seed ginger examination. [source] Selective amplification of bacterial RNA: use of a DNA primer containing mismatched bases near its 3, terminus to reduce false-positive signalsLETTERS IN APPLIED MICROBIOLOGY, Issue 3 2000K. Koo A reverse transcription PCR (RT,PCR) method designed to reduce false-positive results due to the co-amplification of contaminating genomic DNA is reported. Feasibility of the method was evaluated using 16S rRNA sequences specific to Bacillus cereus. A DNA oligonucleotide primer, consisting of 22-bases containing three consecutive mismatched bases near its 3, terminus (primer B16RT), was used for reverse transcription and in subsequent cDNA amplification. Specific rRNA was reverse transcribed at low temperature (40 °C or 45 °C) in the presence of primer B16RT. As subsequent PCR using primer B16RT at high (62 °C) annealing temperatures is not nearly as efficient as amplification using the specific primer, amplification of genomic DNA was hindered relative to the amplification of cDNA. The method was readily adapted to the selective amplification of mRNA of the Listeria monocytogenes listeriolysin O (hly) gene. [source] The natural furanone (5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone disrupts quorum sensing-regulated gene expression in Vibrio harveyi by decreasing the DNA-binding activity of the transcriptional regulator protein luxRENVIRONMENTAL MICROBIOLOGY, Issue 10 2007Tom Defoirdt Summary This study aimed at getting a deeper insight in the molecular mechanism by which the natural furanone (5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone disrupts quorum sensing in Vibrio harveyi. Bioluminescence experiments with signal molecule receptor double mutants revealed that the furanone blocks all three channels of the V. harveyi quorum sensing system. In further experiments using mutants with mutations in the quorum sensing signal transduction pathway, the compound was found to block quorum sensing-regulated bioluminescence by interacting with a component located downstream of the Hfq protein. Furthermore, reverse transcriptase real-time polymerase chain reaction with specific primers showed that there was no effect of the furanone on luxRVh mRNA levels in wild-type V. harveyi cells. In contrast, mobility shift assays showed that in the presence of the furanone, significantly lower levels of the LuxRVh response regulator protein were able to bind to its target promoter sequences in wild-type V. harveyi. Finally, tests with purified LuxRVh protein also showed less shifts with furanone-treated LuxRVh, whereas the LuxRVh concentration was found not to be altered by the furanone (as determined by SDS-PAGE). Therefore, our data indicate that the furanone blocks quorum sensing in V. harveyi by rendering the quorum sensing master regulator protein LuxRVh unable to bind to the promoter sequences of quorum sensing-regulated genes. [source] Characterization of the PCR inhibitory effect of bile to optimize real-time PCR detection of Helicobacter speciesFEMS IMMUNOLOGY & MEDICAL MICROBIOLOGY, Issue 2 2005Waleed Abu Al-Soud Abstract The inhibitory effect of human and porcine bile samples to detect Helicobacter DNA was studied by adding different concentrations of bile samples to PCR mixtures of six thermostable DNA polymerases containing cagA specific primers and Helicobacter pylori DNA. PCR products were amplified by using the Rotorgene system and SYBR Green I. Among the six DNA polymerases tested, rTth had the lowest sensitivity to bile inhibitors, whereas Taq and Tfl had the highest sensitivity. Bile proteins did not inhibit AmpliTaq DNA polymerase, whereas the fraction containing mainly bile acids and their salts inhibited the amplification capacity of AmpliTaq. Heating human bile at 98 °C and adding casein and formamide to the reaction mixture reduced the PCR inhibitory effect of bile. Therefore, a pre-PCR treatment based on dilution and heating of bile, adding casein and formamide to the reaction mixture of rTth DNA polymerase was found efficient to amplify DNA directly in bile. [source] Ecology and characterization of polyhydroxyalkanoate-producing microorganisms on and in plantsFEMS MICROBIOLOGY ECOLOGY, Issue 1 2009Ilona Gasser Abstract Polyhydroxyalkanoates are energy reserve polymers produced by bacteria to survive periods of starvation in natural habitats. Little is known about the ecology of polyhydroxyalkanoate-producing bacteria. To analyse the occurrence of this specific group on/in seven different plant species, a combined strategy containing culture-dependent and -independent methods was applied. Using microbial fingerprint techniques (single-strand conformation polymorphism analysis with specific primers for phaC gene encoding the key enzyme of the polyhydroxyalkanoate synthesis), a high number of bands were especially found for the rhizosphere. Furthermore, cluster analysis revealed plant species-specific communities. Isolation of bacteria, recognition of brightly refractile cytoplasmatic inclusions, lipophilic stainings and a PCR strategy targeted on the phaC gene were used as a culture-dependent strategy for the detection of polyhydroxyalkanoate-producing bacteria. Results again represent a high degree of plant specificity: the rhizosphere of sugar beet contained the highest number of positive strains. This was confirmed by quantitative PCR: the relative copy number of phaC was statistically and significantly enhanced in all rhizospheres in comparison with bulk soil. New polyhydroxyalkanoate-producing bacterial species were detected: for example, Burkholderia terricola, Lysobacter gummosus, Pseudomonas extremaustralis, Pseudomonas brassicacearum and Pseudomonas orientalis. Our results confirm the hypothesis that the rhizosphere is an interesting hidden reservoir for polyhydroxyalkanoate producers. [source] Loop-mediated isothermal amplification targeting the apxIVA gene for detection of Actinobacillus pleuropneumoniaeFEMS MICROBIOLOGY LETTERS, Issue 1 2009Wang Yang Abstract Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method performed under isothermal conditions with high specificity and efficiency. We developed a diagnostic method based on LAMP for detection of Actinobacillus pleuropneumoniae. Using six specific primers targeting the apxIVA gene, the LAMP assay rapidly amplified the target gene within 30 min, requiring only a laboratory water bath for the reaction to occur. The resulting amplificon was visualized by adding SYBR Green I to the mixture. The results obtained from testing 15 A. pleuropneumoniae reference strains and other seven bacterial species strains showed that the LAMP was as specific as and 10 times more sensitive than nested PCR. Sixty-five tonsil samples were collected from 65 healthy pigs. All the samples were negative for A. pleuropneumoniae by immunomagnetic separation-based (IMS) bacterial isolation, nested PCR and LAMP, respectively. Meanwhile, 115 tonsil samples were also collected from 115 pigs with apparent respiratory problems. Twenty-two were positive by IMS bacterial isolation. All the samples that were positive by IMS bacterial isolation were also positive by nested PCR and LAMP. The LAMP assay demonstrated exceptionally higher sensitivity than nested PCR by picking up 14 additional positive cases (,2 test, P<0.0001); we concluded that LAMP was a highly sensitive and reliable method for detection of A. pleuropneumoniae infection. [source] Comparative study and molecular characterization of ectomycorrhizas in Tilia americana and Quercus pubescens with Tuber brumaleFEMS MICROBIOLOGY LETTERS, Issue 1 2002G Giomaro Abstract Mycorrhizas of Tuber brumale on Quercus pubescens and Tilia americana were obtained in vitro using micropropagated plantlets. Mycelium pure cultures were used for inoculation. Both the mycelium used for the inoculations, as well as the mycorrhizas which were obtained, were identified using several molecular approaches: analysis of the ITS region, polymerase chain reaction (PCR) specific primers and sequencing. The mycorrhizas were described from a morphological standpoint. Some of their biometric characteristics were different in bass-wood than they were in oak, thus showing the influence of the host plant on several of the morphological features believed to be necessary for the identification of the species. Considering the variability of their morphological characteristics, molecular analysis proved to be a necessary tool for the recognition of the mycorrhizas of Tuber spp. [source] Identification of a potential "hotspot" DNA region in the RUNX1 gene targeted by mitoxantrone in therapy-related acute myeloid leukemia with t(16;21) translocationGENES, CHROMOSOMES AND CANCER, Issue 3 2009Tiziana Ottone The translocation t(16;21) involving RUNX1 (AML1) and resulting in the RUNX1-CBFA2T3 fusion is a rare but recurrent abnormality mostly found in therapy-related acute myeloid leukemia (t-AML) associated with agents targeting topoisomerase II (topo II). We characterized, at the genomic level, the t(16;21) translocation in a patient who developed t-AML after treatment of multiple sclerosis with mitoxantrone (MTZ). Long template nested PCR of genomic DNA followed by direct sequencing enabled the localization of RUNX1 and CBFA2T3 (ETO2) breakpoints in introns 5 and 3, respectively. Sequencing of the cDNA with specific primers showed the presence of the expected RUNX1-CBFA2T3 fusion transcript in leukemic cells. The RUNX1 intron 5 breakpoint was located at nucleotide position 24,785. This region contained an ATGCCCCAG nucleotide sequence showing ,90% homology to a "hotspot" DNA region ATGCCCTAG present in intron 6 of PML previously identified in therapy-related acute promyelocytic leukemia cases arising following treatment with MTZ. This study suggests a wider distribution in the human genome, and particularly at genes involved in chromosome translocations observed in t-AML, of DNA regions (hotspot) targeted by specific topo II drugs. © 2008 Wiley-Liss, Inc. [source] Detection of Helicobacter pylori DNA by a Simple Stool PCR Method in Adult Dyspeptic PatientsHELICOBACTER, Issue 4 2005Nazime ABSTRACT Introduction.,Helicobacter pylori is the major agent causing peptic ulcer, gastric cancer and mucosa-associated lymphoid tissue (MALT) gastric lymphoma. A simple stool polymerase chain reaction (PCR) method was performed and compared with the gold standards for the diagnosis of H. pylori infection. Material and methods., A total of 54 adult patients (mean age, 46.41 ± 13.12 years) with dyspeptic symptoms from Gastroenterology at Dokuz Eylül University Hospital between May and November 2003 were included. Two antrum and corpus biopsies were taken from each patient. Infection by H. pylori was defined as positivity and negativity of the gold standards. DNA extraction of stool specimens was done using QIAamp DNA Stool Mini Kit (QIAGEN) and PCR conditions included amplification and reamplification steps using the H. pylori ureA gene specific primers (HPU1, HPU2) and were visualized on 1% agarose gel stained with ethidium bromide. Results., Forty-six of 54 patients (85.2%) were diagnosed positive and eight (14.8%) were negative for H. pylori infection by the gold standard methods. Thirty-two patients were positive (59.3%) and 22 of them (40.7%) were detected negative by stool PCR method. The stool PCR method and gold standard methods showed a statistical difference for the detection of H. pylori infection (p < .0001). Sensitivity, specificity, likelihood ratio, and positive and negative predictive values were 65.22%, 75%, 2.61%, 93.75%, and 27.7%, respectively. Discussion., The PCR on the stool specimens resulted as being a very specific test. We suggest that a simple stool PCR method that we developed can be used to detect H. pylori, virulence genes, and in drug resistance studies either first line diagnostic methods in the laboratory or in the clinical management of dyspeptic patients. [source] PCR-BASED TECHNIQUE FOR IDENTIFICATION AND DETECTION OF TRICHOGRAMMA SPP. (HYMENOPTERA: TRICHOGRAMMATIDAE) WITH SPECIFIC PRIMERSINSECT SCIENCE, Issue 3 2002LI Zheng-xi Abstract The rDNA-ITS2 regions of T. dendrolimi Matsumura and T. ostriniae Pang et Chen (Hymenoptera: Trichogrammatidae) were cloned and sequenced. The homologous sequences available in GenBank were retrieved and analyzed, and then specific primers were designed for molecular identification and detection of T. dendrolimi. Repeated screening showed that PCR amplification by the diagnostic primers enabled the differentiation of not only bulk samples and single adult (male or female), but also eggs and juveniles, which was not possible by conventional methods. The advantage of this system over morphology-based systems is that non-specialists are able to identify individuals or trace specimens efficiently. The derived molecular detection technique was then used to identify 12 specimens collected from different localities on the Chinese mainland; the results showed that this protocol could be applied to molecular monitoring of Trichogramma species in the field. Finally, 1132s of 6 geographical populations of T. dendrolimi (TdCHA, TDJL, TdXZ, TdKH, TdCZ and TdYBL) were cloned and sequenced. The multialignment analysis of intraspecific ITS2 sequences showed that the diagnostic primers have their own theoretical bases. [source] Highly sensitive detection of the MGB1 transcript (mammaglobin) in the peripheral blood of breast cancer patientsINTERNATIONAL JOURNAL OF CANCER, Issue 4 2004Nuno Cerveira Abstract We describe a new one-step RT-PCR assay for the detection of the mammaglobin (MGB1) gene transcript in the peripheral blood of breast cancer patients. With this approach, the MGB1 transcript could be detected in the peripheral blood of 22 of 54 (41%) breast cancer patients prior to any therapy. This method, using specific primers for cDNA synthesis, proved to be more sensitive (10,6 to 10,11, usually 10,7) than previously reported methodologies. This increased sensitivity was achieved without compromising specificity, as the MGB1 transcript was not detected in 38 blood samples of healthy donors and in only 1 of 18 blood samples of patients presenting with hematologic malignancies. A positive correlation was seen between MGB1 positivity and breast cancer stage: 0/3 (0%) in stage 0, 3/13 (23%) in stage I, 6/17 (35%) in stage II, 5/10 (50%) in stage III, 8/11 (73%) in stage IV (p = 0.003). The prognostic and therapeutic implications of MGB1 positivity by one-step RT-PCR in the peripheral blood of breast cancer patients, especially in clinically localized disease (stages I and II), should be evaluated after long-term clinical follow-up of these patients. © 2003 Wiley-Liss, Inc. [source] Development and evaluation of a one-step loop-mediated isothermal amplification for detection of spring viraemia of carp virusJOURNAL OF APPLIED MICROBIOLOGY, Issue 4 2008Z. Liu Abstract Aim:, Spring viraemia of carp virus (SVCV) is the causative agent of SVC disease. The main aim of our study was to develop a one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for rapid, sensitive and effective detection of SVCV. Methods and Results:, A set of four specific primers, two outer and two inner primers were designed based on the SVCV M gene for RT-LAMP assay. The sensitivity and specificity of RT-LAMP were determined and clinical test was performed under optimized amplification conditions (64°C, 60 min). The results showed that the assay has a high specificity and the detection limit was 80 copies using 10-fold series dilutions of SVCV RNA, 10 times more sensitive than nest reverse transcription-polymerase chain reaction. In the detection of 472 fish samples, this assay showed excellent agreement with the standard virus isolation method (, = 0·807). Conclusions:, A sensitive and specific RT-LAMP assay was successfully developed to monitor and detect SVCV. Significance and Impact of the Study:, This work provides a robust method for evaluating the risk of SVCV. Given the advantages of LAMP in the detection of SVCV, this method can be applied to diagnose other viruses, which pose serious threats to the aquaculture industry. [source] Preliminary characterization of lactic acid bacteria isolated from Zlatar cheeseJOURNAL OF APPLIED MICROBIOLOGY, Issue 6 2007K. Veljovic Abstract Aims:, Isolation, characterization and identification of lactic acid bacteria (LAB) from artisanal Zlatar cheese during the ripening process and selection of strains with good technological characteristics. Methods and Results:, Characterization of LAB was performed based on morphological, physiological and biochemical assays, as well as, by determining proteolytic activity and plasmid profile. rep-polymerase chain reaction (PCR) analysis and 16S rDNA sequencing were used for the identification of LAB. PCR analysis was performed with specific primers for detection of the gene encoding nisin production. Strains Lactobacillus paracasei subsp. paracasei, Lactobacillus plantarum, Lactobacillus brevis, Lactococcus lactis subsp. lactis, Enterococcus faecium and Enterococcus faecalis were the main groups present in the Zlatar cheese during ripening. Conclusions:, Temporal changes in the species were observed during the Zlatar cheese ripening. Mesophilic lactobacilli are predominant microflora in Zlatar cheese. Significance and Impact of the Study:, In this study we determined that Zlatar cheese up to 30 days old could be used as a source of strains for the preparation of potential starter cultures in the process of industrial cheese production. As the Serbian food market is adjusting to European Union regulations, the standardization of Zlatar cheese production by using starter culture(s) based on autochtonous well-characterized LAB will enable the industrial production of this popular cheese in the future. [source] Rapid and effective detection of anthrax spores in soil by PCRJOURNAL OF APPLIED MICROBIOLOGY, Issue 4 2003H.I. Cheun Abstract Aims: To detect Bacillus anthracis DNA from soil using rapid and simple procedures. Methods and Results: Various amounts of B. anthracis Pasteur II spores were added artificially to 1 g of soil, which was then washed with ethanol and sterile water. Enrichment of the samples in trypticase soy broth was performed twice. A DNA template was prepared from the second enrichment culture using a FastPrep instrument. The template was then used for nested and real-time polymerase chain reaction (PCR) with B. anthracis -specific primers, to confirm the presence of B. anthracis chromosomal DNA and the pXO1/pXO2 plasmids. Conclusions: One cell of B. anthracis in 1 g of soil could be detected by nested and real-time PCR. The usefulness of the PCR method using field samples was also confirmed. Significance and Impact of the Study: The results indicate that this could be a useful method for detecting anthrax-spore contaminated soil with high sensitivity. Its application could have great impact on the progress of epidemiological surveillance. [source] Screening for soluble methane monooxygenase in methanotrophic bacteria using combined molecular and biochemical methods for hydroxylase detectionJOURNAL OF BASIC MICROBIOLOGY, Issue 1 2003Stephan Grosse Dr. Three well known methanotrophic bacteria (Methylosinus trichosporium OB3b, Methylocystis sp. WI 14, and Methylocystis sp. GB 25) and three newly isolated methanotrophic bacteria (Methylocystis sp. WI 11, Methylocystis sp. X, and FI-9) were screened for sMMO considering the existence of hydroxylase (component A) genes as well as its gene expression. For these purposes monoclonal antibodies that specifically recognize each subunit of the hydroxylase of Methylocystis sp. WI 14 (, -subunit [9E5/F2], , -subunit [4E2/G11], , -subunit [10G3/D7]) were produced. PCR amplification using well known primers showed that the hydroxylase encoding genes appear to be only present in M. trichosporium OB3b, Methylocystis sp. WI 11 and WI 14, and in the isolate FI-9. Western and ELISA analysis using the monoclonal antibodies revealed that all subunits of hydroxylase were present. However, in FI-9, only the , -subunit of the hydroxylase might be expressed. Surprisingly, in Methylocystis sp. GB 25, where no sMMO activity and no amplification with sMMO specific primers was obtained, the antibody 4E2/G11 recognized a protein band with exactly the same molecular mass as the , -subunit of the hydroxylase. Methylocystis sp. X showed no positive reaction in any of the tests. In combination with the detection methods currently used, the described antibodies provide a powerful tool for detecting even partially expressed hydroxylase genes. [source] CDK4 IVS4-nt40G,A and T2D-associated obesity in ItaliansJOURNAL OF CELLULAR PHYSIOLOGY, Issue 2 2009Ramachandran Meenakshisundaram Cell cycle regulators play crucial roles in the preadipocyte proliferation and adipocyte differentiation. Cyclin-dependent kinase 4 (CDK4) mediates with D-type cyclins entry of cells into cell cycle in response to external stimuli. CDK4 plays a role in body weight, adipogenesis, and beta cell proliferation. CDK4 null mice develop type 2 diabetes (T2D). Furthermore, CDK4 variants are associated with obesity-associated tumors/cancer. We aimed at identifying a role of CDK4 IVS4-nt40G,,,A variant in T2D-associated obesity (body mass index, BMI,,,30) by association tests in an Italian T2D subjects dataset. We recruited from Italy 128 unrelated T2D subjects with BMI,<30,kg/m2 and 54 unrelated T2D subjects with BMI,,,30,kg/m2. We performed statistical power calculations in our dataset. DNA samples were directly sequenced with specific primers for CDK4 IVS4-nt40G,,,A variant. We identified a significant association of the G allele with T2D-associated obesity and of the A allele with T2D-associated BMI,<,30. In our study, we found that the CDK4 IVS4-nt40GG genotype is a risk variant for T2D-associated obesity and that the AA genotype is associated with BMI,<,30 in T2D. Hence, CDK4 IVS4-nt40A allele is protective and G allele confers risk for obesity in T2D patients. This study should prompt further work aiming at establishing CDK4 role in contributing to human obesity and T2D-associated obesity. J. Cell. Physiol. 221: 273,275, 2009. © 2009 Wiley-Liss, Inc. [source] OCTN3: A Na+ -independent L -carnitine transporter in enterocytes basolateral membraneJOURNAL OF CELLULAR PHYSIOLOGY, Issue 3 2005J.M. Durán L -carnitine transport has been measured in enterocytes and basolateral membrane vesicles (BLMV) isolated from chicken intestinal epithelia. In the nominally Na+ -free conditions chicken enterocytes take up L -carnitine until the cell to medium L -carnitine ratio is 1. This uptake was inhibited by L -carnitine, D -carnitine, ,-butyrobetaine, acetylcarnitine, tetraethylammonium (TEA), and betaine. L - 3H-carnitine uptake into BLMV showed no overshoot, and it was (i) Na+ -independent, (ii) trans-stimulated by intravesicular L -carnitine, and (iii) cis-inhibited by TEA and cold L -carnitine. L - 3H-carnitine efflux from L - 3H-carnitine preloaded enterocytes was also Na+ -independent, and trans-stimulated by L -carnitine, D -carnitine, ,-butyrobetaine, acetylcarnitine, TEA, and betaine. Both, uptake and efflux of L -carnitine were inhibited by verapamil and unaffected by either extracellular pH or palmitoyl- L -carnitine. RT-PCR with specific primers for the mouse OCTN3 transporter revealed the existence of OCTN3 mRNA in mouse intestine, which was confirmed by in situ hybridization studies. Immunohystochemical analysis showed that OCTN3 protein was mainly associated with the basolateral membrane of rat and chicken enterocytes, whereas OCTN2 was detected at the apical membrane. In conclusion, the results demonstrate for the first time that (i) mammalian small intestine expresses OCTN3 mRNA along the villus and (ii) that OCTN3 protein is located in the basolateral membrane. They also suggest that OCTN3 could mediate the passive, Na+ and pH-independent L -carnitine transport activity measured in the three experimental conditions. © 2004 Wiley-Liss, Inc. [source] Prevalence of Actinobacillus actinomycetemcomitans in an ethnic adult Chinese populationJOURNAL OF CLINICAL PERIODONTOLOGY, Issue 9 2001Kai Soo Tan Abstract Aim: The aim of this study was to determine the prevalence and the structure of the leukotoxin promoter region of Actinobacillus actinomycetemcomitans in an ethnic Chinese population. Method: Subgingival plaque samples were collected from 42 patients with moderate to advanced periodontitis and 50 periodontally healthy patients. A. actinomycetemcomitans was detected directly from the crude subgingival plaque by PCR using leukotoxin gene specific primers. The presence of A. actinomycetemcomitans was determined by a single 285 bp PCR amplicon. Results:A. actinomycetemcomitans was found to be present in the subgingival plaque of 68 out of a total of 92 patients examined (74%). 29 out of the 42 periodontitis patients tested were carriers of A. actinomycetemcomitans (69%). Among the periodontally healthy patients studied, 39 out of 50 subjects possessed the bacteria (78%). PCR analysis of the promoter region of the ltx operon revealed that none of the 42 moderate to advanced periodontitis patients examined harboured A.actinomycetemcomitans strains with the JP2-like promoter of the ltx operon, known to enhance leukotoxin expression. 2 out of the 27 advanced periodontitis patients clinically diagnosed as suffering from rapidly progressive periodontitis were found to be carriers of the mildly toxic strain of A. actinomycetemcomitans with the characteristic 652-like promoter. Conclusions: The high prevalence of A. actinomycetemcomitans, regardless of whether the subgingival samples were analysed from patients with healthy or diseased periodontium suggests that this bacterial species is part of the normal oral flora of ethnic Chinese. Our preliminary results also suggested that subjects who harboured the mildly toxic strain of A. actinomycetemcomitans were potentially susceptible to aggressive forms of periodontitis. Zusammenfassung Das Ziel dieser Studie war es, in einer ethnischen Population von Chinesen die Prävalenz von Actinobacillus actinomycetemcomitans und die Struktur der Leukotoxin-Promoterregion zu bestimmen. Von 42 Patienten mit moderater bis fortgeschrittener Parodontitis und 50 parodontal gesunden Patienten wurden subgingivale Plaqueproben entnommen. A. actinomycetemcomitans wurde direkt in der unbehandelten subgingivalen Plaque durch PCR unter Verwendung eines Leukotoxingen-spezifischen Primers nachgewiesen. Das Vorhandensein von A. actinomycetemcomitans wurde mittels eines einzigen 285 bp-PCR-Amplikons bestimmt. Es wurde A. actinomycetemcomitans bei 68 von 92 untersuchten Patienten (74%) vorgefunden. 29 von 42 getesteten Parodontitispatienten waren Träger von A. actinomycetemcomitans (69%). Unter den Studierten parodontal gesunden Patienten besaßen 39 von 50 Personen das Bakterium (78%). Die PCR-Analyse der Promoterregion des ltx -Operons zeigte, dass keiner der 42 untersuchten Patienten mit moderater bis fortgeschrittener Parodontitis den A. actinomycetemcomitans mit dem JP2-ähnlichen Promoter des ltx -Operons, welches die Leukotoxinexpression verstärkt, besaß. Bei 2 der 27 Patienten mit fortgeschrittener Parodontitis wurde klinisch eine rasch fortschreitende Parodontitis diagnostiziert und es wurde der mit geringer Toxizität versehene Stamm des A. actinomycetemcomitans mit dem charakteristischen 652-ähnlichen Promoter vorgefunden. Bedingt durch die hohe Prävalenz von A. actinomycetemcomitans unabhängig davon, ob die Proben von Patienten mit gesundem oder erkranktem Parodontium stammen, lässt sich annehmen, dass diese Bakterienspezies bei ethnischen Chinesen ein Teil normalen Mundflora ist. Unsere vorläufigen Resultate lassen auch annehmen, dass Personen, die den mit geringer Toxizität versehenen Stamm des A. actinomycetemcomitans tragen eine potentielle Anfälligkeit für aggressive Formen der Parodontitis besitzen. Résumé Le but de l'étude présente a été de déterminer la fréquence globale et la structure de la région promoteur de leukotoxine de l'Actinobacillus actinomycetemcomitans (A.a.) dans une population chinoise. Des échantillons de plaque dentaire sous-gingivale ont été prélevés chez 42 patients avec parodontite modérée à avancée et chez 50 patients sains. L'A.a. a été détecté directement dans la plaque sous-gingivale par PCR en utilisant les sites spécifiques de gènes leukotoxines. La présence de l'A.a. a été déterminée par un amplicon PCR de 285 bp. L'A.a. a été décelé dans la plaque sous-gingivale de 68 des 92 patients examinés (74%). 29 des 42 patients avec parodontite ont été reconnus comme porteurs d'A.a. (69%). Parmi les patients sains étudiés, 39 des 50 sujets étaient porteurs de la bactérie (78%). L'analyse PCR de la région promoteur de operon ltx a révélé que des 42 patients avec parodontite modéréà avancée aucun n'avaient de souche A.a. avec le promoteur ressemblant au JP2 de l'operon ltx, reconnu pour acroître la leukotoxine. 2 des 27 patients avec parodontit avancée souffraient d'une parodontite progressant rapidement et étaient porteurs d'une souche moyennement toxique d'A.a. avec la caractéristique du promoteur ressemblant au 652. La fréquence globale importante d'A.a., sans tenir compte si les échantillons sous-gingivaux ont été analysés de patients avec un parodonte sain ou malade, suggère que ces espèces bactériennes font partie de la flore buccale normale de l'ethnie chinoise. Ces résultats indiquent également que les porteurs de la souche peu toxique d'A.a. seraient potentiellement susceptibles à des formes de parodontite agressive. [source] ,HPV vulvitis' revisited: frequent and persistent detection of novel epidermodysplasia verruciformis-associated HPV genotypesJOURNAL OF CUTANEOUS PATHOLOGY, Issue 3 2008Ming-Tseh Lin Background:, ,Human papillomavirus (HPV) vulvitis' is a disputed entity where most studies examining for genital-mucosal (GM) HPV have been negative. Methods:, Using degenerate and type specific primers for cutaneous (CU), GM and epidermodysplasia verruciformis (EV) HPV types, the prevalence of specific HPV types was investigated in biopsy specimens from 19 women with ,HPV vulvitis', seven with asymptomatic vulvar squamous papillomatosis (ASxVSP), and controls of vulvar fibroepithelial polyps (FEP) (15), vulvar condyloma (10) and normal vulva (NV) (10). Results:, HPV DNA/EV HPV/GM HPV/CU HPV were detected in 84/74/47/5% of vulvitis patients, 78/71/0/28% of ASxVSP, 47/20/20/7% of FEP, 10/10%/0/0 of NV and 100/0/100/10% of condyloma. Fourteen putatively novel HPV genotypes were detected in vulvitis and ASxVSP patients, but not in controls. The two most frequent novel EV HPV, Alb-4 and DL285, were detected in 9/19 (47%) and 5/19 (26%) of vulvitis cases and were persistently identified in serial biopsies. HPV co-infection and Alb-4 infection occurred significantly more frequently in vulvitis patients, particularly those complaining of ,burning' (62/62% vs. 17/7%, p , 0.004). Koilocytosis was identified significantly more frequently in vulvitis compared with non-condyloma controls (81% vs. 40%, p = 0.0001), and its presence correlated with detection of HPV DNA (r = 0.3, p = 0.002). Conclusion:, The high frequency of novel EV HPV in HPV vulvitis and correlation of clinicopathologic findings with HPV DNA suggests that HPV vulvitis may indeed exist. [source] Cell traffic between donor and recipient following rat limb allograftJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 1 2005Keiichi Muramatsu Abstract Although cell traffic from the graft into the recipient and from the recipient into the graft had been noticed in allogeneic organ transplantation, little is known following whole-limb allografting. This study was conducted to define cell migration between donor and recipient. Sixty-seven vascularized hind limb allotransplantations were performed in rat sex-mismatched pairs and the recipient animals were treated with FK506 immunosuppression. The ratio of donor and recipient cells was evaluated by semi-quantitative PCR using the specific primers of the Y-chromosome. Allografted limbs had no rejection episode until the final assessment. The male recipient cells were detected in female limb grafts not at 1 week but at 48 weeks after transplantation. The male donor cells were detected in the humerus and tibia in the female recipient but not in the gastrocnemius muscle and leg skin. Our results demonstrated that recipient-derived cells gradually migrated into the grafted bone, muscle and skin cells with the duration of time. Donor-derived cells migrated into the healthy bones but not into the healthy muscle and skin. Because active regeneration occurs in the grafted limb to compensate graft damage secondary to ischemia and operative intervention, recipient-derived cells may mediate a muscular and dermo-epidermal renewal. © 2004 Orthopaedic Research Society. Published by Elsevier Ltd. All rights reserved. [source] E-selectin and L-selectin polymorphisms in patients with periodontitisJOURNAL OF PERIODONTAL RESEARCH, Issue 1 2009B. Houshmand Background amd Objective:, Periodontitis is a multifactorial disease in which environmental and genetic determinant factors contribute to individual subject's susceptibility. A DNA polymorphism in the regulating region of adhesion molecule genes is suggested to modulate the molecule's physiological effects. The aim of this study was to investigate the genetic association between the E-selectin Ser128Arg and L-selectin Phe206Leu polymorphisms and periodontitis. Material and Methods:, DNA was isolated from the whole blood of 88 patients with periodontitis and 139 healthy individuals. All samples were genotyped for the E-selectin Ser128Arg and L-selectin Phe206Leu polymorphisms using the polymerase chain reaction with sequence specific primers. Results:, Our findings revealed a significant difference in the Ser128Arg polymorphism of E-selectin, but not in the L-selectin polymorphism, between periodontal patients and controls. The 128Arg allele was present more frequently in patients than in healthy individuals (31.25% vs. 12.2%, p < 0.0001). In addition, there was an association between the presence of the 128Arg allele and periodontitis (odds ratio 2.9; 95% confidence interval: 1.75,4.4, p < 0.0001). No significant association was found between the polymorphisms tested and the subgroups of periodontal disease (i.e. chronic periodontitis and aggressive periodontitis). Conclusion:, The findings of this study showed that the Ser128Arg polymorphism of E-selectin might contribute to the susceptibility of Iranian individuals to periodontitis. [source] Up-regulation of the lysyl hydroxylase 2 gene by acetaminophen and isoniazid is modulated by transcription factor c-MybJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 4 2010Masafumi Kubota Abstract Objectives Lysyl hydroxylase 2 (LH2), an isoform of hydroxylase, catalyses the hydroxylation of lysine residues in the telopeptide of collagen to form stable and irreversible cross-linkages in collagen. Increased activity of this enzyme in activated stellate cells in human liver has been proposed to relate to the promotion of hepatic fibrosis. In the present study, we examined the regulation of LH2 expression in drug-induced liver injury in order to clarify the mechanisms behind the hepatic fibrosis caused by certain drugs. Methods The mRNA and protein expression of the target gene were detected by real-time reverse transcription-polymerase chain reaction (RT-PCR) with specific primers and Western blotting with a specific antibody, respectively. Key findings The expression of LH2 was increased in HepG2 cells incubated with acetaminophen and isoniazid. This increase was accompanied by an increase in the expression of c-myeloblastosis viral oncogene homolog (Myb) mRNA. Over-expression of c-Myb in cells transfected with a c-Myb expression plasmid, pMbm I, caused an increase in the expression of LH2 mRNA. Mutation of the Myb-binding site in the promoter region of the LH2 gene resulted in a loss of transcriptional activation in the reporter gene assay. Conclusions These results suggest that c-Myb modulates the expression of the LH2 gene in HepG2 cells incubated with drugs causing hepatic fibrosis [source] Genetic Variability Analysis and Molecular Detection of Fusarium oxysporum f.sp. eustomae Isolated from Eustoma grandiflorum in Northern ItalyJOURNAL OF PHYTOPATHOLOGY, Issue 7-8 2010Yuan Li Abstract A total of 35 isolates of Fusarium oxysporum f.sp. eustomae obtained from diseased Eustoma grandiflorum plants in northern Italy, showing typical Fusarium wilt symptoms, were analysed for their genetic variability and molecular identification. Genetic diversity of the isolates was studied by using random amplified polymorphic DNA (RAPD). This analysis clustered the isolates into three groups at a genetic similarity of 69%. Sequence analysis of RAPD fragments led to the design of a pair of specific primers that amplify a 505-bp SCAR (sequence characterized amplified region) marker (SCAR505) which was used to rapidly detect F. oxysporum f.sp. eustomae on Eustoma grandiflorum plants. In a temperature-controlled chamber, detection of the pathogen by PCR was 100% successful in root and stem samples of infected but still symptomless plants. The diagnostic procedure could be completed in 1 day and allowed rapid and reliable detection of the pathogen in asymptomatic plants in the early stages of disease development. [source] Quantifying Phytophthora medicaginis in Susceptible and Resistant Alfalfa with a Real-Time Fluorescent PCR AssayJOURNAL OF PHYTOPATHOLOGY, Issue 11-12 2003G. J. Vandemark Abstract A real-time fluorescent PCR assay using a set of specific primers and a fluorochrome-labelled probe (TaqMan) was developed to quantify the amount of Phytophthora medicaginis DNA in alfalfa plants that were classified as either resistant or susceptible to the pathogen based on visual assessment of disease response. The assay clearly discriminated among three standard check alfalfa populations with different levels of resistance based on the analysis of DNA extracted from the roots of bulked plant samples. In two independent experiments, the Spearman rank correlation between pathogen DNA content and the number of susceptible plants in a bulked sample was greater than 0.89 and highly significant (P<0.0001). Significantly less pathogen DNA was detected in bulked samples of a highly resistant check population than in bulked samples from more susceptible check populations. Analysis of individual plants indicated that significantly less pathogen DNA was detected in resistant plants than in susceptible plants. Applications of the assay are considered for breeding programs and the study of microbial population dynamics in plants simultaneously infected with different pathogens. [source] Occurrence, Symptom Expression and Characterization of Phytoplasma Associated with Pear Decline Disease in Catalonia (Spain)JOURNAL OF PHYTOPATHOLOGY, Issue 11-12 2003M. Garcia-Chapa Abstract A total area of 1500 ha of commercial plots was surveyed to study the extent of pear decline disease and its relative importance in northeastern Spain. A preliminary evaluation indicated that around 7% of the plots had symptoms of the disease. At the same time, pear decline incidence was evaluated in 45 plots, by visual inspection of 500 trees in each plot. In September, the incidence of trees with symptoms ranged from 8 to 59% depending on the cultivar selected. The presence of pear decline (PD) phytoplasma in these plots was confirmed by polymerase chain reaction (PCR) amplification of phytoplasma DNA with universal or group-specific primers. Restriction fragment length polymorphism analyses also showed the presence of a unique phytoplasma strain. The symptom expression of PD disease in different cultivars was evaluated throughout the year. The relationship between the presence of symptoms and detection of PD by PCR in these cultivars was also studied. Results showed that the nested-PCR, using specific primers to detect the DNA from PD phytoplasma, is the most accurate method to identify the total percentage of affected trees. [source] Detection of Heterobasidion annosum s. l. [(Fr.) Bref.] in Norway Spruce by Polymerase Chain ReactionJOURNAL OF PHYTOPATHOLOGY, Issue 7 2002G. Bahnweg Abstract Internal transcribed spacer (ITS) sequences of the rDNA repeat unit of Heterobasidion annosum were used to design specific primers for the detection and quantification of this important forest pathogen by polymerase chain reaction (PCR). Specificity of detection was cross-checked against a variety of other fungi (saprophytes, root pathogens, mycorrhizal fungi) which may occur in the same environment. As little as 1 pg fungal DNA (equiv. to 10,40 genomes) could be detected in 200 ng spruce root DNA (from 1 mg fresh spruce root). The Heterobasidion -specific primers allowed simultaneous detection of Armillaria spp. in multiplex PCR. The method was successfully applied to increment cores of Norway spruce from the forest region Tharandter Wald (Saxonia, Germany), Oberbärenburg (East Ore Mountains, Saxonia) and Oberschleissheim (north of Munich, Bavaria). [source] | ||||||||||||||||||||||||||||||||||