PCR Amplification (pcr + amplification)

Distribution by Scientific Domains
Distribution within Life Sciences

Kinds of PCR Amplification

  • real-time pcr amplification

  • Terms modified by PCR Amplification

  • pcr amplification products

  • Selected Abstracts


    SPECIFIC DETECTION OF AMANITA PHALLOIDES MYCELIUM AND SPORES BY PCR AMPLIFICATION OF THE GPD (GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE) GENE FRAGMENT

    JOURNAL OF FOOD BIOCHEMISTRY, Issue 3 2000
    OWSKI, ROMAN KOT
    ABSTRACT Oligonucleotide primers designed to flank a 635 bp fragment of the gene encoding glyceraldehyde-3-phosphate dehydrogenase (gpd) from Araanita muscaria were used to amplify the corresponding gpd fragment from Amanita phalloides. The A. phalloides PCR product was cloned, sequenced and found to be 70 - 77% similar to the known basidiomycetes gpd genes within the exon part and 25 - 52% within the intron part. Based on these data, species-specific amplification was achieved using a pair of oligonucleotide primers complementary to the A. phalloides gpd intron sequences. These primers allowed the amplification of the corresponding gpd fragment from the A. phalloides but not from various other basidiomycetes, ascomycetes and human matrices. PCR amplification of the A. phalloides DNA gave the predicted PCR product of 284 bp. The created PCR system is an efficient tool for the specific, rapid and sensitive detection of A. phalloides mycelium and spores. [source]


    DNA Extraction from Olive Oil and PCR Amplification of Microsatellite Markers

    JOURNAL OF FOOD SCIENCE, Issue 1 2005
    Raffaele Testolin And
    ABSTRACT: DNA was extracted from single-cultivar of cold-pressed (virgin) unfiltered and cotton-filtered olive oils that were stored at 4 °C for up to a year using different DNA extraction kits and protocols. DNA was amplified using original and nested primers designed on 6 microsatellites loci of the UDO series. The most consistent results in terms of successful single sequence repeat amplifications were achieved using the Qiagen QIAamp DNA stool extraction kit, slightly modified and applied to oil sample amounts as small as 200 ,L without any pretreatment. The kit allowed getting polymerase chain reaction (PCR) amplicons visible on gel and scorable peaks at the automatic sequencer for all 6 markers analyzed. Less consistent results were achieved with other kits, such as the Promega Wizard Magnetic DNA Purification System for Food, the LB Link-Biotech ExtMan 50,100 Evolution, the Qiagen Plant Mini kit, and the standard cetyltrimethyl-ammonium bromide-based DNA extraction protocol. The integration in the protocols of further tools, such as the hexane-based phase separation, the addition of water or NaCl solutions to the oil, the precipitation and the use of the pellet, and others, did not result in any substantial use. PCR amplifications that gave low DNA yields were improved by adopting the nested PCR technique, which uses the product of the 1st PCR as a template for a 2nd PCR carried out by means of internal primers. Conclusions are drawn as to the applicability of the method to trace the identity of single-cultivar virgin olive oils. Further work is required to check the sensitivity of the method in determining the varietal composition of blended oils, especially in detecting alleles from cultivars present in only small amounts. [source]


    Detection and Identification of Brenneria nigrifluens, the Causal Agent of the Shallow Bark Canker of Walnut by, PCR Amplification

    JOURNAL OF PHYTOPATHOLOGY, Issue 7-8 2008
    S. Loreti
    Abstract A 1 kb DNA band from strains of Brenneria nigrifluens, as shown by amplification of their genomic DNA by polymerase chain reaction (PCR) using minisatellite primer designed on the minisatellite sequence of the M13 phage, was isolated, cloned and sequenced. Specific oligonucleotides (F1,C3) were selected into this 1 kb DNA sequence and used in a PCR assay to detect and identify strains of B. nigrifluens. Several strains of B. nigrifluens were assessed with F1,C3 primers producing a specific band of approximately 250 bp pairs in length. This target was successfully amplified from purified genomic DNA, from bacterial culture and directly from infected walnut bark tissue. No amplification was obtained when the PCR assay was performed on other plant-pathogenic species from the following genera Brenneria, Erwinia, Agrobacterium, Pseudomonas, Ralstonia, Pectobacterium, Xanthomonas and from walnut-associated bacteria, indicating the specificity of these primers. The PCR assay with the primers described here provides a rapid, specific and sensitive diagnostic method for B. nigrifluens and a useful tool for epidemiological studies. [source]


    Sequence-related amplified polymorphism, an effective molecular approach for studying genetic variation in Fasciola spp. of human and animal health significance

    ELECTROPHORESIS, Issue 2 2009
    Qiao-Yan Li
    Abstract In the present study, a recently described molecular approach, namely sequence-related amplified polymorphism (SRAP), which preferentially amplifies ORFs, was evaluated for the studies of genetic variation among Fasciola hepatica, Fasciola gigantica and the "intermediate" Fasciola from different host species and geographical locations in mainland China. Five SRAP primer combinations were used to amplify 120 Fasciola samples after ten SRAP primer combinations were evaluated. The number of fragments amplified from Fasciola samples using each primer combination ranged from 12 to 20, with an average of 15 polymorphic bands per primer pair. Fifty-nine main polymorphic bands were observed, ranging in size from 100 to 2000,bp, and SRAP bands specific to F. hepatica or F. gigantica were observed. SRAP fragments common to F. hepatica and the "intermediate" Fasciola, or common to F. gigantica and the "intermediate" Fasciola were identified, excised and confirmed by PCR amplification of genomic DNA using primers designed based on sequences of these SRAP fragments. Based on SRAP profiles, unweighted pair-group method with arithmetic averages clustering algorithm categorized all of the examined representative Fasciola samples into three groups, representing the F. hepatica, the "intermediate" Fasciola, or the F. gigantica. These results demonstrated the usefulness of the SRAP technique for revealing genetic variability between F. hepatica, F. gigantica and the "intermediate" Fasciola, and also provided genomic evidence for the existence of the "intermediate" Fasciola between F. hepatica and F. gigantica. This technique provides an alternative and a useful tool for the genetic characterization and studies of genetic variability in parasites. [source]


    Identification of shrimp species in raw and processed food products by means of a polymerase chain reaction-restriction fragment length polymorphism method targeted to cytochrome b mitochondrial sequences

    ELECTROPHORESIS, Issue 15 2008
    Ananías Pascoal
    Abstract A novel PCR-RFLP method has been developed for the identification of six commercially relevant penaeid shrimp species in raw and processed food products. The method can be completed within 8,h. To implement the method, PCR amplification with the crustF/crustR primers, targeted to the amplification of a ca. 181,bp region of the cytochrome b (cytb) mitochondrial gene in penaeid shrimps, was coupled to restriction analysis with CviJI, DdeI and NlaIV. The method was also applied successfully to the identification of shrimp species in complex processed foods, including this type of shellfish as an added-value food ingredient. The small size of this molecular target facilitates amplification from fresh, frozen, or precooked samples, where DNA fragmentation may be relevant and fragment size critical. We also report the first cytb mitochondrial sequences described to date for the species Farfantepenaeus notialis, Parapenaeus longirostris and Pleoticus muelleri, and these nearly triplicate current knowledge of reference nucleotide sequences in this mitochondrial region for this group of species. The cytb mitochondrial gene may also be considered as a molecular marker for identification and phylogenetic purposes in penaeid shrimp species. [source]


    Molecular analysis of mutations at the HPRT and TK loci of human lymphoblastoid cells after combined treatments with 3,-azido-3,-deoxythymidine and 2,,3,-dideoxyinosine,

    ENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 4 2002
    Quanxin Meng
    Abstract Combinations of antiretroviral drugs that include nucleoside reverse transcriptase inhibitors (NRTIs) are superior to single-agent regimens in treating or preventing HIV infection, but the potential long-term health hazards of these treatments in humans are uncertain. In earlier studies, our group found that coexposure of TK6 human lymphoblastoid cells to 3,-azido-2,,3,-dideoxythymidine (AZT) and 2,,3,-dideoxyinosine (ddI), the first two NRTIs approved by the FDA as antiretroviral drugs, produced multiplicative synergistic enhancement of DNA incorporation of AZT and mutagenic responses in both the HPRT and TK reporter genes, as compared with single-drug exposures (Meng Q et al. [2000a]: Proc Natl Acad Sci USA 97:12667,12671). The purpose of the current study was to characterize the mutational specificity of equimolar mixtures of 100 ,M or 300 ,M AZT + ddI at the HPRT and TK loci of exposed cells vs. unexposed control cells, and to compare the resulting mutational spectra data to those previously found in cells exposed to AZT alone (Sussman H et al. [1999]: Mutat Res 429:249,259; Meng Q et al. [2000b]: Toxicol Sci 54:322,329). Molecular analyses of HPRT mutant clones were performed by reverse transcription,mediated production of cDNA, PCR amplification, and cDNA sequencing to define small DNA alterations, followed by multiplex PCR amplification of genomic DNA to define the fractions of deletion events. TK mutants with complete gene deletions were distinguished by Southern blot analysis. The observed HPRT mutational categories included point mutations, microinsertions/microdeletions, splicing-error mutations, and macrodeletions including partial and complete gene deletions. The only significant difference or shift in the mutational spectra for NRTI-treated cells vs. control cells was the increase in the frequency of complete TK gene deletions following exposures (for 3 days) to 300 ,M AZT,ddI (P = 0.034, chi-square test of homogeneity); however, statistical analyses comparing the observed mutant fraction values (measured mutant frequency × percent of a class of mutation) between control and NRTI-treated cells for each class of mutation showed that the occurrences of complete gene deletions of both HPRT and TK were significantly elevated over background values (0.34 × 10,6 in HPRT and 6.0 × 10,6 in TK) at exposure levels of 100 ,M AZT,ddI (i.e., 1.94 × 10,6 in HPRT and 18.6 × 10,6 in TK) and 300 ,M AZT,ddI (i.e., 5.6 × 10,6 in HPRT and 34.6 × 10,6 in TK) (P < 0.05, Mann,Whitney U -statistic). These treatment-related increases in complete gene deletions were consistent with the spectra data for AZT alone (ibid.) and with the known mode of action of AZT and ddI as DNA chain terminators. In addition, cotreatments of ddI with AZT led to substantial absolute increases in the mutant fraction of other classes of mutations, unlike cells exposed solely to AZT [e.g., the frequency of point mutations among HPRT mutants was significantly increased by 130 and 323% over the background value (4.25 × 10,6) in cells exposed to 100 and 300 ,M AZT,ddI, respectively]. These results indicate that, at the same time that AZT,ddI potentiates therapeutic or prophylactic efficacy, the use of a second NRTI with AZT may confer a greater cancer risk, characterized by a spectrum of mutations that deviates from that produced solely by AZT. Environ. Mol. Mutagen. 39:282,295, 2002. Published 2002 Wiley-Liss, Inc. [source]


    Homologues of nitrite reductases in ammonia-oxidizing archaea: diversity and genomic context

    ENVIRONMENTAL MICROBIOLOGY, Issue 4 2010
    Rita Bartossek
    Summary Ammonia-oxidizing archaea are frequent and ubiquitous inhabitants of terrestrial and marine environments. As they have only recently been detected, most aspects of their metabolism are yet unknown. Here we report on the occurrence of genes encoding potential homologues of copper-dependent nitrite reductases (NirK) in ammonia-oxidizing archaea of soils and other environments using metagenomic approaches and PCR amplification. Two pairs of highly overlapping 40 kb genome fragments, each containing nirK genes of archaea, were isolated from a metagenomic soil library. Between 68% and 85% of the open reading frames on these genome fragments had homologues in the genomes of the marine archaeal ammonia oxidizers Nitrosopumilus maritimus and Cenarchaeum symbiosum. Extensions of NirK homologues with C-terminal fused amicyanin domains were deduced from two of the four fosmids indicating structural variation of these multicopper proteins in archaea. Phylogenetic analyses including all major groups of currently known NirK homologues revealed that the deduced protein sequences of marine and soil archaea were separated into two highly divergent lineages that did not contain bacterial homologues. In contrast, another separated lineage contained potential multicopper oxidases of both domains, archaea and bacteria. More nirK gene variants directly amplified by PCR from several environments indicated further diversity of the gene and a widespread occurrence in archaea. Transcription of the potential archaeal nirK in soil was demonstrated at different water contents, but no significant increase in transcript copy number was observed with increased denitrifying activity. [source]


    Multiple displacement amplification as a pre-polymerase chain reaction (pre-PCR) to process difficult to amplify samples and low copy number sequences from natural environments

    ENVIRONMENTAL MICROBIOLOGY, Issue 7 2005
    Juan M. Gonzalez
    Summary Microbial assessment of natural biodiversity is usually achieved through polymerase chain reaction (PCR) amplification. Deoxyribonucleic acid (DNA) sequences from natural samples are often difficult to amplify because of the presence of PCR inhibitors or to the low number of copies of specific sequences. In this study, we propose a non-specific preamplification procedure to overcome the presence of inhibitors and to increase the number of copies prior to carrying out standard amplification by PCR. The pre-PCR step is carried out through a multiple displacement amplification (MDA) technique using random hexamers as priming oligonucleotides and ,29 DNA polymerase in an isothermal, whole-genome amplification reaction. Polymerase chain reaction amplification using specific priming oligonucleotides allows the selection of the sequences of interest after a preamplification reaction from complex environmental samples. The procedure (MDA-PCR) has been tested on a natural microbial community from a hypogean environment and laboratory assemblages of known bacterial species, in both cases targeting the small subunit ribosomal RNA gene sequences. Results from the natural community showed successful amplifications using the two steps protocol proposed in this study while standard, direct PCR amplification resulted in no amplification product. Amplifications from a laboratory assemblage by the two-step proposed protocol were successful at bacterial concentrations ,,10-fold lower than standard PCR. Amplifications carried out in the presence of different concentrations of fulvic acids (a soil humic fraction) by the MDA-PCR protocol generated PCR products at concentrations of fulvic acids over 10-fold higher than standard PCR amplifications. The proposed procedure (MDA-PCR) opens the possibility of detecting sequences represented at very low copy numbers, to work with minute samples, as well as to reduce the negative effects on PCR amplifications of some inhibitory substances commonly found in environmental samples. [source]


    Quantification of bacterial subgroups in soil: comparison of DNA extracted directly from soil or from cells previously released by density gradient centrifugation

    ENVIRONMENTAL MICROBIOLOGY, Issue 7 2001
    Sophie Courtois
    All molecular analyses of soil bacterial diversity are based on the extraction of a representative fraction of cellular DNA. Methods of DNA extraction for this purpose are divided into two categories: those in which cells are lysed within the soil (direct extraction) and those in which cells are first removed from soil (cell extraction) and then lysed. The purpose of this study was to compare a method of direct extraction with a method in which cells were first separated from the soil matrix by Nycodenz gradient centrifugation in order to evaluate the effect of these different approaches on the analysis of the spectrum of diversity in a microbial community. We used a method based on polymerase chain reaction (PCR) amplification of a 16S rRNA gene fragment, followed by hybridization of the amplified fragments to a set of specific probes to assess the phylogenetic diversity of our samples. Control parameters, such as the relationship between amount of DNA template and amount of PCR product and the influence of competing DNA on PCR amplification, were first examined. Comparison between extraction methods showed that less DNA was extracted when cells were first separated from the soil matrix (0.4 µg g,1 dry weight soil versus 38,93 µg g,1 obtained by in situ lysis methods). However, with the exception of the ,-subclass of Proteobacteria, there was no significant difference in the spectrum of diversity resulting from the two extraction strategies. [source]


    Identification of potentially toxic environmental Microcystis by individual and multiple PCR amplification of specific microcystin synthetase gene regions

    ENVIRONMENTAL TOXICOLOGY, Issue 3 2005
    Youness Ouahid
    Abstract Reliable cyanotoxin monitoring in water reservoirs is difficult because of, among other reasons, unpredictable changes in cyanobacteria biomass, toxin production, and inadequate sampling frequency. Therefore, it would be useful to identify potentially microcystin-producing strains of cyanobacterial populations in field samples. With this aim, we developed a methodology to distinguish microcystin-producing from non-producing Microcystis strains by amplifying six characteristic segments of the microcystin synthetase mcy cluster, three corresponding to the nonribosomal peptide synthetase, genes mcyA, mcyB, and mcyC, and three to the polyketide synthase, genes mcyD, mcyE, and mcyG. For this purpose five new primer sets were designed and tested using purified DNA, cultured cells, and field colonies as DNA sources. Simultaneous amplification of several genes in multipex PCR reactions was performed in this study. The results obtained showed that: (i) the expected specific amplicons were obtained with all microcystin-producing strains but not with nonproducing strains; (ii) cells could be directly used as DNA templates, 2000 cells being a sufficient number in most cases; (iii) simultaneous amplification of several gene regions is feasible both with cultured cells and with field colonies. Our data support the idea that the presence of various mcy genes in Microcystis could be used as a criterion for ascribing potential toxigenicity to field strains, and the possibility of applying whole-cell assays for the simultaneous amplification of various genes may contribute significantly to simplifying toxigenicity testing. © 2005 Wiley Periodicals, Inc. Environ Toxicol 20: 235,242, 2005. [source]


    Identification of economically important Liriomyza species by PCR-RFLP analysis,

    EPPO BULLETIN, Issue 1 2005
    L. F. F. Kox
    Only adult males of Liriomyza bryoniae, L. huidobrensis, L. sativae and L. trifolii can be identified with certainty on basis of their genitalia. Female adults, pupae and larvae can only be identified on the level of groups of species (L. bryoniae and L. huidobrensis vs. L. sativae and L. trifolii). Species identification in all developmental stages is possible using molecular biological techniques. Our method is a PCR amplification of a 790-bp fragment of mitochondrial cytochrome oxidase II (COII) DNA followed by RFLP analysis. The method was tested on single larvae, pupae and adults and proved to be applicable to these three life stages. The specificity of the assay was assessed by comparing the results of the PCR-RFLP analysis with those of morphological analysis using 60 Liriomyza specimens. Molecular and morphological identification agreed for all specimens analysed. PCR-RFLP is a powerful diagnostic tool for rapid and reliable identification of all life stages of economically important Liriomyza species. [source]


    Detection of Tomato yellow leaf curl Sardinia virus in Tunisia

    EPPO BULLETIN, Issue 2 2003
    I. Fekih-Hassan
    Surveys were undertaken for tomato yellow leaf curl viruses in the main Tunisian tomato-growing areas in fields and plastic houses. Symptoms included yellowing, leaf curling and stunting. Collected samples were submitted to molecular analysis using two approaches: (1) hybridization tests with two DNA probes corresponding to an intergenic region derived from a cloned dimer of an Egyptian full-length TYLCV and to the coat region of a Tunisian TYLCV isolate; (2) PCR amplification coupled to RFLP allowing both identification and clustering of Tunisian isolates. Only Tomato yellow leaf curl Sardinia virus was detected in Tunisia. [source]


    Clonally rearranged T-cell receptor , chain genes in HTLV-I carriers with abnormal, non-flower-like, lymphocytes

    EUROPEAN JOURNAL OF HAEMATOLOGY, Issue 4 2005
    Maria M. Sales
    Abstract:,Background:,The diagnosis of Adult T-cell leukemia/lymphoma ATLL subtypes in human T-lymphotropic virus type I (HTLV-I) carriers based in morphology and immunophenotype of lymphocytes can be challenger. We propose that polymerase chain reaction (PCR) amplification of the rearranged TCR gene in HTLV-I healthy carriers would be a convenient method for establishing the nature of the circulating T lymphocytes in asymptomatic HTLV-I carriers, presenting only mild and inconclusive signals of deviation from normality. Methods:,Using PCR, we analyzed the genetic recombination pattern of the T-cell , -chain receptor gene (TCR - ,) in order to identify clonal expansion of peripheral blood T lymphocytes in 17 HTLV-I-positive healthy carriers and in nine normal HTLV-I-negative blood donors. To evaluate the performance of PCR in detection of clonality, we also analyzed 18 patients with post-thymic/mature T-cell malignancies presenting circulating abnormal lymphocytes. Results:,Seven of the 17 HTLV-I positive individuals presented circulating abnormal lymphocytes; monoclonal or oligoclonal expansion of T-cells was detected in five of the 17 HTLV-I-positive individuals, all of them presenting abnormal lymphocytes. Clonal expansion was not detected in any of the negative controls or in any of the 12 remaining healthy carriers. All patients in the positive control group tested positive by PCR and Southern blots. Southern blots were negative for all 17 healthy carriers. Conclusions:,PCR amplification of segments of rearranged TCR- , is reliable for allowing early detection of small populations of clonal T cells in blood samples from asymptomatic HTLV-I carriers, providing an additional alert in the follow-up of carriers with abnormal circulating lymphocytes. [source]


    Modulation of oat arginine decarboxylase gene expression and genome organization in transgenic Trypanosoma cruzi epimastigotes

    FEBS JOURNAL, Issue 3 2006
    María P. Serra
    We have previously demonstrated that wild-type Trypanosoma cruzi epimastigotes lack arginine decarboxylase (ADC) enzymatic activity as well as its encoding gene. A foreign ADC has recently been expressed in T. cruzi after transformation with a recombinant plasmid containing the complete coding region of the oat ADC gene. In the present study, upon modulation of exogenous ADC expression, we found that ADC activity was detected early after transfection; subsequently it decreased to negligible levels between 2 and 3 weeks after electroporation and was again detected ,,4 weeks after electroporation. After this period, the ADC activity increased markedly and became expressed permanently. These changes of enzymatic activity showed a close correlation with the corresponding levels of ADC transcripts. To investigate whether the genome organization of the transgenic T. cruzi underwent any modification related to the expression of the heterologous gene, we performed PCR amplification assays, restriction mapping and pulse-field gel electrophoresis with DNA samples or chromosomes obtained from parasites collected at different time-points after transfection. The results indicated that the transforming plasmid remained as free episomes during the transient expression of the foreign gene. Afterwards, the free plasmid disappeared almost completely for several weeks and, finally, when the expression of the ADC gene became stable, two or more copies of the transforming plasmid arranged in tandem were integrated into a parasite chromosome (1.4 Mbp) bearing a ribosomal RNA locus. The sensitivity of transcription to ,-amanitin strongly suggests involvement of the protozoan RNA polymerase I in the transcription of the exogenous ADC gene. [source]


    Response of bacterioplankton community structures to hydrological conditions and anthropogenic pollution in contrasting subtropical environments

    FEMS MICROBIOLOGY ECOLOGY, Issue 3 2009
    Rui Zhang
    Abstract Bacterioplankton community structures under contrasting subtropical marine environments (Hong Kong waters) were analyzed using 16S rRNA gene denaturing gradient gel electrophoresis (DGGE) and subsequent sequencing of predominant bands for samples collected bimonthly from 2004 to 2006 at five stations. Generally, bacterial abundance was significantly higher in the summer than in the winter. The general seasonal variations of the bacterial community structure, as indicated by cluster analysis of the DGGE pattern, were best correlated with temperature at most stations, except for the station close to a sewage discharge outfall, which was best explained by pollution-indicating parameters (e.g. biochemical oxygen demand). Anthropogenic pollutions appear to have affected the presence and the intensity of DGGE bands at the stations receiving discharge of primarily treated sewage. The relative abundance of major bacterial species, calculated by the relative intensity of DGGE bands after PCR amplification, also indicated the effects of hydrological or seasonal variations and sewage discharges. For the first time, a systematic molecular fingerprinting analysis of the bacterioplankton community composition was carried out along the environmental and pollution gradient in a subtropical marine environment, and it suggests that hydrological conditions and anthropogenic pollutions altered the total bacterial community as well as the dominant bacterial groups. [source]


    Uncultured Archaea in a hydrothermal microbial assemblage: phylogenetic diversity and characterization of a genome fragment from a euryarchaeote

    FEMS MICROBIOLOGY ECOLOGY, Issue 3 2006
    Hélène Moussard
    Abstract The polychaete Alvinella pompejana lives in organic tubes on the walls of active hydrothermal chimneys along the East Pacific Rise. To examine the diversity of the archaeal community associated with the polychaete tubes, we constructed libraries by direct PCR amplification and cloning of 16S rRNA genes. Almost half of the sequences of the 16S rRNA gene libraries clustered with uncultured archaeal groups. In an effort to access genomic information from uncultured archaeal members we further constructed a fosmid library from the same DNA source. One of the clones, Alv-FOS5, was sequenced completely. Its sequence analysis revealed an incomplete rRNA operon and 32 predicted ORFs. Seventeen of these ORFs have been assigned putative functions, including transcription and translation, cellular processes and signalling, transport systems and metabolic pathways. Phylogenetic analyses of the 16S rRNA gene suggested that Alv-FOS5 formed a new lineage related to members of Deep-Sea Hydrothermal Vent Euryarchaeota group II. Phylogenetic analyses of predicted proteins revealed the existence of likely cases of horizontal gene transfer, both between Crenarchaeota and Euryarchaeota and between Archaea and Bacteria. This study is the first step in using genomics to reveal the physiology of an as yet uncultured group of archaea from deep-sea hydrothermal vents. [source]


    Usefulness of R72H PCR assay for differentiation between Vibrio parahaemolyticus and Vibrio alginolyticus species: validation by DNA,DNA hybridization

    FEMS MICROBIOLOGY LETTERS, Issue 1 2002
    Annick Robert-Pillot
    Abstract We compared the efficiencies of biochemical methods and polymerase chain reaction (PCR) for the identification of Vibrio parahaemolyticus strains. The 122 isolates studied, identified by biochemical tests as V. parahaemolyticus or Vibrio alginolyticus, were tested by R72H PCR assay. The results obtained with the two methods were consistent for 90% of the strains studied. PCR amplification of the R72H fragment generated two unique amplicons, 387 bp and 320 bp in length. For 11% of the strains from seawater, the results of biochemical identification did not correlate with PCR results. DNA,DNA hybridization experiments provided evidence that some strains identified as V. alginolyticus in biochemical tests should be considered members of the V. parahaemolyticus species. We therefore suggest that biochemical tests are not accurate enough for the identification of V. parahaemolyticus isolates and we demonstrate that amplification of the R72H fragment, whether the amplicon is 320 bp or 387 bp long, is a powerful tool for the reliable identification of V. parahaemolyticus. [source]


    Risk factors for inhibitor formation in haemophilia: a prevalent case,control study

    HAEMOPHILIA, Issue 5 2009
    M. V. RAGNI
    Summary., Inhibitor formation is a major complication of haemophilia treatment. In a prevalent case,control study, we evaluated blood product exposure, genotype and HLA type on haemophilia A inhibitor formation. Product exposure was extracted from medical records. Genotype was determined on stored DNA samples by detection of virtually all mutations-SSCP (DOVAM-S) and subcycling PCR. HLA typing was performed by PCR amplification and exonuclease-released fluorescence. Cases experienced higher intensity factor, 455 vs. 200 U per exposure, P < 0.005, more frequent central nervous system (CNS) bleeding, seven of 20 (35.0%) vs. one of 57 (1.7%), P = 0.001 and more commonly from inhibitor families, seven of 20 (35.0%) vs. zero of 57 (0%), P < 0.001, and African-American, 12 of 63 (19.0%) vs. six of 117 (5.1%), P = 0.015. Among the latter, CNS bleeding was more commonly the initial bleed, 60% vs. 0%, P < 0.001, and survival was shorter, 14 vs. 38 yr, P = 0.025. Inhibitor formation was uncommon in those with missense mutations, two of 65 (3.1%) vs. 31 of 119 (26.0%), P = 0.008, and unrelated to factor VIII immunogenic epitope, P = 0.388, or HLA type, P > 0.100. Genotype was not associated with race. Time to immune tolerance was shorter for titres <120 vs. ,120 BU/mL, six vs. 16 months, P < 0.01, but unaffected by tolerizing dose regimen, P > 0.50. Inhibitor formation is associated with high intensity product exposure, CNS bleeding, African-American race and low frequency of missense mutations. The ideal time to initiate prophylaxis to reduce CNS bleeding and inhibitor formation will require prospective studies. [source]


    Photoprotein aequorin as a novel reporter for SNP genotyping by primer extension,application to the variants of mannose-binding lectin gene,

    HUMAN MUTATION, Issue 3 2006
    Panayotis G. Zerefos
    Abstract Mannose-binding lectin (MBL) is a key component of the innate immune system, and its deficiency is associated with increased susceptibility to various infections and autoimmune disorders. Since several nucleotide variations in the mannose-binding lectin 2 gene (MBL2) have been associated with the functional deficiency of MBL, there is a growing need to screen its allelic variants and develop genotyping methods for MBL2. In this context we propose a rapid, robust, cost-efficient, and automatable method for detecting all known allelic variants of MBL2. This report introduces for the first time the photoprotein aequorin as a reporter in genotyping by primer extension (PEXT) reactions. The method involves a single PCR amplification of a genomic region that spans all six variant nucleotide sites, i.e., three structural mutations in exon 1 (c.154C>T, pArg52Cys; c.161A>G, p.Gly54Asp; and c.170A>G, p.Gly57Glu), two single nucleotide polymorphisms (SNPs) at positions c.,619G>C and c.,290G>C (promoter region), and one SNP at position c.,66C>T of the 5, untranslated region. PCR is followed by PEXT reactions for each site. Biotin-dUTP is incorporated in the extended primer. The genotyping primers contain a poly(dA) segment at their 5, end. The products are captured by hybridization on the surface of microtiter wells that are coated with a poly(dT)-albumin. The extended primers only are detected by reaction with a streptavidin-aequorin conjugate. The bound photoprotein aequorin is measured within 3,sec by simply adding Ca2+. We carried out extensive optimization studies of the PEXT reaction and genotyped the six nucleotide variant sites using blood specimens from 27 normal DNA samples. The results of the proposed method agreed entirely with the sequencing data. Hum Mutat 27(3), 279,285, 2006. © 2006 Wiley-Liss, Inc. [source]


    Palindromic AT-rich repeat in the NF1 gene is hypervariable in humans and evolutionarily conserved in primates,

    HUMAN MUTATION, Issue 4 2005
    Hidehito Inagaki
    Abstract Palindromic sequences are dispersed in the human genome and may cause chromosomal translocations in humans. They constitute unsequenced gaps in the human genome because of their resistance to PCR amplification, cloning into vectors, and sequencing. We have overcome these difficulties by using a combination of optimized PCR conditions, cloning in a recombination-deficient E. coli strain, and RNA polymerases in sequencing. Using these methods, we analyzed a palindromic AT-rich repeat (PATRR) in the neurofibromatosis type 1 (NF1) gene on chromosome 17 (17PATRR). The 17PATRR manifests a size polymorphism due to a highly variable length of (AT)n dinucleotide repeats within the PATRR. 17PATRRs can be categorized into two types: a longer one that comprises a nearly or completely perfect palindrome, and a shorter one that represents its deleted asymmetric derivative. In vitro analysis shows that the longer 17PATRR is more likely to form a cruciform structure than the shorter one. Two reported t(17;22)(q11;q11) patients with NF1, whose breakpoints were identified within the 17PATRR, have translocations that are derived from perfect or nearly perfect palindromic alleles. This implies that the symmetric structure of a PATRR can induce a translocation. We identified conserved PATRRs within the NF1 gene in great apes and similar inverted repeats in two Old World monkeys, but not in New World monkeys or other mammals. This indicates that the palindromic region appeared approximately 25 million years ago and elongated during primate evolution. Although such palindromic regions are usually unstable and disappear rapidly due to deletion, the 17PATRR in the NF1 gene was stably conserved during evolution for reasons that are still unknown. Hum Mutat 26(4), 332,342, 2005. © 2005 Wiley-Liss, Inc. [source]


    Isolation and molecular characterization of Musca domestica delta-9 desaturase sequences

    INSECT MOLECULAR BIOLOGY, Issue 6 2002
    A. L. Eigenheer
    Abstract We have isolated fatty acyl-CoA desaturase cDNA (Mdomd9) and genomic sequences from the housefly, Musca domestica. Two ,1.66 kb cDNAs were recovered. They had identical coding regions and 3, untranslated regions (UTRs), but differed in their 5, UTRs. The open reading frame encodes a 380 amino acid (aa) protein with 82% identity to Drosophila melanogaster desat1, and significant (> 50%) identity with other insect delta-9 desaturases. Functional analyses in a yeast expression system confirmed the cDNA encodes a ,9 desaturase. Northern analysis indicated two transcripts of 1.7 and 2.9 kb that hybridized specifically to the open reading frame. PCR amplification of genomic templates revealed three intron sites that are conserved among other insect species. Southern analysis of genomic DNA indicated at least two desaturase gene copies per haploid genome. There is a high degree of polymorphism, most of which appears to be due to variable intron sequences; curiously, individual flies had varying morphs of intron II and intron III. Together, the data suggest that there are more ,9 desaturase alleles within the population studied than there are loci within the genome, and support other studies suggesting that insect fatty acyl-CoA desaturases are a dynamically evolving gene family. [source]


    PCR-BASED TECHNIQUE FOR IDENTIFICATION AND DETECTION OF TRICHOGRAMMA SPP. (HYMENOPTERA: TRICHOGRAMMATIDAE) WITH SPECIFIC PRIMERS

    INSECT SCIENCE, Issue 3 2002
    LI 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]


    Association of HPV16 E6 variants with diagnostic severity in cervical cytology samples of 354 women in a US population

    INTERNATIONAL JOURNAL OF CANCER, Issue 11 2009
    Rosemary E. Zuna
    Abstract It has been suggested that DNA sequence variants of HPV16 contribute to differences in the behavior of individual cervical lesions. To address this question, we have analyzed the association of HPV16 variants with diagnostic severity in 354 HPV16-positive Oklahoman women. HPV16 variant status was determined by PCR amplification and DNA sequencing of the E6 open reading frame. European sequences were identified in 86% of samples and 14% were non-European. Of the 51 non-European cases, 61% were Asian-American, 23% African and 16% were Native American variants. European prototype and related variants were present in comparable numbers (43% each) but the relative proportion of each differed with diagnostic category. In general, the proportion of European variants and non-European variants increased with diagnostic severity while the European prototype decreased. When adjusted for age and race (white, black or Hispanic), the increased risk for carcinoma/severe dysplasia for non-European variants was statistically significant with an odds ratio of 3.8 (1.3,10.7). However, the analogous comparison for the European variants, although also showing increased association with carcinoma/severe dysplasia, did not reach statistical significance (OR = 1.6 (95% CI 0.7,3.6). Overall, HPV16 European sequences (both prototype and related variants), were predominant in Oklahoman women including those with cancers. This suggests that while there appear to be differences among the HPV16-variant categories in risk for progression to invasive cancer, all variant categories are associated with the development of invasive cancer. © 2009 UICC [source]


    Construction of self-cloning industrial brewing yeast with high-glutathione and low-diacetyl production

    INTERNATIONAL JOURNAL OF FOOD SCIENCE & TECHNOLOGY, Issue 6 2008
    Zhao-Yue Wang
    Summary Self-cloning strains of industrial brewing yeast were constructed, in which one allele of ,-acetohydroxyacid synthase (AHAS) gene (ILV2) was disrupted by integrating Saccharomyces cerevisiae genes, ,-glutamylcysteine synthetase gene (GSH1) and copper resistant gene (CUP1) into the locus of ILV2. The self-cloning strains were selected for their resistance to CuSO4 and identified by PCR amplification. The results of AHAS and glutathione (GSH) assay from fermentation with the self-cloning strains in 500-mL conical flask showed that AHAS activity decreased and GSH content increased compared with that of host yeasts. The results of pilot scale brewing in 5-L fermentation tank also indicated that GSH content in beer fermented with self-cloning strains T5-3 and T31-2 was 1.3 fold and 1.5 fold of that of host QY5 and QY31, respectively; and diacetyl content decreased to 64% and 58% of their hosts, respectively. The self-cloning strains do not contain any heterologous DNA, they may be more acceptable to the public. [source]


    Novel classical MHC class I alleles identified in horses by sequencing clones of reverse transcription-PCR products

    INTERNATIONAL JOURNAL OF IMMUNOGENETICS, Issue 6 2003
    C. Chung
    Summary Improved typing of horse classical MHC class I is required to more accurately define these molecules and to extend the number identified further than current serological assays. Defining classical MHC class I alleleic polymorphism is important in evaluating cytotoxic T lymphocyte (CTL) responses in horses. In this study, horse classical MHC class I genes were analyzed based on reverse transcription (RT)-PCR amplification of sequences encoding the polymorphic peptide binding region and the more conserved alpha 3, transmembrane and cytoplasmic regions followed by cloning and sequencing. Primer sets included a horse classical MHC class I-specific reverse primer and a forward primer conserved in all known horse MHC class I genes. Sequencing at least 25 clones containing MHC class I sequences from each of 13 horses identified 25 novel sequences and three others which had been described. Of these, nine alleles were identified from different horses or different RT-PCR and 19 putative alleles were identified in multiple clones from the same RT-PCR. The primer pairs did not amplify putative non-classical MHC class I genes as only classical MHC class I and related pseudogenes were found in 462 clones. This method also identified classical MHC class I alleles shared between horses by descent, and defined differences in alleles between horses varying in equine leukocyte antigen (ELA)-A haplotype as determined by serology. However, horses sharing ELA-A haplotypes defined by serotyping did not always share cDNA sequences, suggesting subhaplotypic variations within serologically defined ELA-A haplotypes. The 13 horses in this study had two to five classical MHC class I sequences, indicating that multiple loci code for these genes. Sequencing clones from RT-PCR with classical MHC class I-specific primers should be useful for selection of haplotype matched and mismatched horses for CTL studies, and provides sequence information needed to develop easier and more discriminating typing procedures. [source]


    A new HpaII PCR-RFLP within the porcine prolactin receptor (PRLR) gene and study of its effect on litter size and number of teats

    JOURNAL OF ANIMAL BREEDING AND GENETICS, Issue 1 2002
    L. PUTNOVÁ
    DNA polymorphism of the porcine prolactin receptor gene (PRLR) was investigated and used to study its effect on litter size and number of teats in pigs. By means of PRLR gene sequence homology in pig, human and other species, primers were designed for PCR amplification within 5, unknown (to date) part of the prolactin receptor gene in pigs. In this part of the gene, a new polymorphism with HpaII restriction endonuclease was detected. AluI polymorphism described before and our new HpaII polymorphism were used to study the associations with reproduction traits. The PCR restriction fragment length polymorphism (PCR-RFLP) method was used to genotype AluI and HpaII loci of the PRLR gene in line A with 83 sows of Landrace breed and in two lines (B and C) with 75 and 86 Large White sows, respectively. Statistical analysis of 1020 litters showed that AluI locus was associated with litter size mainly in Landrace and affected the first parities, while HpaII locus of the gene was associated with the same traits in Landrace and Large White pigs and mainly affected numbers of weaned of pigs. The magnitude of the effect varied by population with the effects exceeding two pigs per litter in Landrace line and 1 pig per litter in Large White populations. Ein neuer HpaII PCR-RFLP innerhalb des porcinen Prolaktionrezeptorgens (PRLR), und Zusammenhänge zur Wurfgröße und Zitzenzahl DNA-Polymorphismen im porcinen Prolaktionrezeptorgen (PRLR) wurden untersucht und für die Analyse von Einflüssen auf Wurfgröße und Zitzenzahl bei Schweinen verwendet. Auf der Basis der PRLR -Gensequenzhomologie zwischen Schwein, Mensch und anderen Spezies wurden Primer für die PCR-Amplifikation aus dem 5, Bereich des Prolaktionrezeptorgens abgeleitet, der bisher beim Schwein noch unbekannt ist. In diesem Teil des Gens wurde mittels HpaII-Restriktionsendonuklease ein neuer Polymorphismus dargestellt. AluI Polymorphismus und der neue HpaII Polymorphismus wurden für Assoziationsstudien in Bezug auf Reproduktionsmerkmale verwendet. Mittels PCR-RFLP wurden in Linie A 83 Sauen der Landrasse und die Linien B und C mit 75 bzw. 86 Large White Sauen unter Verwendung von AluI und HpaII am PRLR -Gen genotypisiert. Die statistische Analyse von 1.020 Würfen zeigte, dass der AluI-Polymorphismus insbesondere in der Landrasse mit der Wurfgröße assoziiert ist, sowie die ersten Trächtigkeiten beeinflusst, während der HpaII Polymorphismus die gleichen Merkmale in der Landrasse und Large White Schweinen und insbesondere die Zahl an abgesetzten Ferkeln beeinflusste. Die Auswirkungen des Effekts variierten innerhalb Population, wobei der Effekt 2 Ferkel je Wurf in der Landrasse-Linie und 1 Ferkel je Wurf in der Large White Populationen überstieg. [source]


    The use of a quantitative real-time polymerase chain reaction assay for identification and enumeration of Lactobacillus buchneri in silage

    JOURNAL OF APPLIED MICROBIOLOGY, Issue 3 2008
    R.J. Schmidt
    Abstract Aims:, To detect and quantify Lactobacillus buchneri in plant samples with the aid of polymerase chain reaction (PCR) methods. Methods and Results:, DNA from silage samples spiked with different amounts of L. buchneri cells was isolated using a lysozyme/sodium dodecyl sulfate lysis and phenol/chloroform extraction method. The DNA served as a template for PCR amplification with primers specific for the bacterium. The primers were developed by comparison of 16S rDNA sequences from different lactic acid bacteria (LAB) and testing for specificity with 11 different strains of LAB. As few as 100 L. buchneri colony-forming units per gram of silage could be detected. Additionally, the technique was successfully applied to quantify the population of L. buchneri in two cultivars of corn with or without inoculation. Conclusions:, The PCR assay provided a specific and rapid tool for identifying and enumerating L. buchneri in silage samples. Significance and Impact of the Study:, The use of microbial inoculants for silage production is a safe and environment friendly practice, but the full potential of such additives can only be achieved with a better understanding of the fate and activity of the microbes involved. The current study describes a methodology to detect and enumerate L. buchneri, a micro-organism used as an inoculant. [source]


    Identification of Bacillus spp. from Bikalga, fermented seeds of Hibiscus sabdariffa: phenotypic and genotypic characterization

    JOURNAL OF APPLIED MICROBIOLOGY, Issue 1 2008
    L.I.I. Ouoba
    Abstract Aims:, To identify Bacillus spp. responsible of the fermentation of Hibiscus sabdariffa for production of Bikalga, an alkaline fermented food used as a condiment in Burkina Faso. Methods and Results:, Seventy bacteria were isolated from Bikalga produced in different regions of Burkina Faso and identified by phenotyping and genotyping using PCR amplification of the 16S-23S rDNA intergenic transcribed spacer (ITS-PCR), repetitive sequence-based PCR (rep-PCR) and DNA sequencing. The isolates were characterized as motile, rod-shaped, endospore forming, catalase positive, Gram-positive bacteria. ITS-PCR allowed typing mainly at species level. Rep-PCR was more discriminative and allowed a typing at ssp. level. The DNA sequencing combined with the Blast search program and fermentation profiles using API 50CHB system allowed an identification of the bacteria as Bacillus subtilis, B. licheniformis, B. cereus, B. pumilus, B. badius, Brevibacillus bortelensis, B. sphaericus and B. fusiformis. B. subtilis were the predominant bacterium (42) followed by B. licheniformis (16). Conclusions:, Various species and ssp. of Bacillus are involved in fermentation of H. sabdariffa for production of Bikalga. Significance and Impact of the study:, Selection of starter cultures of Bacillus for controlled production of Bikalga, selection of probiotic bacteria. [source]


    Characterization of dominant microbiota of a Ghanaian fermented milk product, nyarmie, by culture- and nonculture-based methods

    JOURNAL OF APPLIED MICROBIOLOGY, Issue 6 2006
    M. Obodai
    Abstract Aims:, To characterize the predominant micro-organisms in a Ghanaian traditional fermented dairy product, nyarmie, made from cows' milk, using both culture- and nonculture-based methods. Methods and Results:, Samples of nyarmie were analysed from three production sites in Accra, by determining the counts on selective culture media. The microbial diversity occurring in nyarmie was also evaluated by 16S/18S ribosomal DNA PCR amplification and denaturing gradient gel electrophoresis. Results showed that nyarmie contained lactococci and lactobacilli in the range of 108 and 1010 CFU ml,1, respectively, and yeasts at around 107 CFU ml,1. The pH ranged between 3·49 and 4·25. The predominant lactic acid bacteria (LAB) in nyarmie were Leuconostocmesenteroides ssp. mesenteroides, Streptococcus thermophilus, Lactobacillus delbrueckii ssp. bulgaricus, Lact.helveticus, Lact. delbrueckii ssp. lactis and Lactococcus lactis, while Saccharomyces cerevisiae was the predominant yeast species. Lactobacillus delbrueckii ssp. delbrueckii was not detected by cultivation but its predominance was revealed by PCR-DGGE analysis. Conclusions:, The flora in products from different producers varied in the LAB composition present and may result in variations in product quality. Significance and Impact of the Study:, Development and use of starter cultures for nyarmie may be beneficial in improving the consistency of product quality. [source]


    Direct detection of bacterial pathogens in representative dairy products using a combined bacterial concentration-PCR approach

    JOURNAL OF APPLIED MICROBIOLOGY, Issue 6 2004
    K.A. Stevens
    Abstract Aims:, To develop a simple, rapid method to concentrate and purify bacteria and their nucleic acids from complex dairy food matrices in preparation for direct pathogen detection using polymerase chain reaction (PCR). Methods and Results:, Plain non-fat yogurt and cheddar cheese were each seeded with Listeria monocytogenes or Salmonella enterica serovar. Enteritidis in the range of 101,106 CFU per 11-g sample. Samples were then processed for bacterial concentration using high-speed centrifugation (9700 g) followed by DNA extraction, PCR amplification, and amplicon confirmation by hybridization. Bacterial recoveries after centrifugation ranged from 53 to >100% and 71 to >100% for serovar. Enteritidis and L. monocytogenes, respectively, in the non-fat yogurt samples; and from 77 to >100% and 69 to >100% for serovar. Enteritidis and L. monocytogenes, respectively, in the cheddar cheese samples. There were no significant differences in recovery efficiency at different inocula levels, and losses to discarded supernatants were always <5%, regardless of dairy product or pathogen. Conclusions:, When followed by pathogen detection using PCR and confirmation by amplicon hybridization, detection limits of 103 and 101 CFU per 11-g sample were achieved for L. monocytogenes and serovar. Enteritidis, respectively, in both product types and without prior cultural enrichment. Significance and Impact of the Study:, This study represents progress toward the rapid and efficient direct detection of pathogens from complex food matrices at detection limits approaching those that might be anticipated in naturally contaminated products. [source]