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Specific PCR Primers (specific + pcr_primer)
Selected AbstractsDiagnosis and detection of host-specific forms of Fusarium oxysporum,EPPO BULLETIN, Issue 3-4 2000R. P. Baayen Diagnosis and detection of host-specific forms of Fusarium oxysporum are traditionally based on the combination of diagnostic symptoms on the host with the presence of the fungus in the affected tissues. The classical approach is becoming increasingly problematic because more than one forma specialis may occur on a given host, along with non-pathogenic strains which are common soil and rhizosphere inhabitants. Neither formae speciales nor pathogenic races within formae speciales can be distinguished morphologically. Although united by joint pathogenicity to a given host, strains belonging to the same forma specialis need not be phylogenetically related. Development of diagnostics for host-specific groups in F. oxysporum requires monophyletic target groups. Recent studies on gene-genealogy and AFLP-based phylogenies show that the majority of formae speciales in F. oxysporum are polyphyletic (unnatural) and do not offer any prospects for the development of molecular diagnostics. In contrast, highly specific PCR primers have been developed for formae speciales (or races) that consist of a single clonal lineage, and for monophyletic groups of lineages within a forma specialis. Among others, specific PCR primers have thus been developed for F. oxysporum f. sp. basilici, specific races in F. oxysporum ff. spp. dianthi and gladioli, and for the EPPO A2 (EU II/A1) quarantine fungus F. oxysporum f. sp. albedinis which can reliably replace conventional isolation and pathogenicity testing procedures. [source] Genetic diversity and distribution of periphytic Synechococcus spp. in biofilms and picoplankton of Lake ConstanceFEMS MICROBIOLOGY ECOLOGY, Issue 2 2004Sven Becker Abstract In various water depths of the littoral zone of Lake Constance (Bodensee) cyanobacteria of the Synechococcus -type were isolated from biofilms (periphyton) on three natural substrates and an artificial one (unglazed tiles). From one tile three strains of phycoerythrin (PE)-rich Synechococcus spp. were isolated, the first examples of these organisms in the epibenthos. Phylogenetic inference based on the 16S,23S rRNA intergenic spacer (ITS-1) assigned all periphytic isolates to two clusters of the picophytoplankton clade (evolutionary lineage VI of cyanobacteria). The sequence divergence in the ITS-1 was used to design specific PCR primers to allow direct, culture-independent detection and quantification of isolated Synechococcus strains in natural periphytic and pelagic samples. Denaturing gradient gel electrophoresis (DGGE) analysis revealed depth-related differences of Synechococcus spp. distribution on tiles placed in the littoral zone. Synechococcus genotypes were observed which occurred in both the periphyton (on tiles) and in the pelagic picoplankton. A strain with one of these genotypes, Synechococcus sp. BO 8805, was isolated from the pelagic zone in 1988. Its genotype was found on tiles that had been exposed at different water depths in the littoral zone in spring and autumn of the year 2000. Quantitative analysis with a genotype-specific TaqMan probe and real-time Taq nuclease assays (TNA) confirmed its presence in the pelagic zone, although appearance of this and related genotypes was highly irregular and exhibited strong differences between consecutive years. Our results show that the ability to form significant subpopulations in pelagic and periphytic communities exists in three out of four phylogenetic clusters of Synechococcus spp. in Lake Constance. This versatility may be a key feature in the ubiquity of the evolutionary lineage VI of cyanobacteria. [source] Effects of transgenic glufosinate-tolerant oilseed rape (Brassica napus) and the associated herbicide application on eubacterial and Pseudomonas communities in the rhizosphereFEMS MICROBIOLOGY ECOLOGY, Issue 3 2002Stephen Gyamfi Abstract A containment experiment was carried out in order to evaluate possible shifts in eubacterial and Pseudomonas rhizosphere community structures due to the release of genetically modified Basta-tolerant oilseed rape and the associated herbicide application. Treatments included cultivation of the transgenic plant as well as of the wild-type cultivar in combination with mechanical removal of weeds and the application of the herbicides Basta (active ingredient: glufosinate) and Butisan S (active ingredient: metazachlor). Rhizosphere soil was sampled from early and late flowering plants as well as from senescent plants. A culture-independent approach was chosen to characterize microbial communities based on denaturing gradient gel electrophoresis of 16S rRNA gene fragments amplified from rhizosphere DNA using eubacterial and Pseudomonas -specific PCR primers. Dominant pseudomonads in the rhizosphere were analyzed by sequence analysis. Whole community and Pseudomonas electrophoresis fingerprints revealed slightly altered microbial communities in the rhizosphere of transgenic plants; however, effects were minor as compared to the plant developmental stage-dependent shifts. Both herbicides caused transient changes in the eubacterial and Pseudomonas population structure, whereas differences due to the genetic modification were still detected at the senescent growth stage. The observed differences between transgenic and wild-type lines were most likely due to unintentionally modified plant characteristics such as altered root exudation. [source] Quantification of sequence exchange events between PMS2 and PMS2CL provides a basis for improved mutation scanning of lynch syndrome patients,HUMAN MUTATION, Issue 5 2010Heleen M. van der Klift Abstract Heterozygous mutations in PMS2 are involved in Lynch syndrome, whereas biallelic mutations are found in Constitutional mismatch repair-deficiency syndrome patients. Mutation detection is complicated by the occurrence of sequence exchange events between the duplicated regions of PMS2 and PMS2CL. We investigated the frequency of such events with a nonspecific polymerase chain reaction (PCR) strategy, coamplifying both PMS2 and PMS2CL sequences. This allowed us to score ratios between gene and pseudogene-specific nucleotides at 29 PSV sites from exon 11 to the end of the gene. We found sequence transfer at all investigated PSVs from intron 12 to the 3, end of the gene in 4 to 52% of DNA samples. Overall, sequence exchange between PMS2 and PMS2CL was observed in 69% (83/120) of individuals. We demonstrate that mutation scanning with PMS2 -specific PCR primers and MLPA probes, designed on PSVs, in the 3, duplicated region is unreliable, and present an RNA-based mutation detection strategy to improve reliability. Using this strategy, we found 19 different putative pathogenic PMS2 mutations. Four of these (21%) are lying in the region with frequent sequence transfer and are missed or called incorrectly as homozygous with several PSV-based mutation detection methods. Hum Mutat 31:578,587, 2010. © 2010 Wiley-Liss, Inc. [source] Use of specific PCR primers to identify three important industrial species of Saccharomyces genus: Saccharomyces cerevisiae, Saccharomyces bayanus and Saccharomyces pastorianusLETTERS IN APPLIED MICROBIOLOGY, Issue 2 2010G.V. De Melo Pereira Abstract Aim:, To develop species-specific primers capable of distinguishing between three important yeast species in alcoholic fermentation: Saccharomyces bayanus, Saccharomyces cerevisiae and Saccharomyces pastorianus. Methods and Results:, Two sets of primers with sequences complementary to the HO genes from Saccharomyces sensu stricto species were used. The use of the ScHO primers produced a single amplificon of c. 400 or 300 bp with species S. cerevisiae and S. pastorianus, respectively. The second pair of primers (LgHO) was also constructed, within the HO gene, composed of perfectly conserved sequences common for S. bayanus species, which generate amplicon with 700 bp. No amplification product was observed in the DNA samples from non- Saccharomyces yeasts. Saccharomyces species have also been characterized via electrophoretic karyotyping using pulsed-field gel electrophoresis to demonstrate chromosomal polymorphisms and to determine the evolutionary distances between these species. Conclusions:, We conclude that our novel species-specific primers could be used to rapidly and accurately identify of the Saccharomyces species most commonly involved in fermentation processes using a PCR-based assay. Significance and Impact of the Study:, The method may be used for routine identification of the most common Saccharomyces sensu stricto yeasts involved in industrial fermentation processes in less than 3 h. [source] Multiple displacement amplification as a pre-polymerase chain reaction (pre-PCR) to detect ultra low population of Ralstonia solanacearum (Smith 1896) Yabuchi et al. (1996)LETTERS IN APPLIED MICROBIOLOGY, Issue 5 2009A. Grover Abstract Aims:, To develop a reliable and sensitive protocol for detection of Ralstonia solanacearum using MDA-PCR (Multiple displacement amplification,PCR amplification). Methods and Results:, MDA-PCR technique was performed on pure cell lysates as well as soil samples. Pure cell lysate as well as that of soil DNA was used as template in MDA reaction. MDA of template DNA was carried out in the presence of sample buffer, reaction buffer and enzyme mix (, 29 DNA polymerase and random hexamers). The MDA amplified DNA was used for PCR amplification using R. solanacearum -specific PCR primers. MDA-PCR could detect as low as 1 colony forming unit (CFU ml,1) of bacteria within 8 h including DNA isolation. Conclusion:, MDA followed by standard PCR facilitated the detection of pathogen from very low count samples. The method is of great importance in managing the brown rot disease of potato. Significance and Impact of study:, The ultrasensitive detection technique developed in the present study is sensitive and speedy enough to be included into integrated wilt disease control programmes. [source] Development of 16S rDNA-based PCR assay for detecting Centipeda periodontii and Selenomonas sputigenaLETTERS IN APPLIED MICROBIOLOGY, Issue 6 2000S. Sawada To detect oral motile bacteria directly from dental plaque, specific PCR primers for Centipeda periodontii and Selenomonas sputigena were designed based on the sequence analysis of their 16S rDNA. The primers were specific and sensitive enough to amplify DNA fragments from the available oral bacteria. The detection limit was fewer than 10 bacterial cells per sample. It was also possible to detect these bacteria in dental plaque. The prevalence of these bacteria varied in each sample. The specific primers designed in this study may clarify the epidemiology of periodontal disease. [source] Genotype distribution of Candida albicans isolates by 25S intron analysis with regard to invasivenessMYCOSES, Issue 11-12 2004Z. C. Karahan Candida albicans; Genotyp; Invasivität Summary The aim of this study was to genotype Candida albicans strains isolated from patients with invasive and non-invasive deep-seated infections. For this purpose, 301 C. albicans isolates (81 invasive and 220 non-invasive) were genotyped by using specific PCR primers designed to span the transposable group I intron of the 25S rDNA gene. Fifty-three of the 81 invasive isolates were genotype A (65.4%), eight were genotype B (9.9%) and 20 were genotype C (24.7%), while 98 of the 220 non-invasive isolates were genotype A (44.6%), 46 were genotype B (20.9%) and 76 were genotype C (34.5%). Genotype A was more prevalent among invasive isolates and genotypes B and C were more prevalent among non-invasive isolates (P = 0.0046). Genotypes D and E which represent C. dubliniensis were not found. These results indicate that there may be a relationship between C. albicans genotypes and invasiveness; genotype A being more invasive than others. The presence or absence of the transposable group I intron in the 25S rDNA gene may be important in determining the invasiveness of C. albicans. Zusammenfassung Ziel dieser Arbeit war, Candida albicans -Isolate von Patienten mit invasiven und nicht-invasiven Läsionen zu genotypisieren. Es wurden 301 Isolate (81 invasive und 220 nicht-invasive) untersucht. Die Genotypisierung wurde mittels eines spezifischen PCR Primers, der das Group I-Intron des 25S rDNA Gens umfasst, durchgeführt. 53 der invasiven Isolate waren Genotyp A (65,4%), 8 Genotyp B (9,9%), und 20 Genotyp C (24,7%); anderseits waren 98 der nicht-invasiven Isolate Genotyp A (44,6%), 46 Genotyp B (20,9%), und 76 Genotyp C (34,5%). Genotyp A überwog unter invasiven Isolaten, während die Genotypen B und C unter nicht-invasiven Isolaten vorherrschten (P = 0.0046). Die Genotypen D und E, die Candida dubliniensis umfassen, wurden nicht gefunden. Diese Ergebnisse zeigen, dass es eine Beziehung zwischen C. albicans -Genotypen und Invasivität gibt und dass Genotyp A invasiver ist als andere. Das Group I-Intron des 25S rDNA Gens scheint für die Invasivitätsabschätzung bei C. albicans brauchbar zu sein. [source] | ||||||||||