Community analysis of preservative-treated southern pine (Pinus spp.) using terminal restriction fragment length polymorphism (T-RFLP) analysis. Part 1: Fungal field study

Holzforschung ◽  
2012 ◽  
Vol 66 (4) ◽  
Author(s):  
Grant T. Kirker ◽  
M. Lynn Prewitt ◽  
Tor P. Schultz ◽  
Susan V. Diehl
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
Restriction Fragment ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Fragment Length Polymorphism ◽  
Rflp Analysis ◽  
Total Fungi ◽  
Pinus Spp ◽  
Over Time ◽  
Restriction Fragment Length

AbstractThe effects of chlorothalonil (CTN), butylated hydroxytoluene (BHT), and ammoniacal copper quat (ACQ-C) on the fungal community on southern yellow pine (SYP) were assessed using terminal restriction fragment length polymorphism (T-RFLP) analysis over 15 months. Field stakes, treated with 0.25 and 0.37% ACQ-C, 0.1 and 0.25% CTN, 2% BHT alone, 0.1 and 0.25% CTN combined with 2% BHT, and untreated controls, were installed in two field sites in Mississippi. Stakes were sampled at 90-day intervals and rated for decay damage. Fungal DNA was extracted and amplified by non-specific (total fungi) and specific (Basidiomycete) primers and processes for T-RFLP. a-Diversity (richness and diversity) and b-diversity (similarity between communities) were calculated by means of T-RFLP data. The presence of wood preservatives slowed the initial colonization of field stakes by total fungi, resulting in lower richness and diversity that increased over time; however, preservatives increased the richness and diversity of Basidiomycetes. The b-diversity of treated samples was less similar in the early stages of exposure (3–9 months), but coalesced over time into equilibrium communities that were similar to communities on untreated controls. Basidiomycete species compositions were different among treated samples while control communities shared more than 75% of their species. Correlations were found between depletion of 0.1% CTN and increasing fungal diversity, but no other significant correlations were found.

scholarly journals Improved Genotyping Vaccine and Wild-Type Poliovirus Strains by Restriction Fragment Length Polymorphism Analysis: Clinical Diagnostic Implications

2000 ◽  
Vol 38 (12) ◽  
pp. 4337-4342 ◽  
Author(s):  
Amalia Georgopoulou ◽  
Panayotis Markoulatos ◽  
Niki Spyrou ◽  
Nicholas C. Vamvakopoulos
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
Restriction Fragment ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Fragment Length Polymorphism ◽  
Rflp Analysis ◽  
Clinical Samples ◽  
Polymorphism Analysis ◽  
Rt Pcr ◽  
Restriction Fragment Length

The combination of preventive vaccination and diagnostic typing of viral isolates from patients with clinical poliomyelitis constitutes our main protective shield against polioviruses. The restriction fragment length polymorphism (RFLP) adaptation of the reverse transcriptase (RT)-PCR methodology has advanced diagnostic genotyping of polioviruses, although further improvements are definitely needed. We report here on an improved RFLP procedure for the genotyping of polioviruses. A highly conserved segment within the 5′ noncoding region of polioviruses was selected for RT-PCR amplification by the UC53-UG52 primer pair with the hope that it would be most resistant to the inescapable genetic alteration-drift experienced by the other segments of the viral genome. Complete inter- and intratypic genotyping of polioviruses by the present RFLP method was accomplished with a minimum set of four restriction endonucleases (HaeIII, DdeI, NcoI, andAvaI). To compensate for potential genetic drift within the recognition sites of HaeIII, DdeI, orNcoI in atypical clinical samples, the RFLP patterns generated with HpaII and StyI as replacements were analyzed. The specificity of the method was also successfully assessed by RFLP analysis of 55 reference nonpoliovirus enterovirus controls. The concerted implementation of these conditional protocols for diagnostic inter- and intratypic genotyping of polioviruses was evaluated with 21 clinical samples with absolute success.

scholarly journals Identification of Pneumococcal Serotypes by PCR–Restriction Fragment Length Polymorphism

Diagnostics ◽  
2019 ◽  
Vol 9 (4) ◽  
pp. 196 ◽  
Author(s):  
García-Suárez ◽  
González-Rodríguez ◽  
Cima-Cabal ◽  
Yuste ◽  
Vazquez ◽  
...  
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
Restriction Fragment ◽  
Length Polymorphism ◽  
Dna Sequences ◽  
Fragment Length ◽  
Fragment Length Polymorphism ◽  
Rflp Analysis ◽  
Restriction Enzymes ◽  
Pcr Rflp ◽  
Restriction Fragment Length

Streptococcus pneumoniae shows more than 90 capsular serotypes that can be distinguished by their reactivity against antisera. The main objective of this work was the development of a molecular method for serotyping without the use of antisera. A computer program containing an algorithm was used to search in a database for potentially useful enzymes for Restriction Fragment Length Polymorphism-RFLP typing, in order to maximize the discrimination between different serotypes. DNA sequences of 90 serotypes for the region between dexB and aliA genes were compiled, and a computer screening of restriction enzymes was performed. The wzg–wzh–wzd–wze region and Sse9I restriction predicted unique PCR-RFLP patterns for 39 serotypes and eight serogroups. A second restriction enzyme resolved fragment specific patterns for 25 serotypes. The method was tested with 98 serotype-unknown clinical isolates. PCR-RFLP analysis deduced correct serotypes that were confirmed by Quellung reaction for 78.5% of the isolates.

Community analysis of preservative-treated southern pine (Pinus spp.) using terminal restriction fragment length polymorphism (T-RFLP) analysis. Part 2: Bacteria field study

Holzforschung ◽  
2012 ◽  
Vol 66 (4) ◽  
Author(s):  
Grant T. Kirker ◽  
M. Lynn Prewitt ◽  
Walter J. Diehl ◽  
Susan V. Diehl
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
Restriction Fragment ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Bacterial Communities ◽  
Fragment Length Polymorphism ◽  
Wood Preservatives ◽  
Preservative Treatment ◽  
Over Time ◽  
Restriction Fragment Length

Abstract The effects of wood preservatives on the bacterial community in southern yellow pine were assessed by the molecular method ‘terminal restriction fragment length polymorphism’ (T-RFLP). Stakes, treated with 0.25% and 0.37% ammoniacal copper quat (ACQ-C), 0.1% and 0.25% chlorothalonil (CTN), 0.1% and 0.25% CTN with 2% butylated hydroxytoluene (BHT), and 2% BHT were installed with untreated controls in two field test sites in Mississippi and sampled every 90 days. Bacterial DNA was amplified by means of general 16S rDNA primers. Community data were analyzed to determine the effects of test site, exposure (above or below ground), treatment concentrations, and exposure time on the bacterial communities inhabiting the field stakes. Wood preservatives altered the bacterial communities, which fluctuated in numbers and composition over time. Initial exposure to CTN changed the pattern of species that colonized the stakes, and the bacterial communities did not become more similar to controls after CTN depletion. Bacterial communities on untreated controls were the most similar to each other and changed the least over time. Preservative treatment led to greater population turnover and increased diversity by creating a more unstable bacterial environment, which prevented these communities from reaching equilibrium. Although preservative treatment led to changes over time, there were still many shared species within and between the untreated controls and the different preservative treatments.

scholarly journals Identification of Trichomonadid Protozoa from the Bovine Preputial Cavity by Polymerase Chain Reaction and Restriction Fragment Length Polymorphism Typing

2003 ◽  
Vol 15 (4) ◽  
pp. 390-394 ◽  
Author(s):  
Dawn C. Hayes ◽  
Rebecca R. Anderson ◽  
Richard L. Walker
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
Restriction Fragment ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Fragment Length Polymorphism ◽  
Rflp Analysis ◽  
Rrna Gene ◽  
5.8S Rrna Gene ◽  
5.8S Rrna ◽  
Restriction Fragment Length

Accurate identification of the bovine pathogen Tritrichomonas foetus is sometimes complicated by the presence of other trichomonadid protozoa in clinical samples. A highly specific and reproducible approach for differentiating 3 common types of bovine trichomonadid protozoa found in the bovine preputial cavity, T. foetus, Pentatrichomonas hominis, and a Tetratrichomonas species, was developed using polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis. Universal trichomonadid protozoa primers, TFR1 and TFR2, were used to amplify the 5.8S rRNA gene and internal transcribed spacer regions (ITSRs), and the products were digested with the restriction enzyme HpyCH4IV. Restriction fragment length polymorphism analysis was performed on 55 trichomonad isolates from bovine preputial washing and scraping samples. The RFLP results correlated 100% with 5.8S rRNA gene and ITSR sequence results and PCR results with primers specific for T. foetus. The results of this study demonstrate that PCR and RFLP analysis can be used in lieu of DNA sequencing to identify the specific trichomonadid protozoa isolated from the bovine preputial cavity.

scholarly journals Analysis of the Endophytic Actinobacterial Population in the Roots of Wheat (Triticum aestivum L.) by Terminal Restriction Fragment Length Polymorphism and Sequencing of 16S rRNA Clones

2004 ◽  
Vol 70 (3) ◽  
pp. 1787-1794 ◽  
Author(s):  
Vanessa M. Conn ◽  
Christopher M. M. Franco
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
16S Rrna ◽  
Restriction Fragment ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Fragment Length Polymorphism ◽  
Rflp Analysis ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Restriction Fragment Length

ABSTRACT The endophytic actinobacterial population in the roots of wheat grown in three different soils obtained from the southeast part of South Australia was investigated by terminal restriction fragment length polymorphism (T-RFLP) analysis of the amplified 16S rRNA genes. A new, validated approach was applied to the T-RFLP analysis in order to estimate, to the genus level, the actinobacterial population that was identified. Actinobacterium-biased primers were used together with three restriction enzymes to obtain terminal restriction fragments (TRFs). The TRFs were matched to bacterial genera by the T-RFLP Analysis Program, and the data were analyzed to validate and semiquantify the genera present within the plant roots. The highest diversity and level of endophytic colonization were found in the roots of wheat grown in a dark loam from Swedes Flat, and the lowest were found in water-repellent sand from Western Flat. This molecular approach detected a greater diversity of actinobacteria than did previous culture-dependent methods, with the predominant genera being Mycobacterium (21.02%) in Swedes Flat, Streptomyces (14.35%) in Red Loam, and Kitasatospora (15.02%) in Western Flat. This study indicates that the soil that supported a higher number of indigenous organisms resulted in wheat roots with higher actinobacterial diversity and levels of colonization within the plant tissue. Sequencing of 16S rRNA clones, obtained using the same actinobacterium-biased PCR primers that were used in the T-RFLP analysis, confirmed the presence of the actinobacterial diversity and identified a number of Mycobacterium and Streptomyces species.

scholarly journals Characterization of the Mycobacterium bovis Restriction Fragment Length Polymorphism DNA Probe pUCD and Performance Comparison with Standard Methods

2000 ◽  
Vol 38 (9) ◽  
pp. 3362-3369 ◽  
Author(s):  
Rory O'Brien ◽  
Bret S. Danilowicz ◽  
Louise Bailey ◽  
Orla Flynn ◽  
Eamon Costello ◽  
...  
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
Mycobacterium Bovis ◽  
Restriction Fragment ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Fragment Length Polymorphism ◽  
Rflp Analysis ◽  
Performance Comparison ◽  
High Level ◽  
Restriction Fragment Length

In this study, the newly described Mycobacterium bovisrestriction fragment length polymorphism (RFLP) typing probe pUCD was characterized by sequence analysis and the previously observed polymorphic banding pattern was reproduced with a combination of three oligonucleotide probes in a single, mixed hybridization. In addition, the ability of pUCD to distinguish between 299 M. bovisisolates from the Republic of Ireland was assessed in relation to established methods and a statistical function for objective comparison of RFLP probes was derived. It was found that typing with pUCD alone produced greater discrimination between M. bovis isolates than typing with the commonly used mycobacterial DNA probes IS6110, PGRS, and DR and also by the spoligotyping technique. pUCD and DR in combination produced the highest level of discrimination while maintaining a high level of concordance with known epidemiological data relating to the samples. The reduction of pUCD to the level of oligonucleotides should in future allow pUCD and DR to be included together in a mixed hybridization, thus producing a high level of M. bovis strain type discrimination from a single round of RFLP analysis.

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