Identification of Armillaria species isolated from bigtooth aspen based on rDNA RFLP analysis

1998 ◽  
Vol 28 (1) ◽  
pp. 141-149 ◽  
Author(s):  
T M Frontz ◽  
D D Davis ◽  
B A Bunyard ◽  
D J Royse
Keyword(s):  
Restriction Fragment ◽  
Ribosomal Rna ◽  
Rflp Analysis ◽  
Rrna Gene ◽  
Polymorphism Analysis ◽  
5S Rrna Gene ◽  
Aspen Tree ◽  
Armillaria Gallica ◽  
Polymerase Chain ◽  
Armillaria Species

Restriction fragment length polymorphism analysis (RFLP) of the intergenic region (IGR-1) between the 3 ' end of the 26S ribosomal RNA gene and the 5 ' end of the 5S rRNA gene was used to identify 39 isolates of Armillaria species collected from live or recently dead bigtooth aspen (Populus grandidentata Michx.) trees and sucker sprouts in the Tioga State Forest, Pennsylvania. The unknown isolates were identified by comparing their restriction fragment patterns with 18 isolates of known Armillaria species common to the northeastern United States. Twenty of the unknown isolates (50%) were identified as either Armillaria gallica or Armillaria calvescens. Eighteen (46%) of the isolates were identified as Armillaria ostoyae. One isolate of Armillaria sinapina was obtained from a recently dead aspen tree. One isolate of Armillaria mellea, considered to be the most divergent of the Armillaria species, was obtained from basidiomes fruiting on a recently dead aspen tree near Berwick, Pennsylvania. In some instances, amplification of DNA was possible by adding mycelial scrapes directly to the polymerase chain reaction (PCR) mix, thus precluding the need for DNA extraction. Advancements in RFLP analysis may offer a method able to provide rapid and precise identification of most North American and European Armillaria isolates.

scholarly journals Phytoplasma occurrence in apple trees in the Czech Republic

2011 ◽  
Vol 39 (No. 1) ◽  
pp. 7-12 ◽  
Author(s):  
R. Fialová ◽  
M. Navrátil ◽  
P. Válová
Keyword(s):  
Fragment Length Polymorphism ◽  
Rrna Gene ◽  
Polymorphism Analysis ◽  
Apple Proliferation ◽  
Chain Reaction ◽  
Apple Trees ◽  
The Czech Republic ◽  
High Incidence ◽  
Phytoplasma Infection ◽  
Polymerase Chain

The presence of phytoplasmas in apple trees with proliferation symptoms, rubbery wood symptoms and no symp­toms was determined by using polymerase chain reaction assays with primers amplifying phytoplasma 16S rRNA gene. Phytoplasmas were detected in all trees with proliferation symptoms. Positive tests for phytoplasma in the group of trees with rubbery wood symptoms and of those without symptoms revealed a relatively high incidence of latent phytoplasma infection. Using restriction fragment length polymorphism analysis, phytoplasma of the same identity – apple proliferation phytoplasma (subgroup 16SrX-A) – was recorded in all positively tested trees.  

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 Application of a Newly Developed ARB Software-Integrated Tool for In Silico Terminal Restriction Fragment Length Polymorphism Analysis Reveals the Dominance of a Novel pmoA Cluster in a Forest Soil

2005 ◽  
Vol 71 (3) ◽  
pp. 1671-1673 ◽  
Author(s):  
Peter Ricke ◽  
Steffen Kolb ◽  
Gesche Braker
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
Forest Soil ◽  
Restriction Fragment ◽  
Length Polymorphism ◽  
Fragment Length ◽  
In Silico ◽  
Fragment Length Polymorphism ◽  
Rrna Gene ◽  
Polymorphism Analysis ◽  
Restriction Fragment Length

ABSTRACT TRF-CUT, an ARB-implemented tool, was developed to predict in silico the terminal restriction fragments of aligned small-subunit rRNA gene or functional gene sequences. Application of this new tool to perform directed terminal restriction fragment length polymorphism analysis of pmoA products obtained from a forest soil revealed that novel cluster I methanotrophic bacteria were dominant.

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 Microbial burden and inflammasome activation in amniotic fluid of patients with preterm prelabor rupture of membranes

2020 ◽  
Vol 48 (2) ◽  
pp. 115-131 ◽  
Author(s):  
Kevin R. Theis ◽  
Roberto Romero ◽  
Kenichiro Motomura ◽  
Jose Galaz ◽  
Andrew D. Winters ◽  
...  
Keyword(s):  
Amniotic Fluid ◽  
Ribosomal Rna ◽  
Preterm Labor ◽  
Rrna Gene ◽  
Inflammasome Activation ◽  
Amniotic Cavity ◽  
Valuable Complement ◽  
Polymerase Chain ◽  
Adverse Pregnancy ◽  
Prelabor Rupture Of Membranes

Abstract Background Intra-amniotic inflammation, which is associated with adverse pregnancy outcomes, can occur in the presence or absence of detectable microorganisms, and involves activation of the inflammasome. Intra-amniotic inflammasome activation has been reported in clinical chorioamnionitis at term and preterm labor with intact membranes, but it has not yet been investigated in women with preterm prelabor rupture of membranes (preterm PROM) in the presence/absence of detectable microorganisms. The aim of this study was to determine whether, among women with preterm PROM, there is an association between detectable microorganisms in amniotic fluid and intra-amniotic inflammation, and whether intra-amniotic inflammasome activation correlates with microbial burden. Methods Amniotic fluids from 59 cases of preterm PROM were examined for the presence/absence of microorganisms through culture and 16S ribosomal RNA (rRNA) gene quantitative real-time polymerase chain reaction (qPCR), and concentrations of interleukin-6 (IL-6) and ASC [apoptosis-associated spec-like protein containing a caspase recruitment domain (CARD)], an indicator of inflammasome activation, were determined. Results qPCR identified more microbe-positive amniotic fluids than culture. Greater than 50% of patients with a negative culture and high IL-6 concentration in amniotic fluid yielded a positive qPCR signal. ASC concentrations were greatest in patients with high qPCR signals and elevated IL-6 concentrations in amniotic fluid (i.e. intra-amniotic infection). ASC concentrations tended to increase in patients without detectable microorganisms but yet with elevated IL-6 concentrations (i.e. sterile intra-amniotic inflammation) compared to those without intra-amniotic inflammation. Conclusion qPCR is a valuable complement to microbiological culture for the detection of microorganisms in the amniotic cavity in women with preterm PROM, and microbial burden is associated with the severity of intra-amniotic inflammatory response, including inflammasome activation.

Genetic analysis of nuclear DNA restriction fragment patterns

Genome ◽  
1991 ◽  
Vol 34 (5) ◽  
pp. 693-703 ◽  
Author(s):  
Elizabeth M. Gillet
Keyword(s):  
Polymerase Chain Reaction ◽  
Restriction Fragment Length Polymorphism ◽  
Genetic Analysis ◽  
Restriction Fragment ◽  
Length Polymorphism ◽  
Fragment Length Polymorphism ◽  
Rflp Analysis ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Restriction Fragment Length

Restriction fragment length polymorphism (RFLP) analysis in the broad sense is the analysis of differences in restriction fragment pattern produced by defined target segments within or between cell compartments, cell types, etc., in a single individual or in different individuals. Thus both molecular hybridization and DNA amplification by two-primer extension using the polymerase chain reaction can define target segments for RFLP analysis. The two techniques are outlined with special consideration of characteristics important for genetic analysis. The mode of inheritance of restriction fragment patterns as a prerequisite for their use as genetic markers in inheritance studies is explained, leading to criticism of common usage. The importance of internal restriction sites for the determination of allelic variation is stressed. It is shown that, if target segments are under the control of a single nuclear diploid restriction fragment locus, then complete reconstruction of all parental target segments requires controlled crosses between individuals of like restriction fragment pattern.Key words: genetic analysis, inheritance, restriction fragment length polymorphism, controlled cross, polymerase chain reaction.

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