scholarly journals Seasonal Dynamics and Metagenomic Characterization of Estuarine Viriobenthos Assemblages by Randomly Amplified Polymorphic DNA PCR

2009 ◽  
Vol 75 (8) ◽  
pp. 2259-2265 ◽  
Author(s):  
Rebekah R. Helton ◽  
K. Eric Wommack
Keyword(s):  
Chesapeake Bay ◽  
Oligonucleotide Primer ◽  
Viral Diversity ◽  
Randomly Amplified Polymorphic Dna ◽  
Aquatic Sediments ◽  
Banding Patterns ◽  
Rapd Pcr ◽  
Viral Sequences ◽  
Dna Pcr

ABSTRACT Direct enumeration and genetic analyses indicate that aquatic sediments harbor abundant and diverse viral communities. Thus far, synecological analysis of estuarine sediment viral diversity over an annual cycle has not been reported. This oversight is due in large part to a lack of molecular genetic approaches for assessing viral diversity within a large collection of environmental samples. Here, randomly amplified polymorphic DNA PCR (RAPD-PCR) was used to examine viral genotypic diversity within Chesapeake Bay sediments. Using a single 10-mer oligonucleotide primer for all samples, RAPD-PCR analysis of sediment viral assemblages yielded unique banding patterns across spatial and temporal scales, with the occurrence of specific bands varying among the sample set. Cluster analysis of RAPD-PCR amplicon banding patterns indicated that sediment viral assemblages changed with season and to a lesser extent with geographic location. Sequence analysis of RAPD-PCR amplicons revealed that 76% of sediment viral sequences were not homologous to any sequence in the GenBank nonredundant protein database. Of the GenBank sequence homologs, the majority belonged to viruses within the Podoviridae (24%) and Myoviridae (22%) viral families, which agrees with the previously observed frequencies of these morphological families in Chesapeake Bay sediments. Furthermore, the majority of the sediment viral sequences homologous to GenBank nonredundant protein sequences were phages or prophages (57%). Hence, RAPD-PCR proved to be a reliable and useful approach for characterization of viral assemblages and the genetic diversity of viruses within aquatic sediments.

scholarly journals Characterization of Alstroemeria Species using Random Amplified Polymorphic DNA (RAPD) Analysis

HortScience ◽  
1997 ◽  
Vol 32 (3) ◽  
pp. 482F-482 ◽  
Author(s):  
Deric D. Picton ◽  
Harrison G. Hughes
Keyword(s):  
Rapd Analysis ◽  
Random Amplified Polymorphic Dna ◽  
Randomly Amplified Polymorphic Dna ◽  
Banding Patterns ◽  
Extraction Buffer ◽  
Rapd Pcr ◽  
High Degree ◽  
Species Hybrids ◽  
Color Variants

In this study, 11 species, hybrids, and color variants were characterized using randomly amplified polymorphic DNA (RAPD) analysis. Total genomic DNA was extracted using a 2% CTAB extraction buffer using fresh or frozen leaf material. The DNA was amplified using standard RAPD-PCR protocols utilizing 10-mer primers. All primers utilized exhibited a high degree of polymorphism in their banding patterns among the species and hybrids studied. The primers used produced ≈40 reproducible bands. It was possible to identify and uniquely distinguish all species and hybrids investigated using these bands.

scholarly journals Randomly Amplified Polymorphic DNA PCR as a Tool for Assessment of Marine Viral Richness

2008 ◽  
Vol 74 (9) ◽  
pp. 2612-2618 ◽  
Author(s):  
Danielle M. Winget ◽  
K. Eric Wommack
Keyword(s):  
Chesapeake Bay ◽  
Spatial Dynamics ◽  
Sequence Information ◽  
Randomly Amplified Polymorphic Dna ◽  
Banding Patterns ◽  
Hybridization Probes ◽  
Community Profiling ◽  
Rapd Pcr ◽  
Pcr Products ◽  
Template Dna

ABSTRACT Recent discoveries have uncovered considerable genetic diversity among aquatic viruses and raised questions about the variability of this diversity within and between environments. Studies of the temporal and spatial dynamics of aquatic viral assemblages have been hindered by the lack of a common genetic marker among viruses for rapid diversity assessments. Randomly amplified polymorphic DNA (RAPD) PCR bypasses this obstacle by sampling at the genetic level without requiring viral isolation or previous sequence knowledge. In this study, the utility of RAPD-PCR for assessing DNA viral richness within Chesapeake Bay water samples was evaluated. RAPD-PCR using single 10-mer oligonucleotide primers successfully produced amplicons from a variety of viral samples, and banding patterns were highly reproducible, indicating that each band likely represents a single amplicon originating from viral template DNA. In agreement with observations from other community profiling techniques, resulting RAPD-PCR banding patterns revealed more temporal than spatial variability in Chesapeake Bay virioplankton assemblages. High-quality hybridization probes and sequence information were also easily generated from single RAPD-PCR products or whole reactions. Thus, the RAPD-PCR technique appears to be practical and efficient for routine use in high-resolution viral diversity studies by providing assemblage comparisons through fingerprinting, probing, or sequence information.

Use of PCR-Based Methods for Rapid Differentiation of Lactobacillus delbrueckii subsp.bulgaricus and L. delbrueckii subsp.lactis

1999 ◽  
Vol 65 (10) ◽  
pp. 4351-4356 ◽  
Author(s):  
Sandra Torriani ◽  
Giacomo Zapparoli ◽  
Franco Dellaglio
Keyword(s):  
Numerical Analysis ◽  
Dairy Products ◽  
Lactobacillus Delbrueckii ◽  
Randomly Amplified Polymorphic Dna ◽  
Specific Pcr ◽  
Single Colony ◽  
Rapd Pcr ◽  
Reliable Differentiation ◽  
Dna Pcr ◽  
Proline Iminopeptidase

ABSTRACT Two PCR-based methods, specific PCR and randomly amplified polymorphic DNA PCR (RAPD-PCR), were used for rapid and reliable differentiation of Lactobacillus delbrueckii subsp.bulgaricus and L. delbrueckii subsp.lactis. PCR with a single combination of primers which targeted the proline iminopeptidase (pepIP) gene ofL. delbrueckii subsp. bulgaricus allowed amplification of genomic fragments specific for the two subspecies when either DNA from a single colony or cells extracted from dairy products were used. A numerical analysis of the RAPD-PCR patterns obtained with primer M13 gave results that were consistent with the results of specific PCR for all strains except L. delbrueckii subsp.delbrueckii LMG 6412T, which clustered withL. delbrueckii subsp. lactis strains. In addition, RAPD-PCR performed with primer 1254 provided highly polymorphic profiles and thus was superior for distinguishing individual L. delbrueckii strains.

scholarly journals A Pseudomonas syringae Diversity Survey Reveals a Differentiated Phylotype of the Pathovar syringae Associated with the Mango Host and Mangotoxin Production

2013 ◽  
Vol 103 (11) ◽  
pp. 1115-1129 ◽  
Author(s):  
José A. Gutiérrez-Barranquero ◽  
Víctor J. Carrión ◽  
Jesús Murillo ◽  
Eva Arrebola ◽  
Dawn L. Arnold ◽  
...  
Keyword(s):  
Pseudomonas Syringae ◽  
Randomly Amplified Polymorphic Dna ◽  
Chain Reaction ◽  
Catabolic Diversity ◽  
Rapd Pcr ◽  
Geographical Regions ◽  
Extensive Collection ◽  
Polymerase Chain ◽  
Apical Necrosis ◽  
Dna Pcr

Pseudomonas syringae pv. syringae, the causal agent of bacterial apical necrosis (BAN) in mango crops, has been isolated in different mango-producing areas worldwide. An extensive collection of 87 P. syringae pv. syringae strains isolated from mango trees affected by BAN from different countries, but mainly from Southern Spain, were initially examined by repetitive sequence-based polymerase chain reaction (rep-PCR) to analyze the genetic diversity with an epidemiological aim. rep-PCR was powerful in assessing intrapathovar distribution and also allowing clustering of the P. syringae pv. syringae strains isolated from mango, depending on the isolation area. A clear pattern of clustering was observed for all the P. syringae pv. syringae strains isolated from mango distinct from strains from other hosts, including strains for the same geographical regions as the mango isolates. For this reason, a representative group of 51 P. syringae pv. syringae strains isolated from mango and other hosts, as well as some P. syringae strains from other pathovars, were further characterized to determine their possible genetic, phenotypic, and phylogenetic relationships. Similar to the rep-PCR results, the randomly amplified polymorphic DNA PCR (RAPD-PCR) and catabolic diversity analysis using the Biolog GN2 profile grouped 90% of the mango isolates together in a unique cluster. Interestingly, the majority of P. syringae pv. syringae strains isolated from mango produced mangotoxin. The analysis of the phylogenetic distribution using the multilocus sequence typing analysis strongly supports the existence of a differentiated phylotype of the pathovar syringae mainly associated with the mango host and characterized by the mangotoxin production.

scholarly journals Characterization of verocytotoxin-producingEscherichia coliO157 isolates from patients with haemolytic uraemic syndrome in Western Europe

1995 ◽  
Vol 115 (1) ◽  
pp. 1-3 ◽  
Author(s):  
A. E. Heuvelink ◽  
N. C. A. J. van de Kar ◽  
J. F. G. M. Meis ◽  
L. A. H. Monnens ◽  
W. J. G. Melchers
Keyword(s):  
Haemolytic Uraemic Syndrome ◽  
Western Europe ◽  
Random Amplified Polymorphic Dna ◽  
E Coli ◽  
Pcr Fingerprinting ◽  
Phage Types ◽  
Rapd Pcr ◽  
Typing Methods ◽  
Dna Pcr

SummaryFifty verocytotoxin (VT)-producingEscherichia coli(VTEC) strains of serogroup O157 were characterized by phage typing, polymerase chain reaction (PCR) for VT genes and theE. coliattaching and effacing (eae) gene, and random amplified polymorphic DNA–PCR (RAPD–PCR) fingerprinting. The collection represented isolates obtained from patients with diarrhoea-associated haemolytic-uraemic syndrome (D+ HUS) and their family contacts, isolated in the Netherlands, Belgium and Germany between 1989 and 1993. Based on isolates from separate families (n= 27) seven different phage types were identified, types 2 (44%) and 4 (33%) were predominant. Eighty-five percent of the strains contained only VT2 gene sequences and 15% both VT1 and VT2. All strains of the dominant phage types 2 and 4 carried the VT2 gene. Strains that belonged to the minor phage types 8, 14, 32 carried both VT1 and VT2 genes, with the exception of two isolates identified as phage types 49 and 54 which contained only VT2 genes. All O157 VTEC strains possessed the chromosomally-locatedeaegene, which indicates its usefulness as virulence marker. RAPD–PCR fingerprinting identified four distinct banding patterns, with one profile found among 79% of the strains. Based on the combined results of all typing methods used in this study, the collection of 50 O157 VTEC strains could be divided into nine distinct groups. Strains isolated from different persons within one family could not be distinguished by any of these methods. The data suggest that O157 VTEC strains are members of one clone that has become widely distributed.

Differentiation of faecal Escherichia coli from human and animal sources by random amplified polymorphic DNA-PCR (RAPD-PCR)

2004 ◽  
Vol 50 (1) ◽  
pp. 193-198 ◽  
Author(s):  
D. Venieri ◽  
A. Vantarakis ◽  
G. Komninou ◽  
M. Papapetropoulou
Keyword(s):  
Escherichia Coli ◽  
Rapd Analysis ◽  
Random Amplified Polymorphic Dna ◽  
Hierarchical Cluster ◽  
Group Method ◽  
Randomly Amplified Polymorphic Dna ◽  
Arbitrary Primers ◽  
E Coli ◽  
Rapd Pcr ◽  
Dna Pcr

In this study the assessment of randomly amplified polymorphic DNA (RAPD) analysis was established as a molecular epidemiological tool. RAPD analysis was performed to differentiate faecal Escherichia coli isolates from human and animal sources. E. coli strains (128) were isolated from human and animal faeces (from cattle and sheep). Genomic DNA was extracted and randomly amplified polymorphic DNA-PCR (RAPD-PCR) fingerprinting was performed. Seven arbitrary primers were tested with a view to discriminating between E. coli isolates from humans and E. coli isolates from animals. RAPD profiles were analysed with hierarchical cluster analysis using an unweighted pair group method. RAPD profiles obtained with three of the tested primers (1247, 1290 and 1254) established a distinct differentiation between E. coli isolates from humans and E. coli from animals. Low levels of misclassification and high levels of specificity make RAPD a sensitive, efficient and reliable means of distinguishing closely related strains.

scholarly journals Phenotypic and Genotypic Characterization ofStaphylococcus aureusStrains from Italian Dairy Products

2009 ◽  
Vol 2009 ◽  
pp. 1-7 ◽  
Author(s):  
Stefano Morandi ◽  
Milena Brasca ◽  
Cristian Andrighetto ◽  
Angiolella Lombardi ◽  
Roberta Lodi
Keyword(s):  
Dairy Products ◽  
Animal Species ◽  
Randomly Amplified Polymorphic Dna ◽  
Foodborne Illnesses ◽  
Phenotypic Properties ◽  
Rapd Pcr ◽  
Genes Encoding ◽  
Dna Pcr ◽  
Phenotypic And Genotypic Characterization ◽  
Milk And Dairy Products

Staphylococcus aureusis a known major cause of foodborne illnesses, and milk and dairy products are often contaminated by enterotoxigenic strains of this bacterium. In the present study, 122S. aureusisolates collected from different dairy products were characterised by phenotypic properties, by the distribution of genes encoding staphylococcal enterotoxins (sea,sec,sed,seg,seh,sei,sej, andsel) and by randomly amplified polymorphic DNA PCR (RAPD-PCR). Moreover, strain resistance to vancomycin and methicillin (oxacillin) was studied. The differences in the RAPD-PCR profiles obtained with the primers M13 and AP4 revealed the presence of a great genetic heterogeneity among the differentS. aureusstrains. Using the primer AP4 and M13, eight groups were distinguished by RAPD-PCR cluster analysis, although, except in few cases, it was not possible to correlate the isolates of different animal species (cow or ovine) with the presence ofsegenes. None of the isolates showed resistance to vancomycin or methicillin.

scholarly journals Molecular Characterization of Three Comercial Cultivars and a New Pollinator in Kiwifruit

HortScience ◽  
2006 ◽  
Vol 41 (1) ◽  
pp. 90-95 ◽  
Author(s):  
M.J. Prado ◽  
S. Romo ◽  
M. Novo ◽  
M. Rey ◽  
M.T. Herrera ◽  
...  
Keyword(s):  
Plant Material ◽  
Rapd Markers ◽  
Fragment Length Polymorphism ◽  
Molecular Techniques ◽  
Aflp Markers ◽  
Randomly Amplified Polymorphic Dna ◽  
Banding Patterns ◽  
Specific Protocol ◽  
Technical Features

We investigated the characterization of genotypes of Actinidia deliciosa (Chev.) Liang and Ferguson var. deliciosa by using isozymatic and molecular techniques [randomly amplified polymorphic DNA (RAPD), amplified fragment-length polymorphism (AFLP), standard AFLP, and modified AFLP]. Four genotypes were tested, the female cultivar `Hayward', the traditional New Zealand pollinizers `Matua' and `Tomuri', and a new pollinizer named clone A selected in a breeding program in Spain. PGI and PGM were the only isozymes that allowed us to distinguish the kiwifruit genotypes, although the accessions of `Matua' presented two different banding patterns for both isozymes. All three molecular markers differentiated between the genotypes of kiwifruit tested, although RAPD markers did not allow us to establish differences between accessions of `Matua', while both standard and modified AFLP did. These results, along with those of isozymes, support the hypothesis that the male kiwifruit genotypes present in Europe belong to different clones. None of the markers used showed differences between accessions of `Hayward', which would suggest that it is a uniform cultivar. On the other hand, clone A was a seedling derived from `Hayward' and an unknown pollinizer. The results obtained using AFLP markers strongly suggest that `Tomuri' may have been the male parent of clone A. A specific protocol for kiwifruit characterization based on a modified AFLP technique is also presented, that gave rise to the highest percentage of polymorphism while scoring the lowest number of bands. This, together with the technical features of modified AFLP markers, make them very useful for identifying propagated kiwifruit plant material in commercial nurseries.

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