Genetic Relatedness among Environmental, Clinical, and Diseased-Eel Vibrio vulnificus Isolates from Different Geographic Regions by Ribotyping and Randomly Amplified Polymorphic DNA PCR

1998 ◽  
Vol 64 (9) ◽  
pp. 3403-3410 ◽  
Author(s):  
Covadonga R. Arias ◽  
María Jesús Pujalte ◽  
Esperanza Garay ◽  
Rosa Aznar
Keyword(s):  
Genetic Relatedness ◽  
Vibrio Vulnificus ◽  
Genetic Relationships ◽  
Random Amplified Polymorphic Dna ◽  
Mediterranean Coast ◽  
Universal Primers ◽  
23S Rrna ◽  
Rrna Gene ◽  
Conserved Sequence ◽  
Rapd Pcr

ABSTRACT Genetic relationships among 132 strains of Vibrio vulnificus (clinical, environmental, and diseased-eel isolates from different geographic origins, as well as seawater and shellfish isolates from the western Mediterranean coast, including reference strains) were analyzed by random amplified polymorphic DNA (RAPD) PCR. Results were validated by ribotyping. For ribotyping, DNAs were digested with KpnI and hybridized with an oligonucleotide probe complementary to a highly conserved sequence in the 23S rRNA gene. Random amplification of DNA was performed with M13 and T3 universal primers. The comparison between ribotyping and RAPD PCR revealed an overall agreement regarding the high level of homogeneity of diseased-eel isolates in contrast to the genetic heterogeneity of Mediterranean isolates. The latter suggests the existence of autochthonous clones present in Mediterranean coastal waters. Both techniques have revealed a genetic proximity among Spanish fish farm isolates and a close relationship between four Spanish eel farm isolates and some Mediterranean isolates. Whereas the differentiation within diseased-eel isolates was only possible by ribotyping, RAPD PCR was able to differentiate phenotypically atypical isolates of V. vulnificus. On the basis of our results, RAPD PCR is proposed as a better technique than ribotyping for rapid typing in the routine analysis of new V. vulnificusisolates.

Molecular identification and typing of lactobacilli isolated from kefir grains

2005 ◽  
Vol 73 (1) ◽  
pp. 20-27 ◽  
Author(s):  
Lucrecia Delfederico ◽  
Axel Hollmann ◽  
Mariano Martínez ◽  
N. Gabriel Iglesias ◽  
Graciela De Antoni ◽  
...  
Keyword(s):  
Restriction Analysis ◽  
Random Amplified Polymorphic Dna ◽  
Restriction Enzymes ◽  
23S Rrna ◽  
Pcr Analysis ◽  
Rrna Gene ◽  
Bacterial Isolates ◽  
Good Tool ◽  
Rapd Pcr ◽  
Kefir Grains

Seventeen heterofermentative lactobacilli isolated from kefir grains were characterized by molecular methods. Bacterial isolates were identified by amplification of 16S rRNA gene and analysis by Amplified Ribosomal DNA Restriction Analysis (ARDRA), using the restriction enzymes Hae III, Dde I, and Hinf I. ARDRA analysis of lactobacilli isolates showed, for each enzyme used, a same banding pattern between the heterofermentative lactobacilli and the reference strains Lactobacillus kefir JCM 5818 and Lb. kefir ATCC 8007. Other reference lactobacilli and one homofermentative isolate showed differences in at least one of these patterns. The 16S-23S rRNA spacer region was also used to discriminate the bacterial isolates at the species level. The data obtained from the analysis of spacer region confirmed that sequencing of this genome region is a good tool for a reliable identification of members of Lb. kefir species. Genotyping of isolates was performed by Random Amplified Polymorphic DNA (RAPD-PCR) analysis using M13, Coc, ERIC-2 and 1254 primers. Patterns obtained allowed the differentiation of isolates in three groups. The three clusters showed by RAPD-PCR analysis could be correlated with at least three different strains of Lb. kefir species in the group of heterofermentative lactobacilli isolates obtained from Argentinian kefir grains.

Genetic Relatedness of Salmonella Serovars Isolated from Catfish (Clarias gariepinus) and Tilapia (Tilapia mossambica) Obtained from Wet Markets and Ponds in Penang, Malaysia

2016 ◽  
Vol 79 (4) ◽  
pp. 659-665 ◽  
Author(s):  
TITIK BUDIATI ◽  
GULAM RUSUL ◽  
WAN NADIAH WAN-ABDULLAH ◽  
LI-OON CHUAH ◽  
ROSMA AHMAD ◽  
...  
Keyword(s):  
Genetic Relatedness ◽  
Random Amplified Polymorphic Dna ◽  
Clarias Gariepinus ◽  
Fish Feed ◽  
Commercial Fish ◽  
Tilapia Mossambica ◽  
Rapd Pcr ◽  
Salmonella Serovars ◽  
Relationship Of ◽  
Repetitive Extragenic Palindromic Pcr

ABSTRACT A total of 43 Salmonella enterica isolates belonging to different serovars (Salmonella Albany, Salmonella Agona, Salmonella Corvallis, Salmonella Stanley, Salmonella Typhimurium, Salmonella Mikawasima, and Salmonella Bovis-morbificans) were isolated from catfish (Clarias gariepinus) and tilapia (Tilapia mossambica) obtained from nine wet markets and eight ponds in Penang, Malaysia. Thirteen, 19, and 11 isolates were isolated from 9 of 32 catfish, 14 of 32 tilapia, and 11 of 44 water samples, respectively. Fish reared in ponds were fed chicken offal, spoiled eggs, and commercial fish feed. The genetic relatedness of these Salmonella isolates was determined by random amplified polymorphic DNA PCR (RAPD-PCR) using primer OPC2, repetitive extragenic palindromic PCR (REP-PCR), and pulsed-field gel electrophoresis (PFGE). Composite analysis of the RAPD-PCR, REP-PCR, and PFGE results showed that the Salmonella serovars could be differentiated into six clusters and 15 singletons. RAPD-PCR differentiated the Salmonella isolates into 11 clusters and 10 singletons, while REP-PCR differentiated them into 4 clusters and 1 singleton. PFGE differentiated the Salmonella isolates into seven clusters and seven singletons. The close genetic relationship of Salmonella isolates from catfish or tilapia obtained from different ponds, irrespective of the type of feed given, may be caused by several factors, such as the quality of the water, density of fish, and size of ponds.

scholarly journals Identification of non-fermenting Gram-negative bacteria of clinical importance by an oligonucleotide array

2009 ◽  
Vol 58 (5) ◽  
pp. 596-605 ◽  
Author(s):  
Siou Cing Su ◽  
Mario Vaneechoutte ◽  
Lenie Dijkshoorn ◽  
Yu Fang Wei ◽  
Ya Lei Chen ◽  
...  
Keyword(s):  
Clinical Importance ◽  
Intergenic Spacer ◽  
16S Rrna Genes ◽  
Oligonucleotide Array ◽  
Rrna Genes ◽  
Universal Primers ◽  
23S Rrna ◽  
Rrna Gene ◽  
Nylon Membrane ◽  
Gram Negative

Many species of non-fermenting Gram-negative bacilli (non-fermenters) are important opportunistic and nosocomial pathogens. Identification of most species of non-fermenters by phenotypic characteristics can be difficult. In this study, an oligonucleotide array was developed to identify 38 species of clinically relevant non-fermenters. The method consisted of PCR-based amplification of 16S–23S rRNA gene intergenic spacer (ITS) regions using bacterial universal primers, followed by hybridization of the digoxigenin-labelled PCR products with oligonucleotide probes immobilized on a nylon membrane. A total of 398 strains, comprising 276 target strains (i.e. strains belonging to the 38 species to be identified) and 122 non-target strains (i.e. strains not included in the array), were analysed by the array. Four target strains (three reference strains and one clinical isolate) produced discrepant identification by array hybridization. Three of the four discordant strains were found to be correctly identified by the array, as confirmed by sequencing of the ITS and 16S rRNA genes, with the remaining one being an unidentified species. The sensitivity and specificity of the array for identification of non-fermenters were 100 and 96.7 %, respectively. In summary, the oligonucleotide array described here offers a very reliable method for identification of clinically relevant non-fermenters, with results being available within one working day.

scholarly journals Genetic Diversity of Pierce's Disease Strains and Other Pathotypes of Xylella fastidiosa

2001 ◽  
Vol 67 (2) ◽  
pp. 895-903 ◽  
Author(s):  
Mavis Hendson ◽  
Alexander H. Purcell ◽  
Deqiao Chen ◽  
Chris Smart ◽  
Magalie Guilhabert ◽  
...  
Keyword(s):  
Gel Electrophoresis ◽  
Xylella Fastidiosa ◽  
Random Amplified Polymorphic Dna ◽  
Consensus Sequence ◽  
23S Rrna ◽  
Pierce's Disease ◽  
Pcr Analysis ◽  
Leaf Scorch ◽  
Pierce’S Disease ◽  
Rapd Pcr

ABSTRACT Strains of Xylella fastidiosa isolated from grape, almond, maple, and oleander were characterized by enterobacterial repetitive intergenic consensus sequence-, repetitive extragenic palindromic element (REP)-, and random amplified polymorphic DNA (RAPD)-PCR; contour-clamped homogeneous electric field (CHEF) gel electrophoresis; plasmid content; and sequencing of the 16S-23S rRNA spacer region. Combining methods gave greater resolution of strain groupings than any single method. Strains isolated from grape with Pierce's disease (PD) from California, Florida, and Georgia showed greater than previously reported genetic variability, including plasmid contents, but formed a cluster based on analysis of RAPD-PCR products,NotI and SpeI genomic DNA fingerprints, and 16S-23S rRNA spacer region sequence. Two groupings of almond leaf scorch (ALS) strains were distinguished by RAPD-PCR and CHEF gel electrophoresis, but some ALS isolates were clustered within the PD group. RAPD-PCR, CHEF gel electrophoresis, and 16S-23S rRNA sequence analysis produced the same groupings of strains, with RAPD-PCR resolving the greatest genetic differences. Oleander strains, phony peach disease (PP), and oak leaf scorch (OLS) strains were distinct from other strains. DNA profiles constructed by REP-PCR analysis were the same or very similar among all grape strains and most almond strains but different among some almond strains and all other strains tested. Eight of 12 ALS strains and 4 of 14 PD strains of X. fastidiosa isolated in California contained plasmids. All oleander strains carried the same-sized plasmid; all OLS strains carried the same-sized plasmid. A plum leaf scald strain contained three plasmids, two of which were the same sizes as those found in PP strains. These findings support a division of X. fastidiosaat the subspecies or pathovar level.

scholarly journals Using Random Amplified Polymorphic DNA (RAPD) analysis to investigation of genetic diversity, and relationships among a set of clinical Aspergillus fumigatus isolates

2014 ◽  
Vol 8 (1) ◽  
pp. 46-54
Author(s):  
Batool Omran Theeb ◽  
Abdulkareem Jasim Hashim ◽  
Akeel Hussain Ali Al-Assi
Keyword(s):  
Genetic Diversity ◽  
Aspergillus Fumigatus ◽  
Genetic Relatedness ◽  
Random Amplified Polymorphic Dna ◽  
Geographic Origin ◽  
Close Relation ◽  
Genetic Distances ◽  
Universal Primers ◽  
Characteristic Banding ◽  
High Level

This study is an attempt to determine the genetic diversity and relationships among fourteen local isolate isolated from patients with Aspergillosis (Aspergillus fumigatus) by using the Random Amplified Polymorphic DNA (RAPD) technique. Twelve universal primers used in this study produced 94 bands across fourteen isolates. Of these bands, 67 bands or 71.2% were polymorphic. The size of the amplified bands ranged between 100-2000 bp. The genetic polymorphism value of each primer was determined and ranged between 33-100%. In terms of unique banding patterns, determine the finger print for six isolates the most characteristic banding pattern was for the (AFU1, AFU2, AFU3, AFU4, AFU8 and AFU14) with primer (OP F-16 , OP I-06, OP F-16, OP X-01, OP X-01and OP A-06). Genetic distances ranged from 0.12419 to 0.64404 among A. fumigatus isolates. Cluster analyses were performed to construct a dendrogram among studied A. fumigatus isolates. The cluster analysis places most of the A.fumigatus isolates isolated from patient come from yhe same area into a close relation (subcluster) showing a high level of genetic relatedness and were distinct from isolates from another area (the other subcluster). Interestingly, a number of isolates originating from the same sources did form well defined groups, indicating association between the RAPD patterns and the geographic origin of the isolates. The information generated from this study can be used in the future for controlling of Aspergillosis programs.

scholarly journals Allelic Diversity and Population Structure in Oenococcus oeni as Determined from Sequence Analysis of Housekeeping Genes

2004 ◽  
Vol 70 (12) ◽  
pp. 7210-7219 ◽  
Author(s):  
Blanca de las Rivas ◽  
Ángela Marcobal ◽  
Rosario Muñoz
Keyword(s):  
Genetic Diversity ◽  
Population Structure ◽  
Genetic Relationships ◽  
Allelic Diversity ◽  
Rflp Analysis ◽  
The United States ◽  
Oenococcus Oeni ◽  
23S Rrna ◽  
Rrna Gene ◽  
Single Strain

ABSTRACT Oenococcus oeni is the organism of choice for promoting malolactic fermentation in wine. The population biology of O. oeni is poorly understood and remains unclear. For a better understanding of the mode of genetic variation within this species, we investigated by using multilocus sequence typing (MLST) with the gyrB, pgm, ddl, recP, and mleA genes the genetic diversity and genetic relationships among 18 O. oeni strains isolated in various years from wines of the United States, France, Germany, Spain, and Italy. These strains have also been characterized by ribotyping and restriction fragment length polymorphism (RFLP) analysis of the PCR-amplified 16S-23S rRNA gene intergenic spacer region (ISR). Ribotyping grouped the strains into two groups; however, the RFLP analysis of the ISRs showed no differences in the strains analyzed. In contrast, MLST in oenococci had a good discriminatory ability, and we have found a higher genetic diversity than indicated by ribotyping analysis. All sequence types were represented by a single strain, and all the strains could be distinguished from each other because they had unique combinations of alleles. Strains assumed to be identical showed the same sequence type. Phylogenetic analyses indicated a panmictic population structure in O. oeni. Sequences were analyzed for evidence of recombination by split decomposition analysis and analysis of clustered polymorphisms. All results indicated that recombination plays a major role in creating the genetic heterogeneity of O. oeni. A low standardized index of association value indicated that the O. oeni genes analyzed are close to linkage equilibrium. This study constitutes the first step in the development of an MLST method for O. oeni and the first example of the application of MLST to a nonpathogenic food production bacteria.

Random amplified polymorphic DNA of screwworm fly populations (Diptera: Calliphoridae) from Southeastern Brazil and Northern Argentina

Genome ◽  
1999 ◽  
Vol 42 (4) ◽  
pp. 772-779 ◽  
Author(s):  
Maria Elena Infante-Malachias ◽  
Karla Suemy Clemente Yotoko ◽  
Ana Maria Lima de Azeredo Espin
Keyword(s):  
Gene Flow ◽  
Genetic Relatedness ◽  
Random Amplified Polymorphic Dna ◽  
Polymorphic Marker ◽  
Southeastern Brazil ◽  
Cochliomyia Hominivorax ◽  
Factors Affecting ◽  
Screwworm Fly ◽  
Rapd Pcr ◽  
Frequency Changes

The screwworm fly Cochliomyia hominivorax is one of the most important agents of traumatic myiasis throughout neotropical regions. In this work, we optimized the technique of RAPD-PCR for these species and used it to study the genetic variability among seven populations (six from southeastern Brazil and one from northern Argentina) of C. hominivorax. RAPD fingerprints showed high variation for 12 primers used, revealing 209 presumptive loci of which 198 were polymorphic. Marker pattern relationships for these different populations were used to determine genetic relatedness, as well as to examine potential patterns of gene flow. Our interpretation of Lynch and Milligan's analogue of Wright's F(ST) was that C. hominivorax populations are genetically subdivided (F'(ST) for pooled samples = 0.122). Our data suggested that the subdivision detected in C. hominivorax populations by RAPD can be explained by the interplay of random factors affecting allele frequency changes. These results indicate that the RAPD-PCR technique is useful for revealing genetic variation in screwworm fly populations not detected by others techniques and can represent an efficient method for understanding the genetic structure and population genetic phenomena of this important pest.Key words: Cochliomyia hominivorax, screwworm fly, population genetics, gene flow.

Genetic relationships within and among Iberian fescues (Festuca L.) based on PCR-amplified markers

Genome ◽  
2006 ◽  
Vol 49 (9) ◽  
pp. 1170-1183 ◽  
Author(s):  
Pedro J.G. de Nova ◽  
Marcelino de la Cruz ◽  
Juan V. Monte ◽  
Consuelo Soler
Keyword(s):  
Molecular Markers ◽  
Genetic Relatedness ◽  
Genetic Relationships ◽  
Random Amplified Polymorphic Dna ◽  
Molecular Genetic ◽  
Species Level ◽  
Lolium Rigidum ◽  
Length Polymorphisms ◽  
Amplified Fragment Length Polymorphisms ◽  
Molecular Genetic Variation

The genus Festuca comprises approximately 450 species and is widely distributed around the world. The Iberian Penninsula, with more than 100 taxa colonizing very diverse habitats, is one of its main centers of diversification. This study was conducted to assess molecular genetic variation and genetic relatedness among 91 populations of 31 taxa of Iberian fescues, based on several molecular markers (random amplified polymorphic DNA, amplified fragment length polymorphisms, and trnL sequences). The analyses showed the paraphyletic origin of the broad-leaved (subgenus Festuca , sections Scariosae and Subbulbosae, and subgenus Schedonorus ) and the fine-leaved fescues (subgenus Festuca, sections Aulaxyper, Eskia, and Festuca). Schedonorus showed a weak relationship with Lolium rigidum and appeared to be the most recent of the broad-leaved clade. Section Eskia was the most ancient and Festuca the most recent of the fine-leaved clade. Festuca and Aulaxyper were the most related sections, in concordance with their taxonomic affinities. All taxa grouped into their sections, except F. ampla and F. capillifolia (section Festuca), which appeared to be more closely related to Aulaxyper and to a new independent section, respectively. Most populations clustered at the species level, but some subspecies and varieties mixed their populations. This study demonstrated the value in combining different molecular markers to uncover hidden genetic relationships between populations of Festuca.

Characterization of Staphylococcus aureus Isolates from White-Brined Urfa Cheese

2011 ◽  
Vol 74 (11) ◽  
pp. 1788-1796 ◽  
Author(s):  
KURSAT KAV ◽  
RAMAZAN COL ◽  
MUSTAFA ARDIC
Keyword(s):  
Staphylococcus Aureus ◽  
Tandem Repeats ◽  
Genetic Relatedness ◽  
Food Poisoning ◽  
Rflp Analysis ◽  
23S Rrna ◽  
Enzyme Linked Immunosorbent Assay ◽  
Toxin Gene ◽  
Rrna Gene ◽  
23S Rrna Gene

The aim of this study was to investigate the presence of Staphylococcus aureus and staphylococcal enterotoxin (SE) genes in Urfa cheese samples and to characterize the enterotoxigenic potential of these isolates. From a total of 127 Urfa cheese samples, 53 isolates (from 41.7% of the samples) were identified by a species-specific PCR assay as S. aureus. Of these isolates, 40 (75.5%) gave positive PCR results for the 3′ end of the coa gene. The coa-positive S. aureus strains were characterized for their population levels and enterotoxigenic properties, including slime factor, β-lactamase, antibiotic susceptibilities, production of the classical SEs (SEA through SEE), in both cheese and liquid cultures by enzyme-linked immunosorbent assay (ELISA) and for the presence of specific genes, including classical SE genes (sea through see), mecA, femA, and spa, by PCR. The genetic relatedness among the coa-positive S. aureus isolates was investigated by PCR-based restriction fragment length polymorphism (RFLP) analysis and the 23S rRNA gene spacer. The 23S rRNA gene spacer and coa RFLP analysis using AluI and Hin6I revealed 14 different patterns. SEB, SEC, and SEA and SEE were detected by ELISA in three cheese samples. Fourteen S. aureus strains harbored enterotoxin genes sea through see, and three strains carried multiple toxin genes. The most commonly detected toxin gene was sec (25% of tested strains). Of the 40 analyzed S. aureus strains, 3 (7.5%) were mecA positive. Based on tandem repeats, four coa and spa types were identified. The results of this study indicate that S. aureus and SEs are present at significant levels in Urfa cheese. These toxins can cause staphylococcal food poisoning, creating a serious hazard for public health.

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