Use of PCR–RFLP for genotyping 16S rRNA and characterizing bacteria cultured from halibut fry

2002 ◽  
Vol 48 (5) ◽  
pp. 379-386 ◽  
Author(s):  
Sigmund Jensen ◽  
Øivind Bergh ◽  
Øivind Enger ◽  
Brit Hjeltnes
Keyword(s):  
16S Rdna ◽  
Small Subunit ◽  
Atlantic Halibut ◽  
Normal Flora ◽  
Pathogen Invasion ◽  
Restriction Profiles ◽  
Pathogenic Vibrio ◽  
Efficient Detection ◽  
Pcr Rflp ◽  
Deleya Marina

Small subunit ribosomal genes were explored using PCR–RFLP to facilitate the characterization of bacteria cultured from reared fry of the Atlantic halibut (Hippoglossus hippoglossus). Concern has been expressed about pathogen invasion in larvae lacking a counteracting normal flora that may aid the immune system in producing robust noninfected individuals. In this study, pure cultured representatives of normal flora that were previously found to be antagonistic towards a pathogenic Vibrio sp. were subjected to a whole cell PCR protocol amplifying ~1500 bp of 16S rDNA. Amplified DNA was digested by AluI, BstUI, CfoI, and RsaI, to generate restriction profiles. Before the isolates were characterized, a survey was performed to test the discriminative efficiency of the RFLP. Efficient detection of polymorphism and the resolution of species and subspecies were achieved. Using the RFLP on 103 isolates generated as many as 22 genotypes. Based on the restriction profiles, a taxonomic tree incorporating 19 reference strains was constructed. Partial sequencing found this tree to be dominated by γ-Proteobacteria in clusters of Vibrio-, Pseudomonas-, and Alteromonas-affiliated species. Only nine isolates fell outside these genera, including the three isolates Shewanella alga, Deleya marina, and Marinomonas protea. These species have not previously been reported as halibut flora. The most frequently isolated genotype resembled Vibrio salmonicida.Key words: halibut, Vibrio, RFLP, 16S rDNA, phylogeny.

scholarly journals 16S rDNA Genotyping of Lactic Acid Bacteria Using PCR-RFLP Analysis

2017 ◽  
Vol 64 (7) ◽  
pp. 355-364
Author(s):  
Hideyuki Tamakawa ◽  
Yoshihito Ito
Keyword(s):  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
16S Rdna ◽  
Rflp Analysis ◽  
Pcr Rflp

A rapid method for identification of typical Leuconostoc species by 16S rDNA PCR-RFLP analysis

2003 ◽  
Vol 55 (1) ◽  
pp. 295-302 ◽  
Author(s):  
Jichan Jang ◽  
Bongjoon Kim ◽  
Jongho Lee ◽  
Hongui Han
Keyword(s):  
16S Rdna ◽  
Rapid Method ◽  
Rflp Analysis ◽  
16S Rdna Pcr ◽  
Pcr Rflp

scholarly journals Genotypic and Phenotypic Characterization of Fungi in the Fusarium oxysporum Species Complex from Soybean Roots

2014 ◽  
Vol 104 (12) ◽  
pp. 1329-1339 ◽  
Author(s):  
Margaret L. Ellis ◽  
David R. Cruz Jimenez ◽  
Leonor F. Leandro ◽  
Gary P. Munkvold
Keyword(s):  
Fusarium Oxysporum ◽  
Mating Type ◽  
Species Complex ◽  
Phenotypic Diversity ◽  
Rflp Analysis ◽  
Small Subunit ◽  
Phenotypic Characterization ◽  
Pcr Rflp ◽  
Soybean Roots ◽  
Fusarium Oxysporum Species Complex

Isolates in the Fusarium oxysporum species complex (FOSC) from soybean range from nonpathogenic to aggressive pathogens causing seedling damping-off, wilt, and root rot. The objective of this research was to characterize the genotype and phenotype of isolates within the FOSC recovered predominantly from soybean roots and seedlings. Sequence analyses of the translation elongation factor (tef1α) gene and the mitochondrial small subunit (mtSSU), polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analysis of the intergenic spacer (IGS) region, and identification of the mating type loci were conducted for 170 isolates. Vegetative compatibility (VC) tests were conducted for 114 isolates. Isolate aggressiveness was tested using a rolled towel assay for 159 isolates. Phylogenetic analysis of the tef1α and mtSSU and PCR-RFLP analysis of the IGS region separated the FOSC isolates into five clades, including F. commune. Both mating type loci, MAT1-1 or MAT1-2, were present in isolates from all clades. The VC tests were not informative, because most VC groups consisted of a single isolate. Isolate aggressiveness varied within and among clades; isolates in clade 2 were significantly less aggressive (P < 0.0001) when compared with isolates from the other clades and F. commune. The results from this study demonstrate the high levels of genotypic and phenotypic diversity within the FOSC from soybean but further work is needed to identify characteristics associated with pathogenic capabilities.

scholarly journals Targeting Single-Nucleotide Polymorphisms in the 18S rRNA Gene To Differentiate Cyclospora Species from Eimeria Species by Multiplex PCR

2003 ◽  
Vol 69 (8) ◽  
pp. 4806-4813 ◽  
Author(s):  
Palmer A. Orlandi ◽  
Laurenda Carter ◽  
Anna Marie Brinker ◽  
Alexandre J. da Silva ◽  
Dan-My Chu ◽  
...  
Keyword(s):  
Single Nucleotide Polymorphisms ◽  
Eimeria Species ◽  
Small Subunit ◽  
Primate Species ◽  
Rrna Gene ◽  
Nucleotide Polymorphisms ◽  
Small Subunit Rrna ◽  
Single Nucleotide ◽  
Pcr Rflp ◽  
Parasitic Pathogens

ABSTRACT Cyclospora cayetanensis is a coccidian parasite that causes protracted diarrheal illness in humans. C. cayetanensis is the only species of this genus thus far associated with human illness, although Cyclospora species from other primates have been named. The current method to detect the parasite uses a nested PCR assay to amplify a 294-bp region of the small subunit rRNA gene, followed by restriction fragment length polymorphism (RFLP) or DNA sequence analysis. Since the amplicons generated from C. cayetanensis and Eimeria species are the same size, the latter step is required to distinguish between these different species. The current PCR-RFLP protocol, however, cannot distinguish between C. cayetanensis and these new isolates. The differential identification of such pathogenic and nonpathogenic parasites is essential in assessing the risks to human health from microorganisms that may be potential contaminants in food and water sources. Therefore, to expand the utility of PCR to detect and identify these parasites in a multiplex assay, a series of genus- and species-specific forward primers were designed that are able to distinguish sites of limited sequence heterogeneity in the target gene. The most effective of these unique primers were those that identified single-nucleotide polymorphisms (SNPs) at the 3′ end of the primer. Under more stringent annealing and elongation conditions, these SNP primers were able to differentiate between C. cayetanensis, nonhuman primate species of Cyclospora, and Eimeria species. As a diagnostic tool, the SNP PCR protocol described here presents a more rapid and sensitive alternative to the currently available PCR-RFLP detection method. In addition, the specificity of these diagnostic primers removes the uncertainty that can be associated with analyses of foods or environmental sources suspected of harboring potential human parasitic pathogens.

Physiological and genetic characterization of fluorescentPseudomonasassociated withCantharellus cibarius

2002 ◽  
Vol 48 (8) ◽  
pp. 739-748 ◽  
Author(s):  
J Ignacio Rangel-Castro ◽  
Jolanta J Levenfors ◽  
Eric Danell
Keyword(s):  
16S Rdna ◽  
Fungal Mycelium ◽  
Sequencing Analysis ◽  
Carbon Utilization ◽  
Pseudomonas Spp ◽  
Fluorescent Pseudomonas ◽  
Rdna Sequencing ◽  
Pcr Rflp ◽  
Polymerase Chain

Fluorescent Pseudomonas spp. isolated from fruiting bodies (FB) of Cantharellus cibarius were characterized physiologically and genetically and were compared with fluorescent Pseudomonas from forest soil and with sequences from the GenBank database. Pseudomonas spp. from FB differed physiologically from isolates from soil lacking FB and had some similarities with the strains obtained from soil underneath the FB. Analyses of the polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) patterns and partial sequencing analysis of the 16S-rDNA region indicated that the bacteria isolated from these environments were different. However, there was no specific Pseudomonas genotype restricted to the FB environment. Utilization of the reported fungal exudates trehalose and mannitol may explain how millions of bacteria survive in the C. cibarius FB without deteriorating the fungal mycelium. The importance of the metabolic characterization of bacteria and the possible mechanisms involved in the association with C. cibarius are discussed. Our study showed that standard processes for bacterial identification, e.g., Biolog®and 16S-rDNA are insufficient until databases for different ecosystems are created.Key words: Cantharellus cibarius, fluorescent Pseudomonas, carbon utilization, PCR–RFLP, 16S-rDNA sequencing.

scholarly journals Genomic-Based Restriction Enzyme Selection for Specific Detection of Piscirickettsia salmonis by 16S rDNA PCR-RFLP

2016 ◽  
Vol 7 ◽  
Author(s):  
Dinka Mandakovic ◽  
Benjamín Glasner ◽  
Jonathan Maldonado ◽  
Pamela Aravena ◽  
Mauricio González ◽  
...  
Keyword(s):  
16S Rdna ◽  
Restriction Enzyme ◽  
Specific Detection ◽  
16S Rdna Pcr ◽  
Pcr Rflp ◽  
Piscirickettsia Salmonis ◽  
Selection For

scholarly journals First-tier detection of intragenomic 16S rRNA gene variation in culturable endophytic bacteria from cacao seeds

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e7452
Author(s):  
Cleiziane Bispo da Silva ◽  
Hellen Ribeiro Martins dos Santos ◽  
Phellippe Arthur Santos Marbach ◽  
Jorge Teodoro de Souza ◽  
Valter Cruz-Magalhães ◽  
...  
Keyword(s):  
16S Rdna ◽  
Endophytic Bacteria ◽  
Restriction Analysis ◽  
Natural Populations ◽  
Amplicon Sequencing ◽  
Limiting Factor ◽  
Rrna Gene ◽  
Bacterial Strains ◽  
Restriction Profiles ◽  
Chi Square Analysis

Background Intragenomic variability in 16S rDNA is a limiting factor for taxonomic and diversity characterization of Bacteria, and studies on its occurrence in natural/environmental populations are scarce. In this work, direct DNA amplicon sequencing coupled with frequent-cutter restriction analysis allowed detection of intragenomic 16S rDNA variation in culturable endophytic bacteria from cacao seeds in a fast and attractive manner. Methods Total genomic DNA from 65 bacterial strains was extracted and the 16S rDNA hyper variable V5–V9 regions were amplified for enzyme digestion and direct Sanger-type sequencing. The resulting electropherograms were visually inspected and compared to the corresponding AluI-restriction profiles, as well as to complete genome sequences in databases. Restriction analysis were employed to substitute the need of amplicon cloning and re-sequencing. A specifically improved polyacrylamide-gradient electrophoresis allowed to resolve 5-bp differences in restriction fragment sizes. Chi-square analysis on 2 × 2 contingency table tested for the independence between the ‘number of AluI bands’ and ‘type of eletropherogram’. Results Two types of electropherograms were obtained: unique template, with single peaks per base (clean chromatograms), and heterogeneous template, with various levels of multiple peaks per base (mixed chromatograms). Statistics revealed significant interaction between number of restriction fragments and type of electropherogram for the same amplicons: clean or mixed ones associated to ≤5 or ≥6 bands, respectively. The mixed-template pattern combined with the AluI-restriction profiles indicated a high proportion of 49% of the culturable endophytes from a tropical environment showing evidence of intragenomic 16S rDNA heterogeneity. Conclusion The approach presented here was useful for a rapid, first-tier detection of intragenomic variation in culturable isolates, which can be applied in studies of other natural populations; a preliminary view of intragenomic heterogeneity levels can complement culture-dependent and -independent methods. Consequences of these findings in taxonomic and diversity studies in complex bacterial communities are discussed.

scholarly journals Multilocus Fragment Typing and Genetic Structure of Cryptosporidium parvum Isolates from Diarrheic Preweaned Calves in Spain

2011 ◽  
Vol 77 (21) ◽  
pp. 7779-7786 ◽  
Author(s):  
Joaquín Quílez ◽  
Claudia Vergara-Castiblanco ◽  
Luis Monteagudo ◽  
Emilio del Cacho ◽  
Caridad Sánchez-Acedo
Keyword(s):  
Cryptosporidium Parvum ◽  
Geographical Area ◽  
Small Subunit ◽  
Discriminatory Power ◽  
Northern Spain ◽  
Content Type ◽  
Pcr Rflp ◽  
Fluorescent Tags ◽  
Multilocus Approach ◽  
Cattle Farms

ABSTRACTA collection of 140Cryptosporidium parvumisolates previously analyzed by PCR-restriction fragment length polymorphism (PCR-RFLP) and sequence analyses of the small-subunit (SSU) rRNA and 60-kDa glycoprotein (GP60) genes was further characterized by multilocus fragment typing of six minisatellite (MSB and MS5) and microsatellite (ML1, ML2, TP14, and 5B12) loci. Isolates were collected from diarrheic preweaned calves originating from 61 dairy cattle farms in northern Spain. A capillary electrophoresis-based tool combining three different fluorescent tags was used to analyze all six satellites in one capillary. Fragment sizes were adjusted after comparison with sizes obtained by sequence analysis of a selection of isolates for every allele. Size discrepancies at all but the 5B12 locus were found for those isolates that were typed by both techniques, although identical size differences were reported for every allele within each locus. A total of eight alleles were seen at the ML2 marker, which contributed the most to the discriminatory power of the multilocus approach. Multilocus fragment typing clearly improved the discriminatory power of GP60 sequencing, since a total of 59 multilocus subtypes were identified based on the combination of alleles at the six satellite loci, in contrast to the 7 GP60 subtypes previously reported. The majority of farms (38) displayed a unique multilocus subtype, and individual isolates with mixed multilocus subtypes were seen at 22 farms. Bayesian structure analysis based on combined data for both satellite and GP60 loci suggested the presence of two major clusters among theC. parvumisolates from cattle farms in this geographical area.

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