scholarly journals Genotypic characterisation of the Erwinia genus by PCR-RFLP analysis of rpoS gene

2017 ◽  
Vol 38 (SI 2 - 6th Conf EFPP 2002) ◽  
pp. 288-290 ◽  
Author(s):  
M. Waleron ◽  
K. Waleron ◽  
E. Łojkowska
Keyword(s):  
Restriction Analysis ◽  
Wide Spectrum ◽  
Rflp Analysis ◽  
Pcr Primers ◽  
High Variability ◽  
Rpos Gene ◽  
Pcr Rflp ◽  
Identification Of Species ◽  
Genetic Profiles ◽  
Erwinia Species

Genotypic characterisation of the members of the genus Erwinia, based on the PCR-RFLP analysis of a fragment of the rpoS gene was done. PCR primers deduced from described rpoS gene sequences of E. carotovora allowed the amplification of about 880 bp DNA fragments from all tested Erwinia species. The rpoS fragments, amplified from 20 species of the studied Erwinia genus, were compared by RFLP analysis with 4 enzymes (AluI, Hin6I, HinfI, and Tru1I). Restriction analysis allowed drawing 63 common profiles of RFLP products for all tested Erwinia. From 1 to 3 specific RFLP profiles were identified among most of the species tested. However, in two cases: E. chrysanthemi and E. c. subsp. carotovora 15 and 20 specific RFLP groups were detected, respectively. High variability of genetic profiles of the E. chrysanthemi and E. c. subsp. carotovora can be explained by the wide spectrum of plants, which they infect. The results indicated that rpoS PCR-RFLP analysis is a useful tool for identification of species and subspecies belonging to the former Erwinia genus, as well as for differentiation of strains within E. c. subsp. carotovora and E. chrysanthemi.

The gp63 gene locus, a target for genetic characterization of Leishmania belonging to subgenus Viannia

Parasitology ◽  
1998 ◽  
Vol 117 (1) ◽  
pp. 1-13 ◽  
Author(s):  
K. VICTOIR ◽  
A. L. BAÑULS ◽  
J. AREVALO ◽  
A. LLANOS-CUENTAS ◽  
R. HAMERS ◽  
...  
Keyword(s):  
Gene Locus ◽  
Genetic Characterization ◽  
Restriction Analysis ◽  
Pcr Amplification ◽  
Rflp Analysis ◽  
Restriction Enzymes ◽  
Pcr Rflp ◽  
Natural Isolates ◽  
Phenotype Diversity

In the present study the gp63 gene locus was used as a target for genetic characterization of Leishmania parasites by 2 methods: (i) RFLP analysis with several restriction enzymes (gp63–RFLP), and (ii) intra-genic PCR amplification coupled with restriction analysis (PCR–RFLP). Both methods were applied to a large number of natural isolates belonging to 4 species of the subgenus Viannia, namely L. (V.) braziliensis, L. (V.) peruviana, L. (V.) guyanensis and L. (V.) lainsoni: reference stocks of subgenus Leishmania were included as outgroups. Multilocus isoenzyme typing (MLEE) was used as a reference. On the one hand gp63–RFLP evidenced an extensive polymorphism and revealed specific markers for subgenus, species and geographical populations: congruence with MLEE was demonstrated statistically. The particular interest of gp63–RFLP was illustrated by infra-specific polymorphism, because of the possible relationship with phenotype diversity. On the other hand intra-genic amplification was less resolutive than gp63–RFLP, but also allowed discrimination of the 2 subgenera (PCR alone) and all the species tested in the subgenus Viannia (PCR–RFLP). PCR–RFLP presents an important operational advantage as it allows genetic characterization of minute amounts of parasites, using Leishmania specific primers. The polymorphism revealed by gp63–RFLP and PCR–RFLP illustrates the very high genomic and genetic plasticity of gp63 genes.

Molecular identification of some Hoplolaimus species from the USA based on duplex PCR, multiplex PCR and PCR-RFLP analysis

Nematology ◽  
2009 ◽  
Vol 11 (3) ◽  
pp. 471-480 ◽  
Author(s):  
Robert Robbins ◽  
Allen Szalanski ◽  
Chang-Hwan Bae
Keyword(s):  
Multiplex Pcr ◽  
Dna Sequences ◽  
Rflp Analysis ◽  
Pcr Primers ◽  
Interspecific Variation ◽  
Molecular Techniques ◽  
Specific Pcr ◽  
Pcr Multiplex ◽  
Pcr Rflp ◽  
Species Specific

AbstractTwo different molecular approaches, a multiplex PCR and PCR-RFLP of ITS-rDNA, were developed for the identification of Hoplolaimus species. DNA sequences of H. columbus, H. galeatus, H. concaudajuvencus, H. magnistylus, H. seinhorsti and three undescribed Hoplolaimus species were used to design species-specific primers. Three reverse species-specific PCR primers for H. columbus, H. galeatus and H. magnistylus were developed using the ITS1 region exhibiting interspecific variation. Three species-specific PCR primers in combination with the forward primer, Hoc-1f, produced distinct amplicons of 580 bp for H. columbus, 120 bp for H. galeatus and 340 bp for H. magnistylus. We successfully identified each of three species by multiplex PCR when all three were mixed in a single PCR reaction. Restriction enzyme digests of the PCR amplicon using HaeIII and RsaI permitted discrimination of H. columbus, H. galeatus, H. magnistylus, H. concaudajuvencus, H. sp. 1, H. sp. 2 and H. sp. 3 from each other. These results suggest that these molecular techniques allow for rapid, easy and reliable identification of Hoplolaimus species.

scholarly journals Evaluation of internal transcribed spacer 2-RFLP analysis for the identification of dermatophytes

2010 ◽  
Vol 59 (1) ◽  
pp. 48-54 ◽  
Author(s):  
Thierry De Baere ◽  
Richard Summerbell ◽  
Bart Theelen ◽  
Teun Boekhout ◽  
Mario Vaneechoutte
Keyword(s):  
Internal Transcribed Spacer ◽  
Rflp Analysis ◽  
Its2 Region ◽  
Restriction Digestion ◽  
Internal Transcribed Spacer 2 ◽  
Pcr Rflp ◽  
Identification Technique ◽  
Identification Of Species ◽  
Length Determination ◽  
Taxonomic Approach

A total of 95 isolates, belonging to 33 species of five dermatophyte genera, i.e. Arthroderma (15 species), Chrysosporium (two), Epidermophyton (one), Microsporum (three) and Trichophyton (12), were studied using internal transcribed spacer 2 (ITS2)-PCR-RFLP analysis (ITS2-RFLP), consisting of amplification of the ITS2 region, restriction digestion with BstUI (CG/CG) and restriction fragment length determination by capillary electrophoresis. ITS2-RFLP analysis proved to be most useful for identification of species of the genera Arthroderma, Chrysosporium and Epidermophyton, but could not distinguish between several Trichophyton species. The identification results are in agreement with established and recent taxonomical insights into the dermatophytes; for example, highly related species also had closely related and sometimes difficult-to-discriminate ITS2-RFLP patterns. In some cases, several ITS2-RFLP groups could be distinguished within species, again mostly in agreement with the taxonomic delineations of subspecies and/or genomovars, confirming the relevance of ITS2-RFLP analysis as an identification technique and as a useful taxonomic approach.

scholarly journals Identification of species belonging to the Bifidobacterium genus by PCR-RFLP analysis of a hsp60 gene fragment

2013 ◽  
Vol 13 (1) ◽  
pp. 149 ◽  
Author(s):  
Loredana Baffoni ◽  
Verena Stenico ◽  
Erwin Strahsburger ◽  
Francesca Gaggìa ◽  
Diana Di Gioia ◽  
...  
Keyword(s):  
Rflp Analysis ◽  
Gene Fragment ◽  
Hsp60 Gene ◽  
Pcr Rflp ◽  
Identification Of Species

Rapid and reliable identification of rice genomes by RFLP analysis of PCR-amplified Adh genes

Genome ◽  
2001 ◽  
Vol 44 (6) ◽  
pp. 1136-1142 ◽  
Author(s):  
Song Ge ◽  
Tao Sang ◽  
Bao-rong Lu ◽  
De-yuan Hong
Keyword(s):  
Rflp Analysis ◽  
Restriction Enzymes ◽  
Pcr Primers ◽  
Specific Pcr ◽  
Adh Genes ◽  
Pcr Rflp ◽  
Restriction Sites ◽  
Pcr Products ◽  
Adh Gene ◽  
Genus Oryza

The rice genus (Oryza L.) consists of 24 species with 10 recognized genome types. With the realization of many useful genes in species of wild rice, continuous efforts have been made to understand their genomic composition and relationships. However, the identification of rice genomes has often been difficult owing to complex morphological variation and formation of allotetraploids. Here we propose a rapid and reliable method for identifying rice genomes based on the restriction sites of PCR-amplified Adh genes. The experimental procedure was as follows: (i) amplify a portion of Adh1 and Adh2 genes with the locus-specific PCR primers; (ii) digest PCR products with restriction enzymes that distinguish different genomes; and (iii) run the digested products on 1.4% agarose gel, and photograph. Using various combinations of restriction digestion of the two Adh genes, all of the rice genomes can be identified.Key words: Adh gene, genome, identification, Oryza L., PCR–RFLP.

scholarly journals Genotyping of bacteria belonging to the former Erwinia genus by PCR-RFLP analysis of a recA gene fragment

Microbiology ◽  
2002 ◽  
Vol 148 (2) ◽  
pp. 583-595 ◽  
Author(s):  
Małgorzata Waleron ◽  
Krzysztof Waleron ◽  
Anna J Podhajska ◽  
Ewa Łojkowska
Keyword(s):  
Geographic Origin ◽  
Rflp Analysis ◽  
Erwinia Carotovora ◽  
Pcr Primers ◽  
Reca Gene ◽  
Gene Fragment ◽  
Erwinia Tracheiphila ◽  
Pcr Product ◽  
Pcr Rflp ◽  
Erwinia Stewartii

Genotypic characterization, based on the analysis of restriction fragment length polymorphism of the recA gene fragment PCR product (recA PCR-RFLP), was performed on members of the former Erwinia genus. PCR primers deduced from published recA gene sequences of Erwinia carotovora allowed the amplification of an approximately 730 bp DNA fragment from each of the 19 Erwinia species tested. Amplified recA fragments were compared using RFLP analysis with four endonucleases (AluI, HinfI, TasI and Tru1I), allowing the detection of characteristic patterns of RFLP products for most of the Erwinia species. Between one and three specific RFLP groups were identified among most of the species tested (Erwinia amylovora, Erwinia ananas, Erwinia cacticida, Erwinia cypripedii, Erwinia herbicola, Erwinia mallotivora, Erwinia milletiae, Erwinia nigrifluens, Erwinia persicina, Erwinia psidii, Erwinia quercina, Erwinia rhapontici, Erwinia rubrifaciens, Erwinia salicis, Erwinia stewartii, Erwinia tracheiphila, Erwinia uredovora, Erwinia carotovora subsp. atroseptica, Erwinia carotovora subsp. betavasculorum, Erwinia carotovora subsp. odorifera and Erwinia carotovora subsp. wasabiae). However, in two cases, Erwinia chrysanthemi and Erwinia carotovora subsp. carotovora, 15 and 18 specific RFLP groups were detected, respectively. The variability of genetic patterns within these bacteria could be explained in terms of their geographic origin and/or wide host-range. The results indicated that PCR-RFLP analysis of the recA gene fragment is a useful tool for identification of species and subspecies belonging to the former Erwinia genus, as well as for differentiation of strains within E. carotovora subsp. carotovora and E. chrysanthemi.

scholarly journals Autoscreening of Restriction Endonucleases for PCR-Restriction Fragment Length Polymorphism Identification of Fungal Species, with Pleurotus spp. as an Example

2007 ◽  
Vol 73 (24) ◽  
pp. 7947-7958 ◽  
Author(s):  
Zhi-Hui Yang ◽  
Ji-Xiang Huang ◽  
Yi-Jian Yao
Keyword(s):  
Fragment Length Polymorphism ◽  
Rflp Analysis ◽  
Restriction Enzymes ◽  
Fungal Species ◽  
Restriction Endonucleases ◽  
Its Sequences ◽  
Pcr Rflp ◽  
Average Coefficient ◽  
Identification Of Species ◽  
Restriction Fragment Length

ABSTRACT A molecular method based on PCR-restriction fragment length polymorphism (RFLP) analysis of internal transcribed spacer (ITS) ribosomal DNA sequences was designed to rapidly identify fungal species, with members of the genus Pleurotus as an example. Based on the results of phylogenetic analysis of ITS sequences from Pleurotus, a PCR-RFLP endonuclease autoscreening (PRE Auto) program was developed to screen restriction endonucleases for discriminating multiple sequences from different species. The PRE Auto program analyzes the endonuclease recognition sites and calculates the sizes of the fragments in the sequences that are imported into the program in groups according to species recognition. Every restriction endonuclease is scored through the calculation of the average coefficient for the sequence groups and the average coefficient for the sequences within a group, and then virtual electrophoresis maps for the selected restriction enzymes, based on the results of the scoring system, are displayed for the rapid determination of the candidate endonucleases. A total of 85 haplotypes representing 151 ITS sequences were used for the analysis, and 2,992 restriction endonucleases were screened to find the candidates for the identification of species. This method was verified by an experiment with 28 samples representing 12 species of Pleurotus. The results of the digestion by the restriction enzymes showed the same patterns of DNA fragments anticipated by the PRE Auto program, apart from those for four misidentified samples. ITS sequences from 14 samples (of which nine sequences were obtained in this study), including four originally misidentified samples, confirmed the species identities revealed by the PCR-RFLP analysis. The method developed here can be used for the identification of species of other living microorganisms.

Molecular authentication of Thai medicinal plant Khamin khruea by PCR-RFLP analysis

Planta Medica ◽  
2008 ◽  
Vol 74 (09) ◽  
Author(s):  
P Rojsanga ◽  
W Gritsanapan ◽  
W Leelamanit ◽  
S Sukrong
Keyword(s):  
Medicinal Plant ◽  
Rflp Analysis ◽  
Molecular Authentication ◽  
Pcr Rflp
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