Rapid differentiation of Microsporum dermatophytes based on arbitrarily primed PCR amplification

1997 ◽  
Vol 9 (1) ◽  
pp. 3-6 ◽  
Author(s):  
D. Liu ◽  
S. Coloe ◽  
R. Baird ◽  
J. Pedersen
Keyword(s):  
Pcr Amplification ◽  
Arbitrarily Primed Pcr

Rapid differentiation of phenotypically and genotypically similar Synechococcus elongatus strains by PCR fingerprinting.

Biologia ◽  
2011 ◽  
Vol 66 (2) ◽  
Author(s):  
Gangatharan Muralitharan ◽  
Nooruddin Thajuddin
Keyword(s):  
Indian Subcontinent ◽  
Pcr Amplification ◽  
Synechococcus Elongatus ◽  
Molecular Fingerprints ◽  
Specific Level ◽  
Unicellular Cyanobacterium ◽  
Pcr Fingerprinting ◽  
Perform Cluster Analysis ◽  
Arbitrarily Primed Pcr ◽  
Genotypic Characteristics

AbstractPCR amplification techniques viz., repetitive DNA element PCR (REP-PCR), short tandemly repeated repetitive PCR (STRR-PCR) and arbitrarily primed PCR (RAPD-PCR) were used for the taxonomic discrimination among the strains of the unicellular cyanobacterium Synechococcus elongatus collected across the coastal regions of the Indian subcontinent. These strains showed similar phenotypic and genotypic characteristics. Data obtained from genomic fingerprinting were used to perform cluster analysis and demonstrated ability to differentiate strains at intra-specific level. Polymorphisms of different PCR amplification products can serve as strain-specific molecular fingerprints. In comparison with the STRR and RAPD, the REP primer set generates fingerprints of lower complexity, but still the phenogram clearly differentiated the strains. In conclusion, described PCR fingerprinting methods can be considered as promising tools for the differentiation at the strain level of cyanobacteria from the same species.

scholarly journals Characterization of Serologically NontypeableActinobacillus actinomycetemcomitans Isolates

1998 ◽  
Vol 36 (7) ◽  
pp. 2019-2022 ◽  
Author(s):  
Susanna Paju ◽  
Maria Saarela ◽  
Satu Alaluusua ◽  
Paula Fives-Taylor ◽  
Sirkka Asikainen
Keyword(s):  
Restriction Analysis ◽  
Pcr Amplification ◽  
Actinobacillus Actinomycetemcomitans ◽  
Genotype 3 ◽  
Banding Patterns ◽  
Arbitrarily Primed Pcr

Our previous studies have shown that Actinobacillus actinomycetemcomitans isolates of a given arbitrarily primed PCR (AP-PCR) genotype belong to the same serotype (of serotypes a through e). In the present study we investigated whether the AP-PCR genotypes of nonserotypeable A. actinomycetemcomitans isolates match those of the serotypeable isolates. The isolates were additionally characterized by restriction analysis of the apaH PCR amplification products. The material included 75 nonserotypeable and 18 serotypeable A. actinomycetemcomitans isolates from 34 epidemiologically unrelated subjects. The serotypeable isolates were obtained from subjects who also harbored nonserotypeable isolates. Eight AP-PCR genotypes were distinguished among the isolates; six genotypes matched those detected in our previous studies, whereas two genotypes were new. Intraindividually, the A. actinomycetemcomitans isolates produced identical AP-PCR banding patterns, regardless of whether they were serotypeable or nonserotypeable, in 22 of 23 subjects participating with multiple isolates. AP-PCR genotype 3, corresponding to serotype c, was by far the most common among the nonserotypeable isolates (62% of subjects). Results obtained with the apaH restriction analysis confirmed the results obtained with AP-PCR for 31 of the 34 subjects. The results suggest that nonserotypeable A. actinomycetemcomitans isolates originate from serotypeable isolates, especially from serotype c isolates, and the likelihood of the existence of additional serotypes is small.

scholarly journals gyrA and gyrB Mutations Are Implicated in Cross-Resistance to Ciprofloxacin and Moxifloxacin in Clostridium difficile

2002 ◽  
Vol 46 (11) ◽  
pp. 3418-3421 ◽  
Author(s):  
Larbi Dridi ◽  
Jacques Tankovic ◽  
Béatrice Burghoffer ◽  
Frédéric Barbut ◽  
Jean-Claude Petit
Keyword(s):  
Amino Acid ◽  
Clostridium Difficile ◽  
Ethidium Bromide ◽  
Dna Hybridization ◽  
Pcr Amplification ◽  
Cross Resistance ◽  
Topoisomerase Iv ◽  
Clinical Strains ◽  
Resistant Strains ◽  
Arbitrarily Primed Pcr

ABSTRACT A total of 198 nonrepetitive clinical strains of Clostridium difficile isolated from different French hospitals in 1991 (n = 100) and 1997 (n = 98) were screened for decreased susceptibility to fluoroquinolones by plating onto Wilkins-Chalgren agar containing 16 μg of ciprofloxacin per ml. The frequency of decreased susceptibility was 7% (14 of 198) and was identical for the years 1991 and 1997. Serogroups C, H, D, A9, and K accounted for five, four, two, one, and one of the resistant strains, respectively, one strain being nontypeable. Arbitrarily primed PCR typing showed that all resistant strains had unique patterns except two serotype C strains, which could not be clearly distinguished. All isolates with decreased susceptibility carried a mutation either in gyrA (eight mutations, amino acid changes Asp71→Val in one, Thr82→Ile in six, and Ala118→Thr in one) or in gyrB (six mutations, amino acid changes Asp426→Asn in five and Arg447→Leu in one). These changes are similar to those already described in other species except for Asp71→Val, which is novel, and Ala118→Thr, which is exceptional. Attempts to detect the topoisomerase IV parC gene by PCR amplification with universal parC primers or DNA-DNA hybridization under low-stringency conditions were unsuccessful. The susceptibilities of all resistant strains to ciprofloxacin and ethidium bromide were not affected by the addition of reserpine at 20 μg/ml. In conclusion, decreased susceptibility to fluoroquinolones in C. difficile is rare in France and is associated with the occurrence of a gyrA or gyrB mutation.

scholarly journals Evaluation of Two Commercial Kits and Arbitrarily Primed PCR for Identification and Differentiation ofActinobacillus actinomycetemcomitans,Haemophilus aphrophilus, and Haemophilus paraphrophilus

1999 ◽  
Vol 37 (3) ◽  
pp. 742-747 ◽  
Author(s):  
Başak Doğan ◽  
Sirkka Asikainen ◽  
Hannele Jousimies-Somer
Keyword(s):  
Pcr Amplification ◽  
Clinical Isolates ◽  
Species Differentiation ◽  
Oral Microbiota ◽  
Closely Related Species ◽  
Haemophilus Aphrophilus ◽  
Consistent Performance ◽  
Commercial Kits ◽  
Arbitrarily Primed Pcr ◽  
Reference Strains

The closely related species Actinobacillus actinomycetemcomitans, Haemophilus aphrophilus, andHaemophilus paraphrophilus are common findings in oral microbiota. The aims of this study were to evaluate the applicability of the Rapid NH and API ZYM kits and arbitrarily primed PCR (AP-PCR) in the identification and differentiation of the three species from each other. The material included 62 clinical isolates and three reference strains of A. actinomycetemcomitans representing the 5 serotypes and 18 AP-PCR genotypes. Haemophilus species included 12 clinical isolates and 11 reference strains of H. aphrophilus, H. paraphrophilus, and 5 other species. For the PCR amplification, the oligonucleotide 5′-CAGCACCCAC-3′ was used as a primer. Contrary to the consistent performance of API ZYM, the Rapid NH system was able to identify only 10 of 65 (15%) A. actinomycetemcomitans isolates, whereas allHaemophilus species were correctly identified. The API ZYM test differentiated A. actinomycetemcomitans from H. aphrophilus and H. paraphrophilus by negative β-galactosidase and α-glucosidase reactions and a positive esterase lipase reaction. However, the API ZYM test was unable to differentiateH. aphrophilus from H. paraphrophilus, it also could not differentiate A. actinomycetemcomitans serotypes from each other. Among the H. aphrophilus isolates three AP-PCR genotypes and among H. paraphrophilus isolates only one AP-PCR genotype, distinct from those of A. actinomycetemcomitans, were found. The Rapid NH test showed poor ability to identify clinical isolates of all A. actinomycetemcomitans serotypes. Moreover, AP-PCR genotyping proved to be a rapid method for the species differentiation of A. actinomycetemcomitans, H. aphrophilus, and H. paraphrophilus.

Investigations of Genetic Variation Between Olive (Olea europaea L.) Cultivars Using Arbitrarily Primed Polymerase Chain Reaction (AP-PCR)

2003 ◽  
Vol 58 (11-12) ◽  
pp. 837-842 ◽  
Author(s):  
Feray Kockar ◽  
Rahsan Ilıkcı
Keyword(s):  
Genetic Relationship ◽  
Genomic Dna ◽  
Pcr Amplification ◽  
Dna Fingerprint ◽  
Factors Affecting ◽  
Genomic Dna Isolation ◽  
Amplification Protocol ◽  
Olive Cultivars ◽  
Arbitrarily Primed Pcr ◽  
Relationship Of

Abstract Characterization and selection of olive clones for the production of olive oil is essential in Turkey because of its profitable exploitation. AP-PCR (Arbitrarily-Primed PCR) is a technique that can distinguish the genetic relationship among plant species and other organisms. In this study, AP-PCR approach was used in order to determine the genetic relationship of different six olive clones. The purity of DNA is one of the most important factors affecting the product of the AP-PCR method. In this respect, modified genomic DNA isolation procedure from Oleae europaea clones was developed so that this procedure can be used to obtain plant genomic DNA from diverse aromatic plants, which produce essential oils and secondary metabolites. By following the optimized AP-PCR amplification protocol, unique DNA fingerprint profiles for each olive clone were produced. AP-PCR-generated unique DNA fingerprint profiles can be used in the identification, distribution and diversity of various olive cultivars.

scholarly journals Characterization of Colletotrichum acutatum Causing Anthracnose of Anemone (Anemone coronaria L.)

2000 ◽  
Vol 66 (12) ◽  
pp. 5267-5272 ◽  
Author(s):  
Stanley Freeman ◽  
Ezra Shabi ◽  
Talma Katan
Keyword(s):  
Western Europe ◽  
Pcr Amplification ◽  
Vegetative Compatibility ◽  
Colletotrichum Acutatum ◽  
Vegetative Compatibility Groups ◽  
The Third ◽  
Artificial Inoculations ◽  
Species Specific ◽  
Arbitrarily Primed Pcr

ABSTRACT Anthracnose, or leaf-curl disease of anemone, caused byColletotrichum sp., has been reported to occur in Australia, western Europe, and Japan. Symptoms include tissue necrosis, corm rot, leaf crinkles, and characteristic spiral twisting of floral peduncles. Three epidemics of the disease have been recorded in Israel: in 1978, in 1990 to 1993, and in 1996 to 1998. We characterized 92Colletotrichum isolates associated with anthracnose of anemone (Anemone coronaria L.) for vegetative compatibility (72 isolates) and for molecular genotype (92 isolates) and virulence (4 isolates). Eighty-six of the isolates represented the three epidemics in Israel, one isolate was from Australia, and five isolates originated from western Europe. We divided these isolates into three vegetative-compatibility groups (VCGs). One VCG (ANE-A) included all 10 isolates from the first and second epidemics, and 13 of 62 examined isolates from the third epidemic in Israel, along with the isolate from Australia and 4 of 5 isolates from Europe. Another VCG (ANE-F) included most of the examined isolates (49 of the 62) from the third epidemic, as well as Colletotrichum acutatum from strawberry, in Israel. Based on PCR amplification with species-specific primers, all of the anemone isolates were identified as C. acutatum. Anemone and strawberry isolates of the two VCGs were genotypically similar and indistinguishable when compared by arbitrarily primed PCR of genomic DNA. Only isolate NL-12 from The Netherlands, confirmed as C. acutatum but not compatible with either VCG, had a distinct genotype; this isolate represents a third VCG of C. acutatum. Isolates from anemone and strawberry could infect both plant species in artificial inoculations. VCG ANE-F was recovered from natural infections of both anemone and strawberry, but VCG ANE-A was recovered only from anemone. This study of C. acutatum from anemone illustrates the potential of VCG analysis to reveal distinct subspecific groups within a pathogen population which appears to be genotypically homogeneous by molecular assays.

scholarly journals Randomly Amplified DNA Fingerprinting: A Culmination of DNA Marker Technologies Based on Arbitrarily-Primed PCR Amplification

2002 ◽  
Vol 2 (3) ◽  
pp. 141-150 ◽  
Author(s):  
Julie Waldron ◽  
Cameron P. Peace ◽  
Iain R. Searle ◽  
Agnelo Furtado ◽  
Nick Wade ◽  
...  
Keyword(s):  
Dna Fingerprinting ◽  
Positional Cloning ◽  
Dna Marker ◽  
Pcr Amplification ◽  
Leaf Tissue ◽  
Dna Amplification ◽  
Sequence Information ◽  
Selective Basis ◽  
Genome Sequence Information ◽  
Arbitrarily Primed Pcr

Arbitrarily-primed DNA markers can be very useful for genetic fingerprinting and for facilitating positional cloning of genes. This class of technologies is particularly important for less studied species, for which genome sequence information is generally not known. The technologies include Randomly Amplified Polymorphic DNA (RAPD), DNA Amplification Fingerprinting (DAF), and Amplified Fragment Length Polymorphism (AFLP). We have modified the DAF protocol to produce a robust PCR-based DNA marker technology called Randomly Amplified DNA Fingerprinting (RAF). While the protocol most closely resembles DAF, it is much more robust and sensitive because amplicons are labelled with either radioactive33P or fluorescence in a 30-cycle PCR, and then separated and detected on large polyacrylamide sequencing gels. Highly reproducible RAF markers were readily amplified from either purified DNA or alkali-treated intact leaf tissue. RAF markers typically display dominant inheritance. However, a small but significant portion of the RAF markers exhibit codominant inheritance and represent microsatellite loci. RAF compares favorably with AFLP for efficiency and reliability on many plant genomes, including the very large and complex genomes of sugarcane and wheat. While the two technologies detect about the same number of markers per large polyacrylamide gel, advantages of RAF over AFLP include: (i) no requirement for enzymatic template preparation, (ii) one instead of two PCRs, and (iii) overall cost. RAF and AFLP were shown to differ in the selective basis of amplification of markers from genomes and could therefore be used in complementary fashion for some genetic studies.

Pseudomonas syringae pv. phaseolicola can be separated into two genetic lineages distinguished by the possession of the phaseolotoxin biosynthetic cluster

Microbiology ◽  
2004 ◽  
Vol 150 (2) ◽  
pp. 473-482 ◽  
Author(s):  
José A. Oguiza ◽  
Arantza Rico ◽  
Luis A. Rivas ◽  
Laurent Sutra ◽  
Alan Vivian ◽  
...  
Keyword(s):  
Pseudomonas Syringae ◽  
Dna Sequences ◽  
Dna Hybridization ◽  
Pcr Amplification ◽  
Pathogenicity Island ◽  
Large Plasmid ◽  
Genetic Lineages ◽  
Genomic Divergence ◽  
Arbitrarily Primed Pcr ◽  
Genomic Insertions

The bean (Phaseolus spp.) plant pathogen Pseudomonas syringae pv. phaseolicola is characterized by the ability to produce phaseolotoxin (Tox+). We recently reported that the majority of the Spanish P. syringae pv. phaseolicola population is unable to synthesize this toxin (Tox−). These Tox− isolates appear to lack the entire DNA region for the biosynthesis of phaseolotoxin (argK-tox gene cluster), as shown by PCR amplification and DNA hybridization using DNA sequences specific for separated genes of this cluster. Tox+ and Tox− isolates also showed genomic divergence that included differences in ERIC-PCR and arbitrarily primed-PCR profiles. Tox+ isolates showed distinct patterns of IS801 genomic insertions and contained a chromosomal IS801 insertion that was absent from Tox− isolates. Using a heteroduplex mobility assay, sequence differences were observed only among the intergenic transcribed spacer of the five rDNA operons of the Tox− isolates. The techniques used allowed the unequivocal differentiation of isolates of P. syringae pv. phaseolicola from the closely related soybean (Glycine max) pathogen, P. syringae pv. glycinea. Finally, a pathogenicity island that is essential for the pathogenicity of P. syringae pv. phaseolicola on beans appears to be conserved among Tox+, but not among Tox− isolates, which also lacked the characteristic large plasmid that carries this pathogenicity island. It is proposed that the results presented here justify the separation of the Tox+ and Tox− P. syringae pv. phaseolicola isolates into two distinct genetic lineages, designated Pph1 and Pph2, respectively, that show relevant genomic differences that include the pathogenicity gene complement.

Characterization of a cDNA for Rhesus Monkey Protein C Inhibitor – Evidence for N-Terminal Involvement in Heparin Stimulation

1995 ◽  
Vol 74 (04) ◽  
pp. 1079-1087 ◽  
Author(s):  
Klaus-P Radtke ◽  
José A Fernández ◽  
Bruno O Villoutreix ◽  
Judith S Greengard ◽  
John H Griffin
Keyword(s):  
Rhesus Monkey ◽  
Protein C ◽  
Activated Protein C ◽  
Pcr Amplification ◽  
Amino Acid Sequences ◽  
Heparin Binding ◽  
Reactive Center ◽  
Protein C Inhibitor ◽  
Polymerase Chain

SummarycDNAs for protein C inhibitor (PCI) were cloned from human and rhesus monkey 1 liver RNAs by reverse transcription and polymerase chain reaction (PCR) amplification. Sequencing showed that rhesus monkey and human PCI cDNAs were 93% identical. Predicted amino acid sequences differed at 26 of 387 residues. Pour of these differences (T352M, N359S, R362K, L3631) were in the reactive center loop that is important for inhibitory specificity, and two were in the N-terminal helix (M8T, E13K) that is implicated in glycosaminoglycan binding. PCI in human or rhesus monkey plasma showed comparable inhibitory activity towards human activated protein C in the presence of 10 U/ml heparin. However, maximal acceleration of the inhibition of activated protein C required 5-fold lower heparin concentration for rhesus monkey than for human plasma, consistent with the interpretation that the additional positive charge (E13K) in a putative-heparin binding region increased the affinity for heparin.

Simple and Rapid Detection of Factor V Leiden by Allele-specific PCR Amplification

1996 ◽  
Vol 75 (05) ◽  
pp. 757-759 ◽  
Author(s):  
Rainer Blasczyk ◽  
Markus Ritter ◽  
Christian Thiede ◽  
Jenny Wehling ◽  
Günter Hintz ◽  
...  
Keyword(s):  
Direct Sequencing ◽  
Activated Protein C ◽  
Factor V Leiden ◽  
Pcr Amplification ◽  
Factor V ◽  
Specific Pcr ◽  
Pcr Rflp ◽  
Allele Specific ◽  
Specific Amplification ◽  
Allele Specific Pcr

SummaryResistance to activated protein C is the most common hereditary cause for thrombosis and significantly linked to factor V Leiden. In this study, primers were designed to identify the factor V mutation by allele-specific PCR amplification. 126 patients with thromboembolic events were analysed using this technique, PCR-RFLP and direct sequencing. The concordance between these techniques was 100%. In 27 patients a heterozygous factor VGln506 mutation was detected, whereas one patient with recurrent thromboembolism was homozygous for the point mutation. Due to its time- and cost-saving features allele-specific amplification should be considered for screening of factor VGln506.

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