Actinobacillus actinomycetemcomitansgenetic heterogeneity: amplification of JP2-likeltxpromoter pattern correlated with specific arbitrarily primed polymerase chain reaction (AP-PCR) genotypes from human but not marmoset Brazilian isolates

2002 ◽  
Vol 48 (7) ◽  
pp. 602-610 ◽  
Author(s):  
L Saddi-Ortega ◽  
M A.R Carvalho ◽  
P S Cisalpino ◽  
E S.A Moreira
Keyword(s):  
Polymerase Chain Reaction ◽  
Clinical Manifestations ◽  
Pcr Amplification ◽  
Actinobacillus Actinomycetemcomitans ◽  
Periodontal Pathogen ◽  
Chain Reaction ◽  
Arbitrary Primers ◽  
Close Relationship ◽  
Host Origin ◽  
Polymerase Chain

Specific clonal types of Actinobacillus actinomycetemcomitans, a major human periodontal pathogen, may be responsible for clinical manifestations and the production of leukotoxin virulence factors. Leukotoxicity is associated with genetic polymorphism at the promoter region of the leukotoxin (ltx) gene. Here, we describe the use of arbitrarily primed polymerase chain reaction (AP-PCR) and ltx promoter PCR to molecularly characterise 35 A. actinomycetemcomitans Brazilian isolates: 21 of human origin and 14 from captive marmosets (Callitrix spp., primates commonly used as animal models for periodontal research). The discriminative capacity of each of 12 arbitrary primers was found to be variable, yielding between 3 and 24 PCR amplitypes. Combination of the results for all primers led to characterisation of 14 genotypes that grouped into four major clusters based on genetic similarity. Clusters 2, 3, and 4 were discriminative to host origin. A correlation with periodontal disease was suggested for strains belonging to clusters 3 and 4. The JP2-like PCR amplification pattern, associated with highly leukotoxic strains, was exclusive to human isolates and present in 29% of human isolates where it occurred in close relationship with AP genotypes L and J (cluster 3).Key words: Actinobacillus actinomycetemcomitans, marmosets, AP-PCR, leukotoxin.

scholarly journals Using Polymerase Chain Reaction-based DNA Amplification to Fingerprint North American Potato Cultivars

HortScience ◽  
1996 ◽  
Vol 31 (1) ◽  
pp. 130-133 ◽  
Author(s):  
B. Sosinski ◽  
D.S. Douches
Keyword(s):  
Polymerase Chain Reaction ◽  
North American ◽  
Genetic Relationships ◽  
Pcr Amplification ◽  
Dna Amplification ◽  
Chain Reaction ◽  
Arbitrary Primers ◽  
Polymerase Chain ◽  
Tuber Type

DNA from 46 North American potato (Solanum tuberosum L.) cultivars was examined using the polymerase chain reaction (PCR) with 16 arbitrary primers of 10 nucleotide length (10 mers) to determine the efficiency of randomly amplified polymorphic DNA (RAPD) in delineating cultivars, both sexually derived and clonal variants. The 16 primers yielded 43 useful polymorphisms that were evaluated according to the presence or absence of fragments of equal size. All cultivars were discriminated with as few as 10 primers. The russet sport of Burbank was distinguished from a white-skinned clone by one band. More primers (29) were examined to identify a band polymorphism among six Russet Burbank clonal variants. When the cultivars were grouped by tuber type (excluding the russet clonal variants), three to four primers discriminated these commonly grown cultivars. Determination of cultivar integrity was accomplished with PCR amplification, regardless of tissue source (leaf vs. tuber) for DNA extraction. Cluster analysis based on RAPD markers was performed to examine pedigree relationships of the cultivars. Genetic relationships correlated with some pedigrees; however, many exceptions were noted.

Using a Polymerase Chain Reaction as a Complementary Test to Improve the Detection of Dermatophyte Fungus in Nails

2014 ◽  
Vol 104 (3) ◽  
pp. 233-237 ◽  
Author(s):  
María José Iglesias Sánchez ◽  
Ana María Pérez Pico ◽  
Félix Marcos Tejedor ◽  
María Jesús Iglesias Sánchez ◽  
Raquel Mayordomo Acevedo
Keyword(s):  
Polymerase Chain Reaction ◽  
Dna Sequences ◽  
Classical Method ◽  
Clinical Manifestations ◽  
Culture Method ◽  
Clinical Samples ◽  
Chain Reaction ◽  
Nested Polymerase Chain Reaction ◽  
Traditional Procedure ◽  
Polymerase Chain

Background Dermatomycoses are a group of pathologic abnormalities frequently seen in clinical practice, and their prevalence has increased in recent decades. Diagnostic confirmation of mycotic infection in nails is essential because there are several pathologic conditions with similar clinical manifestations. The classical method for confirming the presence of fungus in nail is microbiological culture and the identification of morphological structures by microscopy. Methods We devised a nested polymerase chain reaction (PCR) that amplifies specific DNA sequences of dermatophyte fungus that is notably faster than the 3 to 4 weeks that the traditional procedure takes. We compared this new technique and the conventional plate culture method in 225 nail samples. The results were subjected to statistical analysis. Results We found concordance in 78.2% of the samples analyzed by the two methods and increased sensitivity when simultaneously using the two methods to analyze clinical samples. Now we can confirm the presence of dermatophyte fungus in most of the positive samples in just 24 hours, and we have to wait for the result of culture only in negative PCR cases. Conclusions Although this PCR cannot, at present, substitute for the traditional culture method in the detection of dermatophyte infection of the nails, it can be used as a complementary technique because its main advantage lies in the significant reduction of time used for diagnosis, in addition to higher sensitivity.

scholarly journals Amplification of human beta-actin gene by the reverse transcriptase-polymerase chain reaction: implications for assessment of RNA from formalin-fixed, paraffin-embedded material.

1996 ◽  
Vol 44 (10) ◽  
pp. 1205-1207 ◽  
Author(s):  
A Dakhama ◽  
V Macek ◽  
J C Hogg ◽  
R G Hegele
Keyword(s):  
Polymerase Chain Reaction ◽  
Pcr Amplification ◽  
Housekeeping Gene ◽  
Chain Reaction ◽  
Viral Genomes ◽  
Negative Results ◽  
Beta Actin ◽  
Target Nucleic Acid ◽  
Formalin Fixed Paraffin Embedded ◽  
Polymerase Chain

The polymerase chain reaction (PCR) is a powerful method that allows enzymatic amplification of rate target nucleic acid sequences. It has been applied to the amplification of viral genomes from paraffin-embedded pathology specimens. However, interpretation of negative results requires amplification of a housekeeping gene such as beta-actin. In the present study we used specific oligonucleotide primers previously designed to amplify both the genomic DNA and the mRNA transcript from paraffin-embedded tissue. These products have predicted sizes of 250 BP and 154 BP, respectively, but our results showed that PCR amplification only (without reverse transcription) unexpectedly generated the 154-BP product. Further investigation of the nature of this product demonstrated that it originated from the amplification of DNA, not RNA. We conclude that the 154-BP product generated by these primers cannot be exclusively considered as beta-actin RNA product and should not be used to assess successful extraction of RNA, to ascertain its integrity, or to normalize for the total amount of RNA assayed by RT-PCR from paraffin-embedded tissue.

Genetic diversity and phylogeny analysis of Azolla based on DNA amplification by arbitrary primers

Genome ◽  
1993 ◽  
Vol 36 (4) ◽  
pp. 686-693 ◽  
Author(s):  
Benoit Van Coppenolle ◽  
Iwao Watanabe ◽  
Charles Van Hove ◽  
Gerard Second ◽  
Ning Huang ◽  
...  
Keyword(s):  
Principal Component Analysis ◽  
Polymerase Chain Reaction ◽  
Principal Component ◽  
Dna Amplification ◽  
Component Analysis ◽  
Genetic Distances ◽  
Group Method ◽  
Chain Reaction ◽  
Arbitrary Primers ◽  
Polymerase Chain

The polymerase chain reaction was used to amplify random sequences of DNA from 25 accessions of Azolla to evaluate the usefulness of this technique for identification and phylogenetic analysis of this aquatic fern. Accessions were selected to represent all known species within the genus Azolla and to encompass the worldwide distribution of the fern. Primers of 10 nucleotides with 70% G + C content were used to generate randomly amplified polymorphic DNA from the symbiotic Azolla–Anabaena complex. Twenty-two primers were used and each primer gave 4–10 bands of different molecular weights for each accession. Bands were scored as present or absent for each accession and variation among accessions was quantified using Nei's genetic distances. A dendrogram summarizing phenetic relationships among the 25 accessions was generated using the unweighted pair-group method with arithmetic mean. Principal component analysis was also used to evaluate genetic similarities. Three distinct groups were identified: group 1 contains five species, group 2 contains the pinnata species, and group 3 contains the nilotica species. The analysis demonstrates that the major groups of Azolla species can be easily distinguished from one an other and, in addition, that closely related accessions within species can be identified. We further found that using 10 primers, a phylogeny that is essentially the same as that derived from 22 primers can be constructed. Our results suggest that total DNA extracted from the Azolla–Anabaena symbionts is useful for classification and phylogenetic studies of Azolla.Key words: Azolla–Anabaena symbiosis, genetic distances, polymerase chain reaction, principal component analysis.

scholarly journals A Self-Contained Portable Polymerase-Chain-Reaction System Integrated with Electromagnetic Mini-Actuators for Bi-Directional Fluid Transport

2011 ◽  
Vol 27 (3) ◽  
pp. 357-364
Author(s):  
B. T. Chia ◽  
S.-A. Yang ◽  
M.-Y. Cheng ◽  
C.-W. Lin ◽  
Y.-J. Yang
Keyword(s):  
Polymerase Chain Reaction ◽  
Fluid Transport ◽  
Point Of Care ◽  
Reaction System ◽  
Thermal Characteristics ◽  
Pcr Amplification ◽  
Chain Reaction ◽  
Rapid Temperature ◽  
Small Footprint ◽  
Polymerase Chain

ABSTRACTIn this paper, the development of a portable polymerase chain reaction (PCR) device is presented. Integrating electromagnetic mini-actuators for bi-directional fluid transport, the proposed device, whose dimension is 67mm × 66mm × 25mm, can be fully operated with a 5V DC voltage. The device consists of four major parts: A disposable channel chip in which PCR mixture is manipulated and reacted, a heater chip which generates different temperature zones for PCR reaction, a linear actuator array for pumping PCR mixture, and a circuit module for controlling and driving the system. The advantages of the device include the rapid temperature responses associated with continuous-flow-type PCR devices, as well as the programmable thermal cycling associated with chamber-type PCR devices. The thermal characteristics are measured and discussed. PCR amplification is successfully performed for the 122 bp segment of MCF-7/adr cell line. Due to its small footprint, this self-contained system potentially can be employed for point-of-care (POC) applications.

scholarly journals Method Comparison of DNA Isolation and Quantification for Fish and Seafood Authenticity Determination

2018 ◽  
Vol 13 (3) ◽  
pp. 115
Author(s):  
Dwiyitno Dwiyitno ◽  
Stefan Hoffman ◽  
Koen Parmentier ◽  
Chris Van Keer
Keyword(s):  
Polymerase Chain Reaction ◽  
Dna Extraction ◽  
Dna Isolation ◽  
Blue Mussel ◽  
Pcr Amplification ◽  
Amplification Efficiency ◽  
Chain Reaction ◽  
Seafood Products ◽  
Polymerase Chain ◽  
Commercial Kit

Fish and seafood products has been commonly targeted for fraudulent activities. For that reason, authentication of fish and seafood products is important to protect consumers from fraudulent and adulteration practices, as well as to implement traceability regulation. From the viewpoint of food safety, authenticity is beneficial to protect public from serious food poisoning incidents, such as due to ingestion of toxic species. Since DNA based identification depends on the nucleic acid polymerase chain reaction (PCR), the quantity and quality/purity of DNA will contribute significantly to the species authentication. In the present study, different DNA extraction and purification methods (3 classical methods and one commercial kit) were compared to produce the better isolated DNA for PCR amplification. Additionally, different methods for the estimation of DNA concentration and purity which is essential for PCR amplification efficiency were also evaluated. The result showed that classical DNA extraction methods (based on TNES-Urea) yielded a higher amount of DNA (11.30-323.60 ng/g tissue) in comparison to commercial kit/Wizard Promega (5.70-83.45 ng/g tissue). Based on the purity of DNA extract (A260/280), classical DNA extraction method produced relatively similar on DNA quality to the commercial kit (1.79-2.12). Interestingly, all classical methods produced DNA with A260/280 ratio of more than 2.00 on the blue mussel, in contrast with commercial kit. The commercial kit also produced better quality of DNA compared to the classical methods, showing the higher efficiency in PCR amplification. NanoDrop is promising as cheap, robust and safe UV-spectrophotometer method for DNA quantification, as well as the purity evaluation.Keywords: seafood authenticity, DNA isolation, polymerase chain reaction, NanoDrop, Picogreen

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