Extraction and random primer PCR detection of genomic DNA of parasitic mites Demodex folliculorum and Demodex brevis(Acari:Demodicidae)
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[Objective] To explore the extraction methods of genomic DNA of parasitic mites Demodex folliculorum (D.f.) and Demodex brevis (D.b.). [Methods] Mites were disrupted by repeated freezing and thawing in liquid nitrogen. Then,the improved DNA extraction method of mini-insects,alkaline lysis and the commercial extraction kit were used respectively to extract genomic DNA of D.f. and D.b.,which had been kept freezing for ﹤5 or 8-10 months. [Results] The results of protein and nucleic acid radiometer showed the purity and quantity of genomic DNA extracted by the commercial kit were obviously better than those by other methods of mini-insects and alkaline lysis. Clear DNA fingerprints were found in the amplification results of random amplified polymorphic DNA (RAPD). There were obviously different banding patterns between D.f. and D.b. The freezing time affected the quantity of DNA extraction,but had only small effect on the purity and RAPD fingerprints of genomic DNA. The same species of mites had similar banding pattern even using different extraction methods. More and clear bands were detected by the commercial extraction kits and improved DNA extraction method of mini-insects. The bands detected by alkaline lysis method were less and obscure. [Conclusions] The application of repeated freezing-thawing in liquid nitrogen acts effectively to disrupt mites. The mites had better not be kept frozen more than 6 months. The commercial extraction kit is an effective approach to extract genomic DNA of mites. RAPD technique provides a simple method for detecting and classifying D.f. and D.b.Keywords:
genomic DNA
Primer (cosmetics)
Lysis buffer
Alkaline lysis
DNA profiling
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Canine demodicosis occurs when Demodex mites colonize hair follicles in large numbers. Their characterization is based on morphologic differences. A molecular genetic analysis within Demodex mite species and between other mite species would better characterize their phylogenetic diversity and could assist in treatment strategies as well as improve our understanding of disease progression. Demodex canis mites were obtained during routine deep scrapings of client-owned dogs and collected in Tris EDTA buffer (TE). The mites were separated from debris with a microhematocrit suction apparatus. Their outer chitin layer was ruptured using chitinase, glass beads, and vortexing. Membranes and proteins were disrupted with Sodium Dodecyl Sulfate (SDS) and proteinase K. Phenol/chloroform extraction, ethanol precipitation, and resuspension in TE further purified the nucleic acids. One hundred ng of mite DNA was amplified by a Random Amplified Polymorphic DNA (RAPD) method as DNA yields were low. The amplified DNA was randomly cloned into plasmids and 10 clones were sequenced. The BLAST program in Macvector version 9.0TM was used to identify sequences that matched insect-type DNAs. Two oligonucleotide primer sets were designed to amplify the mite DNA by polymerase chain reaction (PCR). One sequence was homologous to ubiquitin and the other matched a bacterial species related to those commonly found as commensals in insects. The latter primer set also amplified a similar sequence from flea and mosquito DNA. Neither amplified dog DNA. This method was successful in isolating DNA from Demodex canis mites. Oligonucleotide primers were developed that amplify Demodex sequences and will be useful in analyzing phylogenetic relationships and may assist in understanding demodicosis progression. Extraction and Characterization of DNA from Demodex canis Elizabeth Toops, MS, DVM1 Byron Blagburn, MS, PhD2 Scott Lenaghan, PhD3 Robert. Kennis, MS, DVM, DACVD4 John MacDonald, MEd, DVM, DAVCD4 Christine Dykstra, MS, PhD2 1Pittsburgh Veterinary Specialty and Emergency Center, Pittsburgh Veterinary Dermatology, Pittsburgh, Pennsylvania, USA 2Department of Pathobiology, Auburn University College of Veterinary Medicine, Auburn, Alabama, USA 3Department of Mechanical, Aerospace and Biomedical Engineering, University of Tennessee, Knoxville, Tennessee, USA 4Department of Clinical Sciences, Auburn University College of Veterinary Medicine, Auburn, Alabama, USA
Chitinase
Primer (cosmetics)
Amplicon
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Demodex folliculorum
Population Genetics
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Objective To explore the best method for degenerate primer PCR extraction of genomic DNA from the Pythium sp.GY1938 strain.Methods Five methods were used to extract genomic DNA and their efficiency was separately evaluated with specific and degenerate primer PCR.Results Results of specific primer PCR showed that there were no significant differences in the five methods.However,results of degenerate primer PCR showed that the benzyl chloride method was more effective because it a larger number of clearer bands.Conclusion The benzyl chloride method was best suited to degenerate primer PCR extraction of genomic DNA from the Pythium sp.GY1938 strain.
Primer (cosmetics)
genomic DNA
Primer dimer
Strain (injury)
In silico PCR
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[Objective] Analysis of genomic DNA polymorphism and the related sequence of Demodex folliculorum and D.brevis.[Methods] The genomic DNA of the human Demodex was extracted by using improved DNA extraction method of mini-insects.Random amplified polymorphic DNA(RAPD)was applied to analyze the polymorphism.The related bands were connected with pMD18-T vector,and cloned,sequenced,and identified and analyzed after enzyme digestion.[Results] There were 15 bands obtained in D.folliculorum and 12 in D.brevis.Some bands were shared by the two mites while others were species-specific.The genetic distance between the two Demodex species was 0.5556.After recombination,the sequence of the specific band(about 800 bp)of D.folliculorum was found to be 855 bp(GenBank accession no.FI277970)in size.The 855 bp fragment was confirmed to be characteristic for D.folliculorum according to the PCR result with a specific primer and the result of enzyme digestion analysis.This sequence had 46% similarity to AraC-type DNA-binding domain-containing protein.The shared fragments(about 300 bp)by the two mites were both 341 bp in length(GenBank accession no.FI520176 and FI520175,respectively),and with two different bases at the 84th and 165th sites,which were A/G and C/T permutation,respectively.Their homology was 99.4%,but there was no ORF and high similar sequence.[Conclusions] The 855 bp fragment is specific to D.folliculorum.The 341 bp fragment is shared by D.folliculorum and D.brevis with 99.4% homology.RAPD can be used in genomic DNA polymorphism analysis and species identitfication of D.folliculorum and D.brevis.
Demodex folliculorum
genomic DNA
Homology
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Demodex folliculorum
Animal ecology
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Proteinase K
Lysis buffer
DNA profiling
Isolation
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genomic DNA
Demodex folliculorum
Primer (cosmetics)
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IntroductionPolymerase chain reaction (PCR) is a sensitive and specific molecular-based technique used in identification of causative pathogens in infectious diseases.There are numerous modalities of PCR including, for example, simple conventional PCR, nested PCR, multiplex PCR, random amplified polymorphic DNA (RAPD), and real-time PCR.Microsatellite-based PCR utilizes short nucleotide repeats as primer(s) targeting microsatellites which comprise DNA tandem repeats in the target genome [1].It is considered as a simple, rapid, single-step PCR, and proved to have utility in detection, species differentiation, strain identification and genotyping of several bacteria and fungi [2][3][4].The key factor used in the interpretation of the results of this PCR variety is the band pattern of the amplified products electrophoresed in agarose gel.This pattern depends mainly on polymorphisms in lengths of the target repetitive DNA sequences in tested nucleic acid.Many simple repetitive primers
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Objective To introduce a DNA extraction method from yeasts,Prototheca and filamentous fungi for PCR.Methods Clinical isolated and stored strains including unknown yeasts (5 strains) and filamentous fungi (6 strains),Trichosporon dermatis (1 strain),Malassezia furfur (1 strain),Paecilomyces (1 strain),Phoma (1 strain),Prototheca (1 strain),Candida guilliermondii (5 strains) and Aspergillus fumigatus (2 strains) were tested.Lyticase combined with Biospin Fungus Genomic DNA Extraction Kit were used to extract the DNA according to the manufacturer's protocol. The DNA purity was checked by analyzes the ratio of A260/A280 and its concentration was calculated. The universal fungal primers ITS1/ITS4 were used for PCR reaction, to amplify ITS region of ribosome RNA gene (rDNA),by means of the gained DNA as the templates.Results All of 23 tested fungi's genomic DNA could be extracted successfully,and the ratio of A260/A280 ranged from 1.6 to 2.0.All of the PCR products appeared clear DNA bands after electrophoresis.Conclusions Combination of lyticase with Biospin Fungus Genomic DNA Extraction Kit is a convenient and feasible way to extract genomic DNA from yeasts,Prototheca and filamentous fungi for PCR reaction.
genomic DNA
Paecilomyces
Strain (injury)
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Cloning (programming)
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