scholarly journals Comparison of methods for isolating fungal DNA

2012 ◽  
Vol 29 (Special Issue) ◽  
pp. S76-S85 ◽  
Author(s):  
P. Moťková ◽  
J. Vytřasová
Keyword(s):  
Dna Isolation ◽  
Dna Amplification ◽  
Food Samples ◽  
Plant Leaves ◽  
Pcr Inhibitors ◽  
Detection Techniques ◽  
Cell Wall Disruption ◽  
Fungal Dna ◽  
Commercial Kits ◽  
The Individual

In this study methods of fungal DNA isolation were optimised and compared. The aim of the isolation processes was to obtain DNA of sufficient quality and quantity necessary for its amplification, as most detection techniques require DNA amplification before the proper DNA detection itself. For this purpose, classic methods of DNA extraction were compared and optimised while isolations using commercial kits were also done. The methods were evaluated from several perspectives, with focus especially laid on the isolated DNA not contain PCR inhibitors which would prevent DNA amplification, thus inhibiting the detection itself. For optimising the individual methods, collection strains of the genus Aspergillus were used. After the evaluation, two most suitable methods were selected and chosen for isolating potentially aflatoxigenic moulds taken from food samples. These methods were the commercially supplied kit for isolating DNA from plant leaves from Sigma and a classic method according Cenis in combination with the cell wall disruption by means of liquid nitrogen.

scholarly journals EFFICIENT METHOD OF DNA ISOLATION FROM BULKING SAMPLES OF SEEDLINGS

2021 ◽  
Vol 2021 (3) ◽  
pp. 29-48
Author(s):  
Irina Klimenko ◽  
Alexey Antonov ◽  
Vladimir Dushkin ◽  
Anastasia Shamustakimova ◽  
Yulian Mavlyutov
Keyword(s):  
Dna Isolation ◽  
Low Cost ◽  
Leaf Tissue ◽  
Perennial Grasses ◽  
Genetic Studies ◽  
Modified Method ◽  
Extraction Buffer ◽  
Low Efficiency ◽  
Commercial Kits ◽  
The Individual

Forage annual and perennial grasses are the difficult subject for molecular and genetic studies because of the problem with obtaining qualitative genomic DNA for PCR, due of high content of proteins, polysaccharides and polyphenols. The known methods of DNA extraction or the numerous commercial kits allow isolating purified nucleic acids from the leaf tissue, but characterized by low efficiency at seedlings using. The modified method of DNA isolation, based on the SDS-extraction buffer (sodium dodecil sulfate), is presented in this study. Significant modifications were introduced in the reagents compound and the steps of procedure accordingly to used type of plant tissue and the result was positive at usage on the bulking samples, as well as on the individual genotypes (the only seedling). Reliability of this method and the functionality of the obtained DNA samples were tested in PCR with different molecular markers (SSR, SRAP and PawS) in researches on revealing of forage legume grasses DNA polymorphism. The general advantages of the proposed method are simplicity and effectiveness, the possibility to isolate qualitative DNA without toxic reagents application, as well as relatively low cost and availability of reagents. This method can be useful for studying the genetic biodiversity and for decision the different tasks, required the rapid analysis of large plant populations.

scholarly journals Boiling Extraction Method VS Commercial Kits for Bacterial DNA Isolation from Food Samples

2020 ◽  
Vol 03 (04) ◽  
Author(s):  
Maria-Eleni Dimitrakopoulou ◽  
Venia Stavrou ◽  
Chrysoula Kotsalou ◽  
Apostolos Vantarakis
Keyword(s):  
Extraction Method ◽  
Dna Isolation ◽  
Food Samples ◽  
Bacterial Dna ◽  
Commercial Kits

scholarly journals Comparison of Methods for DNA Isolation from Food Samples for Detection of Shiga Toxin-Producing Escherichia coli by Real-Time PCR

2003 ◽  
Vol 69 (3) ◽  
pp. 1844-1846 ◽  
Author(s):  
Loree C. Heller ◽  
Carisa R. Davis ◽  
K. Kealy Peak ◽  
David Wingfield ◽  
Andrew C. Cannons ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Real Time ◽  
Shiga Toxin ◽  
Real Time Pcr ◽  
Dna Isolation ◽  
Food Samples ◽  
Pcr Detection ◽  
Comparison Of Methods ◽  
Commercial Kits ◽  
Shiga Toxin Genes

ABSTRACT In this study, food samples were intentionally contaminated with Escherichia coli O157:H7, and then DNA was isolated by using four commercial kits. The isolated DNA samples were compared by using real-time PCR detection of the Shiga toxin genes. The four kits tested worked similarly.

Extraction of DNA from a Wide Range of Objects by Treatment with Ammonium Salts

2020 ◽  
Vol 36 (6) ◽  
pp. 98-106
Author(s):  
E.I. Levitin ◽  
B.V. Sviridov ◽  
O.V. Piksasova ◽  
T.E. Shustikova
Keyword(s):  
Dna Extraction ◽  
Dna Isolation ◽  
Tissue Samples ◽  
New Approach ◽  
Cell Wall Disruption ◽  
Wide Range ◽  
Low Salt ◽  
Mammalian Blood ◽  
Plasmid Extraction ◽  
Two Hybrid

Currently, simple, rapid, and efficient techniques for DNA isolation from a wide range of organisms are in demand in biotechnology and bioinformatics. A key (and often limiting) step is the cell wall disruption and subsequent DNA extraction from the disintegrated cells. We have developed a new approach to DNA isolation from organisms with robust cell walls. The protocol includes the following steps: treatment of cells or tissue samples with ammonium acetate followed by cell lysis in low-salt buffer with the addition of SDS. Further DNA extraction is carried out according to standard methods. This approach is efficient for high-molecular native DNA isolation from bacteria, ascomycetes, yeast, and mammalian blood; it is also useful for express analysis of environmental microbial isolates and for plasmid extraction for two-hybrid library screening. express method for DNA isolation; ammonium salt treatment (в русских ключевых такой порядок), osmotic breakage of cells This study was financially supported by the NRC "Kurchatov Institute"-GOSNIIGENETIKA Kurchatov Genomic Center.

An efficient protocol for the isolation of fungal DNA for strains obtained from oil contaminated fields

2012 ◽  
Vol 2 (4) ◽  
pp. 162-165
Author(s):  
Bhavya Ravi ◽  
Madhulika Rai ◽  
Sandhya Mehrotra ◽  
Rajesh Mehrotra
Keyword(s):  
Contaminated Soils ◽  
Petroleum Hydrocarbons ◽  
Dna Isolation ◽  
Cost Effective ◽  
Industrial Applications ◽  
Glass Beads ◽  
Natural Ecosystem ◽  
Dna Yield ◽  
Genomic Study ◽  
Fungal Dna

A natural ecosystem contaminated with petroleum hydrocarbons is likely to favor the growth of taxonomically diverse microbes having the ability to degrade these organic compounds. They can be exploited for purposes like bioremediation of oil contaminated soils and to obtain enzymes like lipases having important industrial applications. In this paper, a novel “IBG” (Improved ‘Bust and Grab’) protocol has been reported for the isolation of fungal DNA from strains collected from oil contaminated fields. Conventional methods for DNA isolation from fungi require the use of enzymes, liquid nitrogen, glass beads etc. The method reported here circumvents the use of enzymes or glass beads and is cost effective and can be used while handling large number of samples. The DNA yield obtained by the IBG protocol is significant and of good quality. The good quality DNA isolated by IBG protocol can be used for the quick and cost effective isolation of fungal genomic DNA facilitating the genomic study of microbes obtained from oil contaminated fields.

scholarly journals Capacity of Nine Thermostable DNA Polymerases To Mediate DNA Amplification in the Presence of PCR-Inhibiting Samples

1998 ◽  
Vol 64 (10) ◽  
pp. 3748-3753 ◽  
Author(s):  
Waleed Abu Al-Soud ◽  
Peter Rådström
Keyword(s):  
Dna Polymerase ◽  
Biological Samples ◽  
Detection Method ◽  
Dna Polymerases ◽  
Dna Amplification ◽  
Nucleic Acid Amplification ◽  
Full Potential ◽  
Thermus Aquaticus ◽  
Pcr Inhibitors ◽  
Thermostable Dna Polymerase

ABSTRACT The PCR is an extremely powerful method for detecting microorganisms. However, its full potential as a rapid detection method is limited by the inhibition of the thermostable DNA polymerase fromThermus aquaticus by many components found in complex biological samples. In this study, we have compared the effects of known PCR-inhibiting samples on nine thermostable DNA polymerases. Samples of blood, cheese, feces, and meat, as well as various ions, were added to PCR mixtures containing various thermostable DNA polymerases. The nucleic acid amplification capacity of the nine polymerases, under buffer conditions recommended by the manufacturers, was evaluated by using a PCR-based detection method for Listeria monocytogenes in the presence of purified template DNA and different concentrations of PCR inhibitors. The AmpliTaqGold and the Taq DNA polymerases from Thermus aquaticus were totally inhibited in the presence of 0.004% (vol/vol) blood in the PCR mixture, while the HotTub, Pwo, rTth, andTfl DNA polymerases were able to amplify DNA in the presence of 20% (vol/vol) blood without reduced amplification sensitivity. The DNA polymerase from Thermotoga maritima(Ultma) was found to be the most susceptible to PCR inhibitors present in cheese, feces, and meat samples. When the inhibitory effect of K and Na ions was tested on the nine polymerases, HotTub from Thermus flavus and rTthfrom Thermus thermophilus were the most resistant. Thus, the PCR-inhibiting effect of various components in biological samples can, to some extent, be eliminated by the use of the appropriate thermostable DNA polymerase.

Methods for Detection of Anticarsia gemmatalis Nucleopolyhedrovirus DNA in Soil

1999 ◽  
Vol 65 (6) ◽  
pp. 2307-2311 ◽  
Author(s):  
R. R. de Moraes ◽  
J. E. Maruniak ◽  
J. E. Funderburk
Keyword(s):  
Laboratory Experiments ◽  
Dna Isolation ◽  
Pcr Amplification ◽  
Soil Samples ◽  
Anticarsia Gemmatalis ◽  
Isolation Method ◽  
Viral Dna ◽  
Pcr Inhibitors ◽  
Magnetic Capture ◽  
Pcr Products

ABSTRACT Two methods, phenol-ether and magnetic capture-hybridization (MCH), were developed and compared with regard to their sensitivities and abilities to extract the DNA of the insect baculovirus Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) from soil and to produce DNA amplifiable by PCR. Laboratory experiments were performed with 0.25 g of autoclaved soil inoculated with different viral concentrations to optimize both methods of baculovirus DNA extraction and to determine their sensitivities. Both procedures produced amplifiable DNA; however, the MCH method was 100-fold more sensitive than the phenol-ether procedure. The removal of PCR inhibitors from the soil appeared to be complete when MCH was used as the viral DNA isolation method, because undiluted aliquots of the DNA preparations could be amplified by PCR. The phenol-ether procedure probably did not completely remove PCR inhibitors from the soil, since PCR products were observed only when the AgMNPV DNA preparations were diluted 10- or 100-fold. AgMNPV DNA was detected in field-collected soil samples from 15 to 180 days after virus application when the MCH procedure to isolate DNA was coupled with PCR amplification of the polyhedrin region.

scholarly journals Prospects for rapid bioluminescent detection methods in the food industry – a review

2011 ◽  
Vol 23 (No. 3) ◽  
pp. 85-92 ◽  
Author(s):  
P. Dostálek ◽  
T. Brányik
Keyword(s):  
Historical Development ◽  
Food Industry ◽  
Food Samples ◽  
Detection Methods ◽  
Rapid Methods ◽  
Detection Techniques ◽  
Atp Assay ◽  
Advantages And Disadvantages ◽  
Bacterial Bioluminescence ◽  
Fluorescent Method

This review surveys rapid bioluminescent detection techniques applied in food industry and discusses the historical development of the rapid methods. These techniques are divided into two groups: methods based on bioluminescent adenosine triphosphate (ATP) assay, and on bacterial bioluminescence. The advantages and disadvantages of these methods are described. The article provides the bibliography of fluorescent method applications in food samples.    

scholarly journals Determining the optimal method for DNA isolation from fruit jams

2018 ◽  
Vol 36 (No. 2) ◽  
pp. 126-132
Author(s):  
Sovová Tereza ◽  
Křížová Barbora ◽  
Ovesná Jaroslava
Keyword(s):  
Dna Extraction ◽  
Dna Isolation ◽  
Extraction Methods ◽  
Food Samples ◽  
Pcr Analysis ◽  
Uv Spectrophotometry ◽  
Pcr Assay ◽  
Optimal Method ◽  
Ctab Method ◽  
Dna Extraction Methods

DNA extraction is a crucial step in PCR analysis especially when analysing food samples that can be degraded and can potentially contain PCR-inhibiting substances. In this study, we compared the suitability of three DNA extraction methods – two kits: DNeasy<sup>®</sup> Plant Mini Kit and NucleoSpin<sup>®</sup> Food, and the CTAB method – for DNA extraction from commercial fruit jams. Fourteen jams with different contents of fruit, sugar and other additives were extracted in triplicate using the above-mentioned methods directly and after a washing step. The concentration and optical density were analysed using UV spectrophotometry and the amplifiability of the obtained DNA was evaluated using a PCR assay targeting a sequence coding for chloroplast tRNA-Leu. Samples isolated using the NucleoSpin<sup>®</sup> Food kit contained non-amplifiable DNA in eight cases, and samples isolated using the CTAB method could not be quantified. The DNeasy<sup>®</sup> Plant Mini Kit thus proved to be the most suitable method, since well-amplifiable DNA was obtained for all the analysed samples.

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