scholarly journals Selection of the most suitable method for the extraction of DNA from foods and feeds for species identification

2010 ◽  
Vol 58 (2) ◽  
pp. 169-174
Author(s):  
Michaela Nesvadbová ◽  
Aleš Knoll ◽  
Anna Vašátková
Keyword(s):  
Species Identification ◽  
Genomic Dna ◽  
Pcr Amplification ◽  
Degraded Dna ◽  
Dna Purification ◽  
Pcr Analysis ◽  
Isolation System ◽  
Dna Yield ◽  
Pcr Products ◽  
Food And Feed

High quality and purity of DNA isolated from food and feed is essential for species identification and has unpredictable influences an effect of analysis. In this study, the efficiency of eight different methods for DNA isolation was investigated. For DNA extraction, the raw chicken meet, ham, sausages, tinned lunch meat, pate, tinned feeds for dogs, complete granulated feeds for dogs and chicken flour were used. Kits of several different producers, i.e.: NucleoSpin Food (Marchery-Nagel), Wizard Genomic DNA Purification Kit (Promega), Invisorb Spin Food Kit I (Invitek), Wizard SV Genomic DNA Purification System (Promega), JetQuick Tissue DNA Spin Kit (Genomed), RNA Blue (Top-Bio), JetQuick Blood & Cell Culture Kit (Genomed), QIAamp DNA Mini Kit and QIAamp DNA Blood Mini Kit (Qiagen) were employed in the study. Gel agarose electrophoresis for primary verification of DNA quality was performed. The isolates were subsequently assessed for quantity and quality using by spectrophotometer Nanodrop 2000 (Thermo Scientific). To verify of template usability and quality of isolated DNA, the polymerase chain reaction (PCR) was used.Differences between isolated DNA from tinned products and meat, ham, sausage, granulated dog feed and chicken flour were found. In tinned food and feed, the DNA was more degraded, DNA content and DNA purity was lower and also PCR amplification was the most difficult. Overall DNA yield and quality have important influence on PCR products amplification. The best results were obtained with NucleoSpin Food and JetQuick Tissue DNA Spin Kit. DNA extracted by these methods proved highest yields, purity and template quality in all foods and feeds and the results of PCR analysis are excellent reproducible. Analyses showed that results depended on different food or feed using and dif­fe­rent isolation system.The results of this work will be utilized to choose the suitable isolating kit for educational course, which is designed for students and also for following research and analyses.

RT-PCR analysis of Na+/H+ exchanger mRNAs in rat medullary thick ascending limb

1995 ◽  
Vol 268 (6) ◽  
pp. F1224-F1228 ◽  
Author(s):  
P. Borensztein ◽  
M. Froissart ◽  
K. Laghmani ◽  
M. Bichara ◽  
M. Paillard
Keyword(s):  
Rat Kidney ◽  
Pcr Amplification ◽  
Restriction Enzymes ◽  
Pcr Analysis ◽  
Thick Ascending Limb ◽  
Rt Pcr ◽  
Medullary Thick Ascending Limb ◽  
Pcr Products ◽  
Polymerase Chain ◽  
Nhe Isoforms

The thick ascending limb (TAL) of rat kidney absorbs bicarbonate secondary to proton secretion, but displays both basolateral and luminal Na+/H+ exchange (NHE) activity. Several NHE genes, including NHE-1, NHE-2, NHE-3, and NHE-4, are expressed in the kidney. To identify the NHE isoforms expressed in the rat medullary TAL (MTAL), we used the reverse transcription-polymerase chain reaction (RT-PCR) to detect the mRNAs for NHE in microdissected MTAL. RT-PCR amplification from total RNA was performed between two specific primers for each NHE isoform. In rat kidney homogenate, the four NHE isoform mRNAs were detected, and the identity of the PCR products was demonstrated by the sizes of the fragments, digestion with restriction enzymes, and Southern blot analysis. In microdissected rat MTAL, NHE-3 was strongly expressed and NHE-1 mRNA was also detected, whereas NHE-2 and NHE-4 mRNAs were not detected. Therefore, NHE-3 could be the apical Na+/H+ exchanger, and NHE-1 could be the basolateral isoform in the MTAL.

Application of Bio-Cell Chip Technology to Rapid Genomic DNA Preparation and PCR Amplification.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4197-4197
Author(s):  
Bora Oh ◽  
Dong Soon Lee ◽  
Tae Young Kim ◽  
Hyun Jung Min
Keyword(s):  
Dna Extraction ◽  
Genomic Dna ◽  
Conflicts Of Interest ◽  
Pcr Amplification ◽  
Oligonucleotide Primer ◽  
Cylindrical Shape ◽  
Pdms Layer ◽  
Cell Chip ◽  
Chip Technology ◽  
Pcr Products

Abstract Abstract 4197 Background Though primary cells obtained from patients are necessary to scientists, it has been problems that their amount is not sufficient to perform various assays and that the cell specimens are often impaired, or their constituents or products are destroyed during the transport to the scientists. To address these problems, we developed a bio-cell chip, a chip having hundreds kind of cells arrayed and immobilized on a small slide. We arrayed the cells in a tiny space by spotting them onto a supporting matrix, so that hundreds of assays can be conducted with only a small amount of sample and several hundreds kinds of samples can be assayed under the same condition. We developed the direct extraction of DNA from bio-cell chip and successfully amplified the extracted DNA. Here, we introduce simple and convenient method of DNA extraction utilizing bio-cell chip, which can handle hundreds of specimen at one time. Methods Peripheral blood anticoagulated with EDTA or sodium citrate were centrifuged at 2000 rpm for 20 minutes, and leukocyte layers were transferred to 1.5 ml tube by careful pipetting. Buffy coats are adjusted to the cell count of 1,000 cells/ μl to 10,000 cells/ μl with Phosphate Buffered Saline. One microliter of cell suspension are loaded onto each of 92 well bio cell-chip and air-dried. To investigate the duration of DNA stability of cells loaded on bio cell-chip, loaded bio cell-chip were stored at room temperature for 5day, 15day, and 1 month before DNA extraction. Cell-chip preparation was done as previously published. Briefly, the substrate for the bio cell-chip was a slide glass on which a 1 mm thick perforated poly- dimethylsiloxane (PDMS) layer was bonded to form 16×6 wells array for subsequent cell seeding. Each cylindrical shape with 1.5 mm in diameter and positioned within a lattice pattern with its own indexing number printed on the slide glass. Maximum volume that can be loaded in each cylinder was 10ul. For DNA extraction, 2 ul of BR-A buffer (GenScript BloodReadyTMMultiplex PCR System) was dropped onto each of the wells of bio-cell chip and dried cells in the bottom of wells were mixed with BR-A buffer solution thoroughly by pipetting up and down for 10-15 times. Then, BR-A buffer additionally dropped into each well up to 20ul volume finally and mixed well. One or multiple gene targets can be amplified using a pair of primers or multiple pairs of primers from the mixture of buffer with cells without DNA purification. Then, PCR Amplification was carried out using the MJ Research DNA thermal cycler programmed to denature DNA at 95°C for 30s, and 40cycles of annealing at 62°C and extension at 72°C for 30sec in a 20μl mixture containing 1U BioTherm‘DNA Polymerase (Genecraft, GERMANY), 1μl(10pM/ul) of each oligonucleotide primer, 2.5mM dNTP, and genomic DNA. Results and conclusion The PCR amplification results were successful using BioTherm‘DNA Polymerase. When we carried out PCR amplification using the PCR Premix (BloodReadyTM PCR System), we had no PCR products. The target PCR products were observed from genomic DNA sample with above 100 cells. Also, PCR amplification for genomic DNA from stored cell for 5day, 15 day, and 1 month was successful. With this extraction method, PCR was successful and not only minimize the simplification of experimental procedure but also save the time. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Comparison Between Currently Used Blood Samples And New Saliva Dna Collection Method For Quality Of Genomic Dna And Genotyping

2012 ◽  
Vol 18 (1) ◽  
pp. 19-23
Author(s):  
Loredana Mariana Aftenie ◽  
Irina Franciuc ◽  
Alina Martinescu ◽  
Adina Honcea
Keyword(s):  
Genomic Dna ◽  
Response Rate ◽  
Saliva Sample ◽  
Pcr Amplification ◽  
Good Alternative ◽  
Pcr Analysis ◽  
Blood Samples ◽  
Biospecimen Collection ◽  
Whole Saliva

AbstractObtaining blood biospecimens presents logistical and financial challenges. As a result, saliva biospecimen collection is becoming more frequent because of the ease of collection and lower cost. This article describes an assessment of two different methods for collecting samples: whole blood and whole saliva samples used further for DNA extraction and HLA genotyping in immunogenic disease on a group of patients registered at our Molecular Genetics Laboratory Faculty of Medicine “Ovidius” University Constanţa. Our data show that only 81% of the requested participants delivered a blood sample, whereas 19% delivered a saliva sample because they refuse the first sampling method. Analysis of purified genomic DNA by Nano Photometer and agarose gel electrophoresis revealed that blood and saliva samples resulted in DNA with the best quality. PCR analysis showed that DNA from 100% of the blood samples and 93% of the saliva samples could be subsequently amplified. Our study shows that the response rate of self-collection saliva samples had to be considering for the patients that have a low response rate of blood sampling. The quality of genomic DNA from saliva samples was comparable with blood samples as assessed by purity, concentration, yield and PCR amplification. We conclude that the use of saliva samples is a good alternative to blood samples to obtain genomic DNA of high quality and it will considerably increase the participant’s response rate for genetic studies.

scholarly journals Genomic DNA PCR analysis to assess xenograft development in mouse mammary gland

BioTechniques ◽  
2020 ◽  
Vol 68 (4) ◽  
pp. 219-222
Author(s):  
Etienne Aujean ◽  
Johann Laubier ◽  
Nicolas Brun ◽  
Laurence Finot ◽  
Eric Chanat ◽  
...  
Keyword(s):  
Genomic Dna ◽  
Pcr Amplification ◽  
Mouse Mammary Gland ◽  
Pcr Analysis ◽  
Mammary Fat Pad ◽  
Mammary Stem ◽  
Droplet Pcr ◽  
Functional Capacities ◽  
Dna Pcr ◽  
Transplantation Model

The mouse transplantation model remains the most relevant methodology to assess the functional capacities of mammary cells and is particularly appropriate for investigations regarding mammary stem cells, whatever the species studied. Following xenotransplantation in mice mammary fat pad, the development of the xenograft is commonly evaluated by immunohistology. Here, we present a simple and rapid method to control the species specificity of a xenograft based on genomic DNA PCR amplification. DNA is extracted from the fixed samples intended for histology, thus allowing the reuse of precious samples. Standard and digital droplet PCR (requiring low DNA quantities) methods have been used to make the present method suitable for the analysis of xenotransplanted samples.

scholarly journals Comparison of 12 DNA extraction kits for vertebrate samples

2020 ◽  
pp. 67-77
Author(s):  
I. Martincová ◽  
T. Aghová
Keyword(s):  
Dna Extraction ◽  
Genomic Dna ◽  
Dna Purification ◽  
Research Projects ◽  
High Quality ◽  
Sample Weight ◽  
Crucial Step ◽  
Dna Yield ◽  
Vertebrate Tissue ◽  
Biology Research

Comparison of 12 DNA extraction kits for vertebrate samples Martincová, I. Aghová, T. Abstract Obtaining high quality DNA extractions is a crucial step for molecular biology research projects. At present, numerous protocols are available for vertebrate tissue extractions. In the present study we compared eleven column–based protocols and one HotSHOT protocol using similar conditions (i.e., type of sample, weight of starting material). We evaluated time of extraction, quality and quantity of DNA yield, and price of extraction for a single sample. Based on our analysis, the most successful kits for producing DNA with the highest concentration and purity are the JetQuick® Genomic DNA Purification Kit (Genomed) and the NucleoSpin® Tissue (Macherey–Nagel). Nevertheless, it is highly recommended to test various extraction kits with specific samples to find the optimal kit in all aspects of time, quality and cost for a particular project.

scholarly journals COMPARISON OF SIX COMMERCIALLY-AVAILABLE DNA POLYMERASES FOR DIRECT PCR

2013 ◽  
Vol 55 (6) ◽  
pp. 401-406 ◽  
Author(s):  
Masashi Miura ◽  
Chihiro Tanigawa ◽  
Yoshito Fujii ◽  
Satoshi Kaneko
Keyword(s):  
Dna Polymerase ◽  
Genomic Dna ◽  
Dna Polymerases ◽  
Pcr Amplification ◽  
Dna Purification ◽  
Blood Components ◽  
Direct Pcr ◽  
Taq Polymerase ◽  
Serological Studies ◽  
Original Amount

SUMMARY The use of a “direct PCR” DNA polymerase enables PCR amplification without any prior DNA purification from blood samples due to the enzyme's resistance to inhibitors present in blood components. Such DNA polymerases are now commercially available. We compared the PCR performance of six direct PCR-type DNA polymerases (KOD FX, Mighty Amp, Hemo KlenTaq, Phusion Blood II, KAPA Blood, and BIOTAQ) in dried blood eluted from a filter paper with TE buffer. GoTaq Flexi was used as a standard DNA polymerase. PCR performance was evaluated by a nested PCR technique for detecting Plasmodium falciparum genomic DNA in the presence of the blood components. Although all six DNA polymerases showed resistance to blood components compared to the standard Taq polymerase, the KOD FX and BIOTAQ DNA polymerases were resistant to inhibitory blood components at concentrations of 40%, and their PCR performance was superior to that of other DNA polymerases. When the reaction mixture contained a mild detergent, only KOD FX DNA polymerase retained the original amount of amplified product. These results indicate that KOD FX DNA polymerase is the most resistant to inhibitory blood components and/or detergents. Thus, KOD FX DNA polymerase could be useful in serological studies to simultaneously detect antibodies and DNA in eluents for antibodies. KOD FX DNA polymerase is thus not limited to use in detecting malaria parasites, but could also be employed to detect other blood-borne pathogens.

scholarly journals The Quality of DNA Isolated from Processed Food and Feed via Different Extraction Procedures

Molecules ◽  
2019 ◽  
Vol 24 (6) ◽  
pp. 1188 ◽  
Author(s):  
Zora Piskata ◽  
Eliska Servusova ◽  
Vladimir Babak ◽  
Michaela Nesvadbova ◽  
Gabriela Borilova
Keyword(s):  
Meat Products ◽  
Pcr Amplification ◽  
Single Species ◽  
Extraction Methods ◽  
Optimal Choice ◽  
Pcr Analysis ◽  
Processed Food ◽  
Extraction Procedures ◽  
Heat Treated ◽  
Food And Feed

The extraction of DNA is a critical step for species identification by PCR analysis in processed food and feed products. In this study, eight DNA extraction procedures were compared—DNeasy Blood and Tissue Kit, DNeasy mericon Food Kit, chemagic DNA Tissue 10 Kit, Food DNA Isolation Kit, UltraPrep Genomic DNA Food Mini Prep Kit, High Pure PCR Template Preparation Kit, phenol—chloroform extraction, and NucleoSpin Food—Using self-prepared samples from both raw and heat-processed and/or mechanically treated muscles and different types of meat products and pet food (pork, beef, and chicken). The yield, purity, and suitability of DNA for PCR amplification was evaluated. Additionally, comparisons between the effectiveness of various extraction methods were made with regard to price, and labor- and time-intensiveness. It was found that the DNeasy mericon Food Kit was the optimal choice for the extraction of DNA from raw muscle, heat-treated muscle, and homemade meat products from multiple and single species.

Effect of TP53 mutation status on survival in the MAGIC trial.

2015 ◽  
Vol 33 (3_suppl) ◽  
pp. 71-71 ◽  
Author(s):  
Elizabeth Catherine Smyth ◽  
Sanna Hulkki Wilson ◽  
Matthew Guy Nankivell ◽  
David Gonzalez de Castro ◽  
Andrew Wotherspoon ◽  
...  
Keyword(s):  
Genomic Dna ◽  
Tp53 Mutation ◽  
Pcr Amplification ◽  
Ffpe Tissue ◽  
Degraded Dna ◽  
P Value ◽  
Sequencing Analysis ◽  
Mutation Status ◽  
Tp53 Mutations ◽  
Tp53 Status

71 Background: In oesophagogastric cancer TP53 mutation may be prognostic and/or predict for chemoresistance, however available data are conflicting. We hypothesised that TP53 status would impact on survival for patients (pts) randomised to surgery alone or perioperative ECF chemotherapy in the MRC MAGIC trial. Methods: Genomic DNA FFPE tissue sections were extracted with QIAmp DNA FFPE Tissue Kit (Qiagen, Hilden, Germany). Mutations in exons 4-9 were screened for by fluorescent PCR -amplification of genomic DNA, followed by Capillary Electrophoresis-Single Strand Conformational Analysis (CE-SSCA). Mutations were characterised by bi -directional Sanger sequencing analysis performed on an independent PCR -reaction and the sequencing results were compared to the reference sequences in the COSMIC database. TP53 status was correlated with demographics and survival. Results: TP53 results were available on 154 pts (50% of pts with available DNA). The remainder failed CE-SSCA due to degraded DNA. Pts with/without TP53 data had similar demographics and survival. TP53 mutation was detected in 51/154 (33%) samples. Exon 4, 5, 6, 7 and 8 mutations occurred in 4%, 42%, 8%, 36% and 10% of specimens with a TP53 result. Pts with TP53 mutations were more likely to have an oesophageal or junctional tumor (vs. gastric) than TP53 wild type (WT) pts (38% vs. 17% respectively p = 0.016). Survival from surgery was comparable for pts with mutant and WT TP53 tumors in both arms of the trial (See Table). P-value for interaction between treatment group and TP53 status was 0.776, indicating no interaction was present. Conclusions: In the MAGIC trial, TP53 mutation status was not prognostic, and did not predict for lack of benefit from chemotherapy. Further study is required in order to evaluate the effect of differing TP53 mutations on treatment and survival. [Table: see text]

scholarly journals Presence of plasmid-mediated quinolone resistance (PMQR) genes in non-typhoidal Salmonella strains with reduced susceptibility to fluoroquinolones isolated from human salmonellosis in Gyeonggi-do, South Korea from 2016 to 2019

Gut Pathogens ◽  
2021 ◽  
Vol 13 (1) ◽  
Author(s):  
Sohyun Lee ◽  
Nanjoo Park ◽  
Sujung Yun ◽  
Eunseon Hur ◽  
Jiwon Song ◽  
...  
Keyword(s):  
South Korea ◽  
Pcr Amplification ◽  
Public Health Problem ◽  
Test Method ◽  
Quinolone Resistance ◽  
Human Salmonellosis ◽  
Pcr Products ◽  
Antimicrobial Susceptibility Tests ◽  
Susceptibility Tests ◽  
Sequence Types

AbstractNon-typhoidal salmonellosis remains a pressing public health problem worldwide. Quinolones, particularly fluoroquinolones, are widely used to treat various infections, including non-typhoidal salmonellosis, which can be a serious illness. The emergence of fluoroquinolone-resistant Salmonella has resulted in treatment failure and high mortality rates. In this study, we estimated the presence of plasmid-mediated quinolone resistance (PMQR) genes in Salmonella enterica isolated from human salmonellosis patients in South Korea from 2016 to 2019. We evaluated the association of these genes with fluoroquinolone susceptibility. Antimicrobial susceptibility tests for Salmonella isolates were performed using the Vitek II system, and the minimum inhibitory concentrations (MIC) of ciprofloxacin and levofloxacin were determined using the E-test method. Plasmid-mediated quinolone resistance (PMQR) genes were detected by PCR amplification and quinolone resistance-determining regions (QRDRs) of the gyrA and parC genes were analyzed following Sanger sequencing of the PCR products. Thirty-four Salmonella strains with reduced susceptibility to fluoroquinolones (ciprofloxacin MIC ≥ 0.125 µg/mL and levofloxacin MIC ≥ 0.25 µg/mL) were selected from 208 human clinical Salmonella isolates. Among them, 22 Salmonella strains harbored one PMQR gene (qnrA, qnrB, or qnrS), and three Salmonella strains carried two PMQR genes (qnrS and aac(6′)-Ib-cr or qnrA and qnrB). qnrS was the most common PMQR gene. Serotyping revealed that Salmonella 4,[5]12:i:- (32.4%, 11/34) and Salmonella Typhimurium (29.4%, 10/34) were the two most predominant serovars, and Multi-locus sequence typing (MLST) showed that ST19 and ST34 were the most frequent sequence types. In conclusion, qnr gene-positive Salmonella 4,[5],12:i:- and Salmonella Typhimurium were the main serovars responsible for reduced susceptibility to fluoroquinolones. Therefore, our findings suggest that PMQR-positive Salmonella strains, which can be isolated from various samples including human, food, and the environment, should be carefully monitored.

scholarly journals Single and Double Infections with Wolbachia in the Parasitic Wasp Nasonia vitripennis Effects on Compatibility

Genetics ◽  
1996 ◽  
Vol 143 (2) ◽  
pp. 961-972 ◽  
Author(s):  
Marie-Jeanne Perrot-Minnot ◽  
Li Rong Guo ◽  
John H Werren
Keyword(s):  
Genomic Dna ◽  
Rapid Development ◽  
Parasitic Wasp ◽  
Pcr Amplification ◽  
Bacterial Strains ◽  
Nasonia Vitripennis ◽  
Larval Diapause ◽  
Wide Range ◽  
Reproductive Incompatibility ◽  
Infected Line

Abstract Wolbachia are cytoplasmically inherited bacteria responsible for reproductive incompatibility in a wide range of insects. There has been little exploration, however, of within species Wolbachia polymorphisms and their effects on compatibility. Here we show that some strains of the parasitic wasp Nasonia vitripennis are infected with two distinct bacterial strains (A and B) whereas others are singly infected (A or B). Double and single infections are confirmed by both PCR amplification and Southern analysis of genomic DNA. Furthermore, it is shown that prolonged larval diapause (the overwintering stage of the wasp) of a double-infected strain can lead to stochastic loss of one or both bacterial strains. After diapause of a double-infected line, sublines were produced with AB, A only, B only or no Wolbachia. A and B sublines are bidirectionally incompatible, whereas males from AB lines are unidirectionally incompatible with females of A and B sublines. Results therefore show rapid development of bidirectional incompatibility within a species due to segregation of associated symbiotic bacteria.

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