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.

scholarly journals Comparative investigation of DNA extraction methods in black gram (Vigna mungo (L.)

2019 ◽  
Author(s):  
M. . Prakash ◽  
B. . Priyadharshini ◽  
M. . Vignesh ◽  
R. . Anandan
Keyword(s):  
Dna Extraction ◽  
Extraction Method ◽  
Large Scale ◽  
Dna Isolation ◽  
Extraction Methods ◽  
Comparative Investigation ◽  
Black Gram ◽  
Dna Extraction Method ◽  
Ctab Method ◽  
Dna Extraction Methods

Isolation of intact, double stranded, pure and non- contaminated genomic DNA is prerequisite for large scale genotyping analysis including DNA-banks. Three methods of DNA isolation (Dellaporta, CTAB and Hi-PurAg DNA isolation kits) from 25 black gram genotypes were compared in terms of the yield, purity, integrity, and stability of extracted DNA. Purity and quantification of isolated DNA samples was confirmed by using the UV nano-spectrophotometer at OD260/280 and the same is confirmed based by agarose gel electrophoresis. The CTAB method showed the best results followed by Hi-PurAg and Dellaporta method. The CTAB DNA extraction method was found to be the most efficient DNA extraction method, capable of providing high quality, pure and stable DNA and could be used for various molecular related works. All the 25 black gram genotypes for this research gave good yield of DNA from the established modified CTAB protocol.

Detection of tet(M) Gene from Raw Milk by Rapid DNA Extraction Followed by a Two-Step PCR with Nested Primers

2004 ◽  
Vol 67 (12) ◽  
pp. 2833-2838 ◽  
Author(s):  
B. T. CENCI-GOGA ◽  
S. CROTTI ◽  
S. COSTARELLI ◽  
C. RONDINI ◽  
M. KARAMA ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Direct Detection ◽  
Raw Milk ◽  
Extraction Methods ◽  
Food Samples ◽  
Public Health Issue ◽  
M Gene ◽  
Working Day ◽  
Bacterial Genes ◽  
Dna Extraction Methods

The likelihood that milk and milk products may act as a vehicle for antibiotic-resistant bacterial genes has become a concern to the food industry and a public health issue, and the demand for rapid tests has increased. The purity of DNA extracted from food samples is a key issue in the sensitivity and usefulness of biological analyses, such as PCR for pathogens and nonpathogens. A rapid, phenol-chloroform free method based on a modification of a sodium iodide DNA extraction, followed by a two-step PCR was developed for direct detection of the tet(M) gene in milk samples within a single working day. This study compares the proposed method with a traditional phenol solvent extraction method and with a commercial kit (QIAamp DNA blood mini kit, Qiagen). The three DNA extraction methods were used to ensure access to the tet(M) gene from 1 ml of raw milk, inoculated with a strain of Enterococcus faecalis, which carries the tet(M) gene. The proposed method, followed by a two-step PCR with nested primers specific for the tet(M) gene, was able to reach a detection limit below 10 CFU/ml in less than 4 h, including the two amplification cycles, thus outperforming in sensitivity and rapidity both the traditional and the commercial method.

Evaluation of Three Real-Time PCR Methods for Detection of Salmonella from Cloves

2017 ◽  
Vol 80 (6) ◽  
pp. 982-989 ◽  
Author(s):  
Aparna Tatavarthy ◽  
Laila Ali ◽  
Vikas Gill ◽  
Lijun Hu ◽  
Xiaohong Deng ◽  
...  
Keyword(s):  
Real Time ◽  
Dna Extraction ◽  
Real Time Pcr ◽  
Extraction Methods ◽  
Pcr Assay ◽  
Wide Range ◽  
The Mean ◽  
Salmonella Serotypes ◽  
Pathogen Dna ◽  
Dna Extraction Methods

ABSTRACTThe purpose of the study was to evaluate three real-time PCR platforms for rapid detection of Salmonella from cloves and to compare three different DNA extraction methods. Six trials were conducted with two clove cultivars, Ceylon and Madagascar, and three Salmonella serotypes, Montevideo, Typhimurium, and Weltevreden. Each trial consisted of 20 test portions. The preenrichment cultures were used to perform PCR for comparison of the effectiveness of U.S. Food and Drug Administration, Pacific Regional Laboratory Southwest (FDA-PRLSW), Applied Biosystems Inc. (ABI) MicroSEQ, and GeneDisc platforms for detection of Salmonella. Three DNA extraction methods were used: standard extraction method for each PCR platform, boil preparation, and LyseNow food pathogen DNA extraction cards. The results from real-time PCR correlated well with FDA Bacteriological Analytical Manual culture assay results, with a wide range of cycle threshold (CT) values among the three PCR platforms for intended positive samples. The mean CT values for MicroSEQ (16.36 ± 2.78) were significantly lower than for PRLSW (20.37 ± 3.45) and GeneDisc (23.88 ± 2.90) (P &lt; 0.0001). Pairwise comparisons between PCR platforms using different DNA extraction methods indicate that the CT values are inversely proportional to the relative DNA quantity (RDQ) yields by different platform-extraction combinations. The pairing of MicroSEQ and boil preparation generated the highest RDQ of 120 and the lowest average CT value of 14.48, whereas the pairing of GeneDisc and LyseNow generated the lowest RDQ of 0.18 and the highest average CT of 25.97. Boil preparation yielded higher RDQ than the other extraction methods for all three PCR platforms. Although the MicroSEQ platform generated the lowest CT values, its sensitivity was compromised by narrow separations between the positive and negative samples. The PRLSW platform generated the best segregation between positive and negative groups and is less likely to produce false results. In conclusion, FDA-PRLSW was the most efficient PCR assay for Salmonella detection, and boil preparation was the best method for DNA extraction.

Evaluation of DNA extraction methods and dilution treatment for detection and quantification of Acanthamoeba in water and biofilm by real-time PCR

2010 ◽  
Vol 62 (9) ◽  
pp. 2141-2149 ◽  
Author(s):  
Ching-Wen Chang ◽  
Ying-Chieh Wu
Keyword(s):  
Real Time ◽  
Dna Extraction ◽  
Real Time Pcr ◽  
Pathogenic Bacteria ◽  
Acanthamoeba Castellanii ◽  
Extraction Methods ◽  
Cell Number ◽  
Pcr Analysis ◽  
Human Pathogens ◽  
Dna Extraction Methods

Acanthamoeba, human pathogens and natural hosts of pathogenic bacteria, may be accurately detected and quantified by real-time PCR if Acanthamoeba DNA are properly extracted and PCR inhibitors are effectively eliminated. However, the optimization of DNA extraction methods has not been reported for Acanthamoeba. This study compared the effectiveness of two DNA extraction/purification methods (FastDNA® Spin Kit for soil and Wizard® SV genomic DNA Purification System) by using trophozoites and cysts of Acanthamoeba castellanii and water and biofilm samples of cooling towers. DNA of A. castellanii extracted with the FastDNA® Kit and quantified by TaqMan PCR resulted in a lower variation (CV of Ct &lt; 3%), greater linearity (R2=0.99), and higher slopes (1.177–1.187 log fg DNA/log cell number) as compared to that by the Wizard® Kit. For field testing, the number of Acanthamoeba-positive samples and the Acanthamoeba DNA quantity were both greater with the FastDNA® Kit than with the Wizard® Kit (P=0.016 and &lt;0.0001, respectively). Beneficial effects with dilutions of extracted DNA were also revealed with the FastDNA® Kit (P=0.0003). In conclusion, DNA extraction by the FastDNA® Kit coupled with dilution of extracted DNA and PCR analysis are recommended for detecting and quantifying environmental Acanthamoeba.

scholarly journals Comparison of Three Mycobacterial DNA Extraction Methods from Extrapulmonary Samples for PCR Assay

2013 ◽  
Vol 6 (1) ◽  
pp. 9-11
Author(s):  
Khandaker Shadia ◽  
Shaheda Anwar ◽  
Sayera Banu ◽  
Ahmed Abu Saleh ◽  
Md Ruhul Amin Miah
Keyword(s):  
Dna Extraction ◽  
Extrapulmonary Tuberculosis ◽  
Extraction Methods ◽  
Molecular Diagnostic ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Polymerase Chain ◽  
Lysis Buffer ◽  
Dna Extraction Methods ◽  
Positive Results

Sensitivity of the molecular diagnostic tests of extrapulmonary tuberculosis largely depends upon the efficiency of DNA extraction methods. The objective of our study was to compare three methods of extracting DNA of Mycobacterium tuberculosis for testing by polymerase chain reaction. All three methods; heating, heating with sonication and addition of lysis buffer with heating and sonication were implicated on 20 extrapulmonary samples. PCR positivity was 2 (10%), 4 (20%) and 7 (35%) in the samples extracted by heating, heat+sonication and heat+sonication+lysis buffer method respectively. Of the extraction methods evaluated, maximum PCR positive results were achieved by combined heat, sonication and lysis buffer method which can be applied in routine clinical practice. DOI: http://dx.doi.org/10.3329/imcj.v6i1.14711 Ibrahim Med. Coll. J. 2012; 6(1): 9-11

Effect of Source of DNA on the Quantitative Analysis of Genetically Engineered Traits Using Digital PCR and Real-Time PCR

2017 ◽  
Vol 100 (2) ◽  
pp. 492-498 ◽  
Author(s):  
Tigst Demeke ◽  
Jemima Malabanan ◽  
Michelle Holigroski ◽  
Monika Eng
Keyword(s):  
Real Time ◽  
Dna Extraction ◽  
Real Time Pcr ◽  
Digital Pcr ◽  
Extraction Methods ◽  
Absolute Quantification ◽  
Genetically Engineered ◽  
Rt Pcr ◽  
Ctab Method ◽  
Dna Extraction Methods

Abstract Seven commercially available DNA extraction kits were compared with a cetyltrimethylammonium bromide (CTAB)method to determine the suitability of the extracted DNA for RainDrop digital PCR (dPCR) and real-time PCR (RT-PCR) quantification of OXY235 canola, FP967 flax, and DP305423 soybean (spiked at the 0.1% level). For the kits, the highest amount of DNA extracted from a 0.2 g sample was obtained using OmniPrep for Plant for flax andDNeasy mericon Food for canola and soybean. For canola, DNA extracted with the Fast ID Genomic DNA Extraction Kit, FastDNA Spin Kit, GM Quicker 2, NucleoSpin Food, and DNeasy mericon Food was suitable for dPCR and RT-PCR. For flax, DNA extracted with Fast ID, FastDNA Spin Kit, OmniPrep for Plant, and NucleoSpin Food was suitable for RT-PCR. However, only Fast ID yielded DNA suitable for dPCR. For soybean, DNA extracted with five and six of the seven DNA extraction kits was suitable for dPCR and RT-PCR, respectively. Overall, Fast ID provided reliable results regardless of species or analysis method used. Canola, flax, and soybean DNA extracted with the CTAB method and then purified were suitable for both dPCR and RT-PCR. This is the first report showing the effect of different DNA extraction methods on the absolute quantification of genetically engineered traits using dPCR.

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