scholarly journals EFFICIENCY OF TRADITIONAL DNA EXTRACTION METHOD IN PCR DETECTION OF PORCINE DNA IN MEAT MIXTURES

2020 ◽  
Vol 82 (5) ◽  
Author(s):  
Zaliha Suadi ◽  
Lesley Maurice Bilung ◽  
Kasing Apun ◽  
Aida Azrina Azmi
Keyword(s):  
Dna Extraction ◽  
Extraction Method ◽  
Isoamyl Alcohol ◽  
Cost Effective ◽  
Raw Meat ◽  
Dna Extraction Method ◽  
Meat Samples ◽  
Species Specific ◽  
Cyt B Gene

Through the advancement of biotechnology, DNA-based methods are the most effective techniques in species identification, as they are rapid and have higher stability in harsh conditions compared to protein-based methods. This study was conducted to determine the efficiency of the traditional DNA extraction method, phenol/chloroform/isoamyl alcohol (PCIA), and comparing it with the commercially available kit by evaluating the purity, concentration, and suitability for amplification of porcine DNA in raw chicken and beef mixtures. The quantity and quality of the DNA extracts were assessed using a UV-Vis spectrophotometer. Polymerase chain reaction (PCR) was performed using species-specific primers targeting mitochondrial DNA cytochrome b (cyt b) gene of chicken (227-bp), beef (274-bp), and pork (398-bp), to confirm the template usability and quality of the DNA extracts. High DNA concentrations and purity were obtained from meat samples extracted using the PCIA method. The visualization of pork DNA on 2% agarose gel was able to detect pork contamination in raw meat mixtures up to minute proportion (1%). The existence of pork in chicken and beef was indicated with the presence of a specific 398-bp DNA band. Thus, the PCIA method can be recommended as a cost-effective and an excellent alternative to more expensive extraction kits in detecting pork DNA in raw meat mixtures.

scholarly journals A modified SDS-based DNA extraction method from raw soybean

2019 ◽  
Vol 39 (2) ◽  
Author(s):  
Yimiao Xia ◽  
Fusheng Chen ◽  
Yan Du ◽  
Chen Liu ◽  
Guanhao Bu ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Extraction Method ◽  
Pcr Amplification ◽  
Isoamyl Alcohol ◽  
Monitoring Methods ◽  
Detection Techniques ◽  
Dna Yield ◽  
Seed Flour ◽  
Dna Extraction Method ◽  
Lysis Buffer

Abstract Soybean is the most important genetically modified (GM) oilseed worldwide. Regulations relating to the approval of biotech soybean varieties and product labeling demand accurate and reliable detection techniques to screen for GM soya. High-quality extracted DNA is essential for DNA-based monitoring methods. Thus, four widely used protocols (SDS, CTAB, DP305, and DNeasy Plant Mini Kit) were compared in the present study to explore the most efficient DNA extraction method for raw soya matrix. The SDS-based method showed the highest applicability. Then crucial factors influencing DNA yield and purity, such as SDS lysis buffer component concentrations and organic compounds used to isolate DNA, were further investigated to improve the DNA obtained from raw soybean seeds, which accounts for the innovation of this work. As a result, lysis buffer (2% SDS (w/v), 150 mM NaCl, 50 mM Tris/HCl, 50 mM EDTA, pH 8.0) and organic reagents including chloroform/isoamyl alcohol (24:1, v/v) (C: I), isopropanol, and ethanol corresponding to the extraction and first and second precipitation procedures, respectively, were used in the optimized SDS method. The optimized method was verified by extracting approximately 2020–2444 ng DNA/mg soybean with A260/280 ratios of 1.862–1.954 from five biotech and non-biotech soybean varieties. Only 0.5 mg of soya was required to obtain enough DNA for PCR amplification using the optimized SDS-based method. These results indicate that the screening protocol in the present study achieves the highest suitability and efficiency for DNA isolation from raw soya seed flour.

scholarly journals Total Genomic DNA Extraction Studies from Seaweeds

2020 ◽  
Vol 4 (1) ◽  
pp. 10
Author(s):  
Made Pharmawati ◽  
Wildan Mujahidul Basyar ◽  
Ida Ayu Astarini
Keyword(s):  
Dna Extraction ◽  
Genomic Dna ◽  
Extraction Method ◽  
Genetic Improvement ◽  
Food Industry ◽  
Carotenoid Biosynthesis ◽  
Marine Resources ◽  
Basic Requirement ◽  
Dna Extraction Method

One of marine resources that has high value is seaweed.  Seaweed is a carrageenan producer used in the food industry. Seaweed contains many minerals, vitamins and proteins that are useful for health. Carotenoids are pigments found in seaweed that function as antioxidants.   The genes involved in carotenoid biosynthesis have been studied and provide opportunities for genetic improvement of seaweed.  DNA is a basic requirement in molecular analysis. Therefore, a suitable method of DNA extraction from seaweed is needed.  The aim of this research was to investigate DNA extraction method from several seaweed species and test the DNA quality through PCR-RAPD.  Seaweed samples were collected from Pantai Bumi Perkemahan Taman Nasional Bali Barat and DNA was extracted using Doyle and Doyle’s method with modifications.  PCR-RAPD was conducted using primer UBC127 and OPD 11 to test the quality of DNA.  Results showed that 3 hours incubation in 60ºC had the best result of DNA extraction.  However, the quality of DNA was low, as indicated by inconsistent PCR-RAPD products.   Further optimization in DNA extraction is needed to obtain high quality DNA for genetic analysis.

eDNA extraction: phenol-chloroform-isoamyl alcohol DNA purification from filters stored in Longmire buffer v1

2021 ◽  
Author(s):  
Ana Laca ◽  
Abigail Wells ◽  
Linda Park
Keyword(s):  
Dna Extraction ◽  
Extraction Method ◽  
Isoamyl Alcohol ◽  
Dna Purification ◽  
Phase Lock ◽  
Dna Extraction Method ◽  
Aqueous Layer

This is an organic DNA extraction method for filters preserved in 2 ml of Longmire buffer that uses a phase lock to allow easy decanting of the aqueous layer instead of pipetting.

scholarly journals A comparison of different DNA extraction methods and molecular techniques for the detection and identification of foodborne pathogens

2021 ◽  
Vol 7 (3) ◽  
pp. 304-319
Author(s):  
Spyridon Andreas Papatheodorou ◽  
◽  
Panagiotis Halvatsiotis ◽  
Dimitra Houhoula ◽  
Keyword(s):  
Dna Extraction ◽  
Foodborne Pathogens ◽  
Extraction Method ◽  
Pathogenic Bacteria ◽  
Low Cost ◽  
Isoamyl Alcohol ◽  
Extraction Methods ◽  
Molecular Techniques ◽  
Bacterial Dna ◽  
Dna Extraction Method

<abstract> <p>Foodborne infections continue to plague Europe. Food safety monitoring is in crisis as the existing techniques for detecting pathogens do not keep up with the global rising of food production and consumption. Thus, the development of innovative techniques for detecting and identifying pathogenic bacteria has become critical. The aim of the present study was firstly to develop an innovative simple and low cost method of extracting bacterial DNA from contaminated food and water samples with <italic>Salmonella enteric</italic> subsp. <italic>enteric</italic> serovar Typhimurium and <italic>Listeria monocytogenes</italic> and its comparison with two commercial DNA extraction kits (Qiagen, Macherey-Nagel). Finally, pathogens' detection using two molecular techniques (PCR-electrophoresis, LAMP), in order to evaluate the best combination of DNA extraction and identification based on their sensitivity, cost, rapidity and simplicity. Considering the above criteria, among them, best was proved an in-house bacterial DNA extraction method, based on the chloroform-isoamyl alcohol protocol, with certain modifications. This technique showed statistically similar results in terms of sensitivity, compared to the commercial kits, while at the same time maintained high rapidity and much lower cost. Lastly, between the molecular techniques, LAMP was found more promising considering its simplicity, high rapidity and sensitivity. Conclusively, the in-house DNA extraction method along with the LAMP technique, was proven to be the best among the presented combinations.</p> </abstract>

scholarly journals Fecal genomic DNA extraction method impacts outcome of MinION based metagenome profile of tuberculosis patients.

2021 ◽  
Author(s):  
Sukanya Sahu ◽  
Sandeep Kaushik ◽  
Bidhan Goswami ◽  
Arunabha Dasgupta ◽  
Hritusree Guha ◽  
...  
Keyword(s):  
Microbial Diversity ◽  
Dna Extraction ◽  
Gut Microbiome ◽  
Extraction Method ◽  
Critical Role ◽  
Isoamyl Alcohol ◽  
Fecal Samples ◽  
Sequencing Platform ◽  
Dna Extraction Method ◽  
Generation Sequencing

In the present era, emergence of next generation sequencing approaches has revolutionized the field of gut microbiome study. However, the adopted DNA extraction step used in metagenomics experiments and its efficiency may play a critical role in their reproducibility and outcome. In this study, fecal samples from active and non-tuberculosis subjects (ATB/NTB, n=7) were used. Fecal samples of a subgroup of these subjects were subjected to Mechanical enzymatic lysis (MEL) and Phenol: Chloroform: Isoamyl Alcohol (PCIA) methods of DNA extraction and a third-generation sequencing platform i.e., MinION was employed for microbiome profiling. Findings of this study demonstrated that DNA extraction method significantly impacts the DNA yield and microbial diversity. Irrespective of the adopted method of DNA extraction, ATB patients showed altered microbial diversity compared to NTB controls. Also, the fecal microbial diversity details are better captured in samples processed by MEL method and may be suitable to be adopted for high-throughput gut microbiome studies.

scholarly journals Choice of DNA extraction method affects DNA metabarcoding of unsorted invertebrate bulk samples

2018 ◽  
Vol 2 ◽  
Author(s):  
Markus Majaneva ◽  
Ola H. Diserud ◽  
Shannon H.C. Eagle ◽  
Mehrdad Hajibabaei ◽  
Torbjørn Ekrem
Keyword(s):  
Dna Extraction ◽  
Extraction Method ◽  
Target Material ◽  
Cost Effective ◽  
Community Analysis ◽  
Extraction Methods ◽  
Dna Recovery ◽  
Dna Yield ◽  
Dna Extraction Method ◽  
Dna Metabarcoding

Characterisation of freshwater benthic biodiversity using DNA metabarcoding may allow more cost-effective environmental assessments than the current morphological-based assessment methods. DNA metabarcoding methods where sorting or pre-sorting of samples are avoided altogether are especially interesting, since the time between sampling and taxonomic identification is reduced. Due to the presence of non-target material like plants and sediments in crude samples, DNA extraction protocols become important for maximising DNA recovery and sample replicability. We sampled freshwater invertebrates from six river and lake sites and extracted DNA from homogenised bulk samples in quadruplicate subsamples, using a published method and two commercially available kits: HotSHOT approach, Qiagen DNeasy Blood &amp; Tissue Kit and Qiagen DNeasy PowerPlant Pro Kit. The performance of the selected extraction methods was evaluated by measuring DNA yield and applying DNA metabarcoding to see if the choice of DNA extraction method affects DNA yield and metazoan diversity results. The PowerPlant Kit extractions resulted in the highest DNA yield and a strong significant correlation between sample weight and DNA yield, while the DNA yields of the Blood &amp; Tissue Kit and HotSHOT method did not correlate with the sample weights. Metazoan diversity measures were more repeatable in samples extracted with the PowerPlant Kit compared to those extracted with the HotSHOT method or the Blood &amp; Tissue Kit. Subsampling using Blood &amp; Tissue Kit and HotSHOT extraction failed to describe the same community in the lake samples. Our study exemplifies that the choice of DNA extraction protocol influences the DNA yield as well as the subsequent community analysis. Based on our results, low specimen abundance samples will likely provide more stable results if specimens are sorted prior to DNA extraction and DNA metabarcoding, but the repeatability of the DNA extraction and DNA metabarcoding results was close to ideal in high specimen abundance samples.

eDNA extraction: phenol-chloroform-isoamyl alcohol DNA purification from filters stored in Longmire buffer v1

2021 ◽  
Author(s):  
Abigail Wells ◽  
Linda Park
Keyword(s):  
Dna Extraction ◽  
Extraction Method ◽  
Isoamyl Alcohol ◽  
Dna Purification ◽  
Phase Lock ◽  
Dna Extraction Method ◽  
Aqueous Layer

This is an organic DNA extraction method for filters preserved in 2 ml of Longmire buffer that uses a phase lock to allow easy decanting of the aqueous layer instead of pipetting.

Quality Improvement of the DNA extracted by boiling method in Gram negative bacteria

2017 ◽  
Vol 6 (04) ◽  
pp. 5347 ◽  
Author(s):  
Omar B. Ahmed* ◽  
Anas S. Dablool
Keyword(s):  
Dna Extraction ◽  
Control Method ◽  
Extraction Procedure ◽  
Cost Effective ◽  
High Yield ◽  
Gram Negative Bacteria ◽  
Bacterial Dna ◽  
Gram Negative ◽  
E Coli

Several methods of Deoxyribonucleic acid (DNA) extraction have been applied to extract bacterial DNA. The amount and the quality of the DNA obtained for each one of those methods are variable. The study aimed to evaluate bacterial DNA extraction using conventional boiling method followed by alcohol precipitation. DNA extraction from Gram negative bacilli was extracted and precipitated using boiling method with further precipitation by ethanol. The extraction procedure performed using the boiling method resulted in high DNA yields for both E. coli and K. pneumoniae bacteria in (199.7 and 285.7μg/ml, respectively) which was close to control method (229.3 and 440.3μg/ml). It was concluded that after alcohol precipitation boiling procedure was easy, cost-effective, and applicable for high-yield quality of DNA in Gram-negative bacteria.

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