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 ◽  
...  

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.

2007 ◽  
Vol 10 (7) ◽  
pp. 1122-1125 ◽  
Author(s):  
Li Maoteng ◽  
Liu Jianmin ◽  
Zhangyi . ◽  
Wang Pei ◽  
Gan Lu ◽  
...  

2021 ◽  
Author(s):  
Ana Laca ◽  
Abigail Wells ◽  
Linda Park

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.


2021 ◽  
Author(s):  
Rachel L. Byrne ◽  
Derek Cocker ◽  
Ghaith Alyayyoussi ◽  
Madalitso Mphasa ◽  
Mary Charles ◽  
...  

ABSTRACTBackgroundEnvironmental water samples are increasingly recognised as an important reservoir of antimicrobial resistance (AMR) genes. Polymerase chain reaction (PCR) and next generation sequencing (NGS) offer a potentially inclusive surveillance platform for a wide range of AMR genes. However, molecular methods are dependent upon the extraction of DNA of high yield and quality. Current options for DNA extraction from complex environmental matrices for downstream molecular applications are either expensive or low yielding. We present here a novel magnetic bead-based DNA extraction method, for the detection of antimicrobial resistance genes (ARGs) from river water in Malawi, named MagnaExtract.MethodsMagnaExtract involves initial filtration of 250ml freshwater, followed by an overnight incubation of the filter in 15ml buffered peptone water (BPW), common procedure in microbiology laboratories. 200µl is then taken for a boil (95°C) and spin step and mixed with magnetic beads to bind DNA. Following washes with ethanol, the DNA is eluted in nuclease-free water. To determine the effectiveness of this method, 98 freshwater samples were collected from two rivers in Southern Malawi, and DNA was isolated using the MagnaExtract method, two commercial Qiagen (Germany) kits; PowerWater and DNeasy Blood and tissue, alongside a boil and spin of BPW, and a boil and spin from bacterial isolate grown on agar media. All samples were screened with a high-resolution melt (HRM) PCR panel previously validated for the detection of third generation cephalosporin and carbapenem ARGs. We compared the DNA yield obtained using all extraction methods, as well as the identification of each ARG.ResultsDNA yield using MagnaExtract was statistically greater than both boil and spin methods and DNeasy Blood & Tissue (Qiagen, Germany). DNA yield was slightly lower than using PowerWater (Qiagen) but the difference was not statistically significant. MagnaExtract was the only method to identify ARGs in all 98 water samples compared with PowerWater (n=82), DNeasy (n=95) boilate of BPW (n=75) and boilate of bacterial isolate (n=87). The most commonly detected ARG was OXA-48 (n=93). In addition, we found overnight incubation in non-selective enrichment broth (BPW) to promote the growth of bacteria harbouring extended spectrum beta lactamase (ESBL) genes and reduction in the detection of carbapenemase genes.ConclusionThe MagnaExtract approach offers a simple, affordable, high yielding DNA extraction method for the detection of ARGs isolated from river water samples.


2021 ◽  
Vol 7 (3) ◽  
pp. 304-319
Author(s):  
Spyridon Andreas Papatheodorou ◽  
◽  
Panagiotis Halvatsiotis ◽  
Dimitra Houhoula ◽  

<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>


2021 ◽  
Author(s):  
Sukanya Sahu ◽  
Sandeep Kaushik ◽  
Bidhan Goswami ◽  
Arunabha Dasgupta ◽  
Hritusree Guha ◽  
...  

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.


2020 ◽  
Vol 82 (5) ◽  
Author(s):  
Zaliha Suadi ◽  
Lesley Maurice Bilung ◽  
Kasing Apun ◽  
Aida Azrina Azmi

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.


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