scholarly journals Strategy for DNA extraction and detection from insect pests in stored home grain samples RESEARCH

2021 ◽  
Vol 01 (1) ◽  
pp. 24-32
Author(s):  
Ibrahim Abbasi
Keyword(s):  
Dna Extraction ◽  
Insect Pests ◽  
Pcr Amplification ◽  
Extraction Methods ◽  
Alternative Methods ◽  
Coi Gene ◽  
Pest Species ◽  
Detection Techniques ◽  
Specificity And Sensitivity ◽  
Washing Method

Stored grains are subjected to infestations with more than 60 species of insects, that responsible for millions of dollars’ loss and cause several health problems including allergies and gastrointestinal disorders. Traditional detection techniques are laborious, expensive and not sensitive to detect insect contamination at the egg and larvae stages. Therefore, alternative methods are needed for rapid and sensitive detection. One obvious approach is to develop a molecular approach utilizing genetic information of the potential insect species that infest grains for amplification of specific target gene fragment utilizing polymerase chain reaction [PCR]. In the present study, a number of known infested grain samples were used in standardizing a method to isolate larvae and adult insects that were based on centrifugation washing method and a filtration washing method. The isolated insects were subjected to DNA extraction and PCR amplification of defined regions of cytochrome oxidase I (COI) gene followed by sequencing to identify the di"erent pest species. For PCR amplification new primers were designed and for this purpose the obtained COI sequences from di"erent insects were aligned to design two sets of primers (named: COI-PCR4 and COI-PCR5) specific for the indicated insect mitochondrial COI gene. The designed primers were tested for their specificity and sensitivity. The suitability of PCR primers and DNA extraction methods were evaluated on eleven samples of commercial grains utilizing each primer set with the two extraction methods.

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 ALKALINE LYSIS AS AN EFFICIENT AND ECONOMICAL ALTERNATIVE FOR DNA EXTRACTION FROM HORSEHAIR HAIR SAMPLES: COMPARISON BETWEEN DIFFERENT METHODS

2021 ◽  
Vol 9 (02) ◽  
pp. 836-841
Author(s):  
Myriam Janeth Ortega Torres ◽  
◽  
Jessica Almeida Braga ◽  
Camilo Torres ◽  
Ahmed Sami Shaker ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Hypervariable Region ◽  
Pcr Amplification ◽  
Extraction Methods ◽  
Forensic Population ◽  
Alkaline Lysis ◽  
Hair Samples ◽  
Genomic Studies ◽  
Dna Extraction Methods ◽  
Short Time

Studies related to DNA extraction are becoming more ambitious in the sense that large studies are intended to be carried out with minimum DNA sources. The DNA extracted must be of quality for genetic, forensic, population and genomic studies, these samples must be easy to obtain and product of efficient manipulation.Samples obtained from horsehair are an important technical challenge since they constitute the preferred non-invasive sample for genetic studies in horses, which has been shown to obtain reliable results in a short time. In this sense, working into effective techniques to optimizeDNA extraction of scarse samples is a pertinent task. In this study, different DNA extraction methods were evaluated from mane samples obtained from a population of wild horses from the Region of Arauca in eastern Colombia.Three DNA extraction methods were evaluated (phenol chloroform, alkaline lysis and twocommercial DNA extraction kit), DNA concentration, purity and qualityweredeterminate and PCR amplification product were obtain using primers for a hypervariable region of DNA mitochondrial. DNA preparation from hair roots using alkaline lysis was the most economical and efficient method with which it was possible to obtain high quality and quantity DNA.

scholarly journals Sticky problems: extraction of nucleic acids from molluscs

2021 ◽  
Vol 376 (1825) ◽  
Author(s):  
Coen M. Adema
Keyword(s):  
Nucleic Acids ◽  
Dna Extraction ◽  
Extraction Methods ◽  
Biological Properties ◽  
Alternative Methods ◽  
28S Rrna ◽  
Future Directions ◽  
Dna And Rna ◽  
Standard Quality ◽  
Dna Extraction Methods

Traditional molecular methods and omics-techniques across molluscan taxonomy increasingly inform biology of Mollusca. Recovery of DNA and RNA for such studies is challenged by common biological properties of the highly diverse molluscs. Molluscan biomineralization, adhesive structures and mucus involve polyphenolic proteins and mucopolysaccharides that hinder DNA extraction or copurify to inhibit enzyme-catalysed molecular procedures. DNA extraction methods that employ the detergent hexadecyltrimethylammoniumbromide (CTAB) to remove these contaminants importantly facilitate molecular-level study of molluscs. Molluscan pigments may stain DNA samples and interfere with spectrophotometry, necessitating gel electrophoresis or fluorometry for accurate quantification. RNA can reliably be extracted but the ‘hidden break’ in 28S rRNA of molluscs (like most protostomes) causes 18S and 28S rRNA fragments to co-migrate electrophoretically. This challenges the standard quality control based on the ratio of 18S and 28S rRNA, developed for deuterostome animals. High-AT content in molluscan rRNA prevents the effective purification of polyadenylated mRNA. Awareness of these matters aids the continuous expansion of molecular malacology, enabling work also with museum specimens and next-generation sequencing, with the latter imposing unprecedented demands on DNA quality. Alternative methods to extract nucleic acids from molluscs are available from literature and, importantly, from communications with others who study the molecular biology of molluscs. This article is part of the Theo Murphy meeting issue ‘Molluscan genomics: broad insights and future directions for a neglected phylum’.

scholarly journals Molecular characterization and identification of three stored grain pests based on mitochondrial cytochrome C oxidase subunit I (COI) gene sequences

2019 ◽  
Vol 47 (1) ◽  
pp. 1-11
Author(s):  
Abu Faiz Md Aslam ◽  
Sharmin Sultana ◽  
Faria Farhana Rain ◽  
Sumita Rani Das ◽  
Ayesha Siddika ◽  
...  
Keyword(s):  
Insect Pests ◽  
Sitophilus Oryzae ◽  
Dna Barcode ◽  
Control Measures ◽  
Coi Gene ◽  
Composition Analysis ◽  
Callosobruchus Chinensis ◽  
Pest Species ◽  
Stored Grain ◽  
Stored Grain Pests

Stored grain pests are discovered in food as immature stages, which further complicates the identification process. A DNA barcode dataset of some important pests that can be used for easy and confirm identification in stages of life is constructed. COI genes of three stored grain insect pests i.e,, Sitophilus oryzae, Callosobruchus chinensis and Oryzaephilus surinamensis were sequenced. The sequenced genes were submitted to NCBI GenBank and obtained accession numbers MG967331.1, MG967332.1, MG967333.1 and MK041216.1. BLAST analysis showed 99 to 100% homology with existing GenBank sequences. The nucleotide composition analysis revealed that the value of A+T (64.8%) is greater than G+C (35.2%). Genetic distance among four sequences of three store pests were ranged from 0.00293-0.32807. Phylogenetic analysis showed that these three species are originated from different clades. Haplotype analysis of mitochondrial COI gene of the stored grain insect pests showed high genetic diversity among them. C. chinensis, O. surinamensis and S. oryzae were separated from their common ancestor by 80, 73 and 64 mutational steps. These information may be helpful for attempting any successful control measures against the pest species. In conclusion, present author established the first DNA barcode dataset of three store grain pests and confirmed its efficiency for identifying these pests. Bangladesh J. Zool. 47(1): 1-11, 2019

Alternative Methods of extracting and Amplifying Dna from lichens

2000 ◽  
Vol 32 (2) ◽  
pp. 189-196 ◽  
Author(s):  
Maria P. Martín ◽  
Katarina Winka
Keyword(s):  
Dna Extraction ◽  
Pcr Amplification ◽  
Alternative Methods ◽  
High Quality

AbstractWe have investigated whether DNA extraction protocols designed specifically for fungi and/or lichens perform better on lichens than do corresponding protocols designed for plants and insects. Two different PCR-amplification protocols were used to evaluate the quality of the DNA extracted with each method. The DNA extractions with highest quality were obtained with the protocols designed for insects and plants, and the most successful amplifications were obtained with Ready-To-Go PCR Beads. This indicates that fungal or lichen specific protocols might not be necessary for successful extraction of high quality DNA from lichens.

scholarly journals Saliva leukocytes rather than saliva epithelial cells represent the main source of DNA

2016 ◽  
Vol 24 (1) ◽  
pp. 31-44 ◽  
Author(s):  
Corina Maria Cianga ◽  
Ion Antohe ◽  
Mihaela Zlei ◽  
Daniela Constantinescu ◽  
Petru Cianga
Keyword(s):  
Epithelial Cells ◽  
Dna Extraction ◽  
Peripheral Blood ◽  
Hematological Malignancies ◽  
Mononuclear Cells ◽  
Pcr Amplification ◽  
Hla Typing ◽  
Alternative Methods ◽  
Peripheral Blood Flow ◽  
Immunosuppressed Patients

Abstract Introduction. Several alternative methods to peripheral blood DNA extraction have been implemented so far. Saliva seems to represent a very advantageous type of sample, easy to harvest and able to generate DNA yields comparable to those extracted from blood mononuclear cells. Material and methods. 8 patients suspected of ankylosing spondylitis, 9 patients with various hematological malignancies, displaying post-chemotherapy leucopenia and 30 healthy volunteers were included in our study. DNA was extracted with various commercially available kits and used for HLA typing either by PCR amplification, or by PCR followed by hybridization. Results. Our data regarding HLA typing support already published results regarding the good DNA quality that allows its use in various molecular biology techniques. However, when attempting to use saliva from immunosuppressed patients for DNA extraction we have generated very low yields, comparable again with the ones obtained from peripheral blood. Flow cytometry and immunocytochemistry investigations confirmed the low number of leukocytes present in the saliva of these patients, while the number of epithelial cells was virtually unchanged. Conclusions. The main source of saliva DNA seems to be represented by leukocytes present in this fluid and not by the epithelial cells. Under these circumstances, for immunosuppressed patients saliva cannot represent an alternative to blood when attempting DNA extraction.

scholarly journals Comparison of Three DNA Extraction Methods for Detection of Erysipelothrix Rhusiopathiae in Chicken Blood by Polymerase Chain Reaction

2009 ◽  
Vol 21 (3) ◽  
pp. 354-358 ◽  
Author(s):  
Kazuki Harada ◽  
Mariko Uchiyama ◽  
Teruyuki Hoshi ◽  
Toshio Takahashi
Keyword(s):  
Polymerase Chain Reaction ◽  
Dna Extraction ◽  
Extraction Methods ◽  
Erysipelothrix Rhusiopathiae ◽  
Chain Reaction ◽  
Detection Techniques ◽  
Chicken Isolate ◽  
Polymerase Chain ◽  
Dna Extraction Methods ◽  
Chicken Blood

A previously reported Erysipelothrix-specific polymerase chain reaction (PCR) was used to detect Erysipelothrix bacteremia in chickens. The sensitivity of PCR using 3 DNA extraction methods (boiling method, commercial gene matrix, and DNA extractor kit) was compared by using a serial 10-fold dilution of a chicken isolate of Erysipelothrix rhusiopathiae strain in chicken blood. Of the techniques used, the DNA extractor kit, followed by PCR, provided the most sensitive method for the detection of the E. rhusiopathiae strain in chicken blood (approximately 100 CFU/0.1 ml of blood). Two E. rhusiopathiae infection experiments were then attempted. In a total of 10 inoculated chickens, bacteremia developed in 9 chickens, consisting of all 5 chickens used in the first trial (ranging from 5.1 × 101 to 2.0 × 103 CFU/0.1 ml of blood) and 4 of the 5 chickens used in the second trial (ranging from 1.0 × 100 to 3.3 × 102 CFU/0.1 ml of blood). In the second trial, the 3 detection techniques were applied to the chickens with bacteremia, and the organism could be detected by using the DNA extractor kit in blood specimens from the 3 chickens exhibiting bacteremia of ≥4.2 × 101 CFU/0.1 ml of blood. This observation suggests that most E. rhusiopathiae–infected chickens develop more critical bacteremia than the detectable level by PCR with the DNA extractor kit, and the PCR detection method can be used as a first-line screening of avian erysipelas.

scholarly journals At the crossroads of botanical collections and molecular genetics laboratory: a preliminary study of obtaining amplifiable DNA from moss herbarium material

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e9109
Author(s):  
Marta Saługa
Keyword(s):  
Dna Extraction ◽  
Extreme Environments ◽  
Pcr Amplification ◽  
Extraction Methods ◽  
Herbarium Specimens ◽  
Polar Regions ◽  
Moss Species ◽  
Dna Yield ◽  
Herbarium Collections ◽  
Dna Extraction Methods

Background Research focused on extreme environments is often associated with difficulties in obtaining fresh plant material. Herbaria may provide great support as they house large collections of specimens from different parts of the world. Accordingly, there is also a growing interest in methods using herbarium specimens in molecular studies. Much of the literature on herbarium DNA is aimed to improve extraction and PCR amplification and is focused mostly on vascular plants. Here, I provide a brief study of DNA extraction efficiency from moss herbarium specimens, emphasizing the importance of herbaria as an invaluable source of material from hard-to-access geographical areas, such as the Antarctic region. Methods The presented study is based on herbarium collections of 25 moss species collected in the austral polar regions between 1979 and 2013. The majority of samples were obtained using the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany). The remaining, smaller part was extracted using an adapted CTAB-based approach. The performance of DNA extraction methods in terms of PCR amplification success was measured by testing several DNA fragments of various size. Furthermore, in order to estimate of DNA fragmentation level, an automated on-chip electrophoresis system was used. Results Results reveal that DNA purity and the length of the target genetic region are the fundamental agents which drive the successful PCR reaction. Conversely, the DNA yield and specimen age seem to be less relevant. With this study, I present also an optimized CTAB-based approach which may effectively suppress inhibitors in the herbarium DNA. This method can be considered a cheaper alternative to column-based technology, particularly useful for dealing with a large number of samples. Results of this study confirmed previous reports and contribute to filling the existing gap in molecular analyses which involve the use of herbarium collections of mosses.

scholarly journals Development of a Competent and Trouble Free DNA Isolation Protocol for Downstream Genetic Analyses in Glycine Species

2016 ◽  
Vol 4 (8) ◽  
pp. 700 ◽  
Author(s):  
Muhammad Amjad Nawaz ◽  
Faheem Shehzad Baloch ◽  
Hafiz Mamoon Rehman ◽  
Bao Le ◽  
Fahima Akther ◽  
...  
Keyword(s):  
Molecular Biology ◽  
Dna Extraction ◽  
Genomic Dna ◽  
Dna Isolation ◽  
Plant Molecular Biology ◽  
Pcr Amplification ◽  
Cost Effective ◽  
Extraction Methods ◽  
Glycine Species ◽  
Genomic Dna Isolation

Extraction of deoxyribose nucleic acid (DNA) from plants is preliminary step in molecular biology. Fast and cost effective genomic DNA isolation from Glycine species for downstream application is a major bottleneck. Here we report a high throughput and trouble free method for genomic DNA extraction from leaf and seeds of Glycine species with high quality and quantity. Protocol reports the optimization by employing different concentrations of CTAB and PVP in extraction buffer. Efficiency of optimized protocol was compared with frequently used DNA extraction methods. Wide adoptability and utility of this protocol was confirmed by DNA extraction from leaves as well as seeds of G. max, G. soja, G. tomentella and G. latifolia. Extracted DNA was successfully subjected to PCR amplification of five microsatellite markers and four putative glycosyltransferase genes. DNA extraction protocol is reproducible, trouble free, rapid and can be adopted for plant molecular biology applications.

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