A Syringe-Based and Centrifugation-Free DNA Extraction Procedure for the Rapid Detection of Bacteria

Several bacteria are known to cause food poisoning; therefore, diagnostic systems that detect bacteria are essential. Nucleic acid-based testing methods that involve polymerase chain reaction (PCR) amplification are of great interest due to their high sensitivity and specificity. Herein, we developed a syringe-based one-step DNA extraction device that streamlines the extraction of genomic DNA (gDNA) from bacteria within 2 min, enabling versatile application of nucleic acid-based testing in the field. Notably, the bolt-nut structured case coupled with the syringe enables control of the volume of solution dispensed for enabling DNA extraction without the need for bulky centrifuge equipment. Using the proposed system, the gDNA of a model bacterium, Escherichia coli, was extracted at a good quantity and quality and amplified via PCR. The DNA extracted was comparable to that extracted via a centrifugation-based procedure. In addition, bacteria that were artificially spiked in common samples, including a work cloth, a work bench, and meat, were successfully detected with high accuracy.

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Faculty Opinions recommendation of One-step detection of c-kit point mutations using peptide nucleic acid-mediated polymerase chain reaction clamping and hybridization probes.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1012550.186179 ◽

2003 ◽

Author(s):

Richard L Stevens

Keyword(s):

Polymerase Chain Reaction ◽

Nucleic Acid ◽

Peptide Nucleic Acid ◽

Point Mutations ◽

Chain Reaction ◽

Step Detection ◽

Hybridization Probes ◽

One Step ◽

Polymerase Chain

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Method Comparison of DNA Isolation and Quantification for Fish and Seafood Authenticity Determination

SQUALEN Bulletin of Marine and Fisheries Postharvest and Biotechnology ◽

10.15578/squalen.v13i3.370 ◽

2018 ◽

Vol 13 (3) ◽

pp. 115

Author(s):

Dwiyitno Dwiyitno ◽

Stefan Hoffman ◽

Koen Parmentier ◽

Chris Van Keer

Keyword(s):

Polymerase Chain Reaction ◽

Dna Extraction ◽

Dna Isolation ◽

Blue Mussel ◽

Pcr Amplification ◽

Amplification Efficiency ◽

Chain Reaction ◽

Seafood Products ◽

Polymerase Chain ◽

Commercial Kit

Fish and seafood products has been commonly targeted for fraudulent activities. For that reason, authentication of fish and seafood products is important to protect consumers from fraudulent and adulteration practices, as well as to implement traceability regulation. From the viewpoint of food safety, authenticity is beneficial to protect public from serious food poisoning incidents, such as due to ingestion of toxic species. Since DNA based identification depends on the nucleic acid polymerase chain reaction (PCR), the quantity and quality/purity of DNA will contribute significantly to the species authentication. In the present study, different DNA extraction and purification methods (3 classical methods and one commercial kit) were compared to produce the better isolated DNA for PCR amplification. Additionally, different methods for the estimation of DNA concentration and purity which is essential for PCR amplification efficiency were also evaluated. The result showed that classical DNA extraction methods (based on TNES-Urea) yielded a higher amount of DNA (11.30-323.60 ng/g tissue) in comparison to commercial kit/Wizard Promega (5.70-83.45 ng/g tissue). Based on the purity of DNA extract (A260/280), classical DNA extraction method produced relatively similar on DNA quality to the commercial kit (1.79-2.12). Interestingly, all classical methods produced DNA with A260/280 ratio of more than 2.00 on the blue mussel, in contrast with commercial kit. The commercial kit also produced better quality of DNA compared to the classical methods, showing the higher efficiency in PCR amplification. NanoDrop is promising as cheap, robust and safe UV-spectrophotometer method for DNA quantification, as well as the purity evaluation.Keywords: seafood authenticity, DNA isolation, polymerase chain reaction, NanoDrop, Picogreen

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Simple ‘universal’ DNA extraction procedure compatible with direct PCR amplification

Cellular and Molecular Life Sciences ◽

10.1007/bf01919509 ◽

1996 ◽

Vol 52 (4) ◽

pp. 295-295 ◽

Cited By ~ 1

Author(s):

D. Goldenberger ◽

I. Perschil ◽

M. Ritzler ◽

M. Altwegg

Keyword(s):

Dna Extraction ◽

Extraction Procedure ◽

Pcr Amplification ◽

Direct Pcr

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Molecular Analysis of Sentinel Lymph Nodes in Patients With Breast Cancer Using One-Step Nucleic Acid Amplification (OSNA): Does Its High Sensitivity Lead to Axillary Overtreatment?

European Journal of Surgical Oncology ◽

10.1016/j.ejso.2019.11.079 ◽

2020 ◽

Vol 46 (2) ◽

pp. e44

Author(s):

Kim Hintzen ◽

Lisa De Rooij ◽

Nelleke Schouten ◽

James van Bastelaar ◽

Suzanne Corvers ◽

...

Keyword(s):

Breast Cancer ◽

Nucleic Acid ◽

Lymph Nodes ◽

Molecular Analysis ◽

High Sensitivity ◽

Sentinel Lymph Nodes ◽

Nucleic Acid Amplification ◽

One Step

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A Set of Plasmid-Based Modules for Easy Switching of C-Terminal Epitope Tags in Saccharomyces cerevisiae

Microorganisms ◽

10.3390/microorganisms9122505 ◽

2021 ◽

Vol 9 (12) ◽

pp. 2505

Author(s):

Hiroki Hayashi ◽

Tsutomu Kishi

Keyword(s):

Dna Sequences ◽

Affinity Purification ◽

Pcr Amplification ◽

Restriction Enzymes ◽

Epitope Tagging ◽

One Step ◽

Pcr Method ◽

Polymerase Chain ◽

The One ◽

Step Polymerase Chain Reaction

Epitope tagging is a powerful strategy for analyzing the functions of targeted proteins. The use of this strategy has become more convenient with the development of the epitope switch, which is another type of epitope tagging designed to convert the previously tagged epitopes on the chromosome to other epitopes of interest. Various modules for C-terminal epitope switching have been developed and amplified using the one-step polymerase chain reaction (PCR) method before transformation. However, PCR amplification occasionally generates mutations that affect the fidelity of epitope switching. Here, we constructed several plasmids to isolate modules for epitope switching through digestion by restriction enzymes. The isolated modules contained DNA sequences for homologous recombination, various epitopes (13×Myc, 6×HA, GFP, Venus, YFP, mCherry, and CFP), and a transformation marker (Candida glabrata LEU2). The restriction enzyme-digested plasmids were used to directly transform the cells for epitope switching. We demonstrate the efficient and accurate switching of the MX6 module-based C-terminal tandem affinity purification tags to each aforementioned epitope. We believe that our plasmids can serve as powerful tools for the functional analysis of yeast proteins.

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Detection of Enterotoxigenic Clostridium perfringens in Raw Beef by Polymerase Chain Reaction

Journal of Food Protection ◽

10.4315/0362-028x-58.2.154 ◽

1995 ◽

Vol 58 (2) ◽

pp. 154-159 ◽

Cited By ~ 12

Author(s):

LUIS A. BAEZ ◽

VIJAY K. JUNEJA

Keyword(s):

Polymerase Chain Reaction ◽

Clostridium Perfringens ◽

Direct Detection ◽

Food Poisoning ◽

Pcr Amplification ◽

Oligonucleotide Primer ◽

Enterotoxin Gene ◽

Chain Reaction ◽

Polymerase Chain ◽

Labeled Probe

A polymerase chain reaction (PCR) procedure was developed for direct detection of Clostridium perfringens strains with potential for food poisoning in raw beef samples. An oligonucleotide primer pair was used to amplify a 364 base pair sequence internal to the C. perfringens enterotoxin gene. One milliliter portions of the meat homogenates were inoculated into cooked meat medium (CMM) or reduced Fluid Thioglycollate (FTG) medium and incubated at 37°C. Portions sampled at 2, 4, 6, 8 and 24 h of enrichment were assayed for detection of the enterotoxin sequence by PCR. Amplification of the 364 bp sequence could be detected in 6 h by agarose gel electrophoresis and as early as 2 h by hybridization to a 150 bp digoxigenin (DIG)-labeled probe. To increase the sensitivity of the detection assay a commercial chromosomal deoxyribonucleic acid (DNA) extraction assay was compared with a nested PCR approach. Both methods allowed detection of less than 1 log10 colony forming units (CFU)/g of C. perfringens strains harboring the enterotoxin gene, with no interference with the background microflora present in the raw ground beef.

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Impact of two different commercial DNA extraction methods on BK virus viral load

Microbiologia Medica ◽

10.4081/mm.2016.4825 ◽

2016 ◽

Vol 31 (1) ◽

Author(s):

Massimiliano Bergallo ◽

Ilaria Galliano ◽

Elisa Loiacono ◽

Francesca Ferro ◽

Paola Montanari ◽

...

Keyword(s):

Viral Load ◽

Nucleic Acid ◽

Dna Extraction ◽

Quantitative Polymerase Chain Reaction ◽

Extraction Procedure ◽

Bk Virus ◽

Human Polyomavirus ◽

Acid Extraction ◽

Seroprevalence Rate ◽

The Impact

Background and aim: BK virus, a member of human polyomavirus family, is a worldwide distributed virus characterized by a seroprevalence rate of 70-90% in adult population. Monitoring of viral replication is made by evaluation of BK DNA by quantitative polymerase chain reaction. Many different methods can be applied for extraction of nucleic acid from several specimens. The aim of this study was to assess the impact of two different DNA extraction procedure on BK viral load. <br />Materials and methods: DNA extraction procedure including the Nuclisens easyMAG platform (bioMerieux, Marcy l’Etoile, France) and manual QIAGEN extraction (QIAGEN Hilden, Germany). BK DNA quantification was performed by Real Time TaqMan PCR using a commercial kit. <br />Result and discussion: The samples capacity, cost and time spent were compared for both systems. In conclusion our results demonstrate that automated nucleic acid extraction method using Nuclisense easyMAG was superior to manual protocol (QIAGEN Blood Mini kit), for the extraction of BK virus from serum and urine specimens.

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DNA Extraction from Several Cacti

HortScience ◽

10.21273/hortsci.35.6.1124 ◽

2000 ◽

Vol 35 (6) ◽

pp. 1124-1126 ◽

Cited By ~ 8

Author(s):

Candelario Mondragon-Jacobo ◽

Natalia Doudareva ◽

Bruce P. Bordelon

Keyword(s):

Dna Extraction ◽

Pcr Amplification ◽

Hexadecyltrimethylammonium Bromide ◽

Digestion Time ◽

Chain Reaction ◽

High Quality ◽

Banding Patterns ◽

Extraction Buffer ◽

Ctab Method ◽

Polymerase Chain

A method for extraction of high quality DNA from four Opuntia sp. and other cacti using a hexadecyltrimethylammonium bromide (CTAB) method is described. These plants typically contain high levels of mucilages, complex polysaccharide compounds that bind water, thus preventing DNA extraction by common miniprep methods. The method involves adjusting the amount of tissue used according to species and age, followed by processing in an extraction buffer to separate coarse material. Extended centrifugation and digestion time in a separation buffer with CTAB (2%) was used. Exposing tissue to both buffers maintained polysaccharides in solution and allowed easier recovery of the aqueous phase that contains the DNA. We found that 5-8 g were needed to obtain up to 153 μg·g-1 of DNA from tender tissue. Old tissue yielded 26% less. Extraction of DNA from 5-g samples of tender tissue of the ornamental cacti Stenocereus sp., Cleistocactus sp., and Echinocereus sp. was successful. For these species, average yields ranged from 25 to 53 μg per sample. The DNA obtained was suitable for polymerase chain reaction (PCR) amplification, producing clear, distinctive, and reproducible banding patterns useful for a variety of applications.

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DNA extraction from resin producing Boswellia tree

10.21203/rs.2.1645/v1 ◽

2019 ◽

Author(s):

Abdul Latif Khan ◽

Adil Khan ◽

Sajjad Asaf ◽

Ahmed Al-Harrasi ◽

Ahmed Al-Rawahi

Keyword(s):

Dna Extraction ◽

Pcr Amplification ◽

Secondary Compounds ◽

Ammonium Bromide ◽

Commiphora Wightii ◽

Optimized Protocol ◽

Polymerase Chain ◽

Cetyl Trimethyl Ammonium ◽

Passiflora Foetida ◽

Laborious Method

Abstract The wild nature of plant and the presence of the secondary compounds e.g., Resin and phenolic compound make DNA extraction problematic. Some of them will co-precipitate with DNA during extraction and inhibit further enzymatic modification of the DNA. Furthermore large amounts of complex polysaccharides also make extraction of usable DNA impossible. Previously reported protocols yielded highly viscous and slimy DNA preparations that were not amenable to further analysis. We tried many commonly used protocols but were unable to isolate high quality DNA from the gum containing plant Bosweliia sacra. We did extensive optimization of the Cetyl Trimethyl Ammonium Bromide \(CTAB) protocol established by Doyle in 1987 to extract DNA which is feasible for PCR amplification. In this study, we have specifically tested the previously developed CTAB based protocols developed by I Haque adapted for Commiphora wightii and Bipin Deochand Lade protocol developed for Passiflora foetida. Both are developed for plants, which contain high amount of secondary metabolites, polysaccharides and phenolic compounds. Furthermore, we investigate the possibility to extract DNA from resin producing plants which is feasible for downstream application. To our knowledge, this is the first optimized protocol which a rapid and less laborious method for the extraction of DNA from Boswellia species \(Boswellia sacra and Boswellia elongata found in Oman and Yemen respectively) which are resin \(Luban) producing plant. The isolated DNA by the currently optimized protocol were suitable for polymerase chain reaction \(PCR) mediated amplification for applications genetic diversity and DNA barcoding.

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