scholarly journals A quantitative comparison of two kits for DNA extraction from canned tuna

2019 ◽  
Vol 88 (3) ◽  
pp. 315-322
Author(s):  
Eliška Servusová ◽  
Vladimír Babák ◽  
Zora Piskatá ◽  
Pavel Krčmář
Keyword(s):  
Real Time ◽  
Dna Extraction ◽  
Species Identification ◽  
Real Time Pcr ◽  
Dna Analysis ◽  
Extraction Methods ◽  
Thunnus Albacares ◽  
Species Determination ◽  
Katsuwonus Pelamis ◽  
Canned Tuna

The most common methods that can be used for species identification of tuna include methods based on detection of species-specific DNA via the polymerase chain reaction (PCR) method. The problem with DNA detection in processed products is the possibility of DNA fragmentation during the technological process. The quantity and quality of extracted DNA is a crucial step for species identification based on the DNA analysis. In this study, two DNA extraction methods (DNeasy Blood & Tissue Kit and DNeasy mericon Food Kit) for tuna DNA isolation were compared. Eight food products of canned tuna (three of them were declared as Thunnus albacares and five products were declared as Katsuwonus pelamis) with a different addition of various ingredients were tested. Furthermore, three different times of proteolysis (30 min, 60 min, overnight) for each sample and each extraction kit were evaluated. The DNA concentration was determined by a Qubit dsDNA HS Assay Kit fluorescence method and quantified using a Qubit fluorometer. The DNA purity was evaluated using the A260/A280 ratio of absorbances measured on a spectrophotometer. The main indicator of DNA quality and quantity was its amplifiability in the subsequent real-time PCR for Thunnus species, Thunnus albacares and Katsuwonus pelamis. Based on the results, both kits can be used for tuna species determination in highly heat-treated products with different composition, nevertheless, the DNeasy mericon Food Kit provided better statistical values in some parameters. The effect of different times of proteolysis was significant in most of the samples with regard to the crossing point values determined by real-time PCR.

scholarly journals Evaluation of DNA extraction methods for quantifying Helicobacter pylori in water with PCR inhibitors

2021 ◽  
Author(s):  
Eun-Sook Lee ◽  
So-Yang Cha ◽  
Jong-Soon Jung
Keyword(s):  
Helicobacter Pylori ◽  
Real Time ◽  
Dna Extraction ◽  
Water Samples ◽  
Real Time Pcr ◽  
Heavy Rain ◽  
Extraction Methods ◽  
Pcr Inhibitors ◽  
H Pylori ◽  
Dna Extraction Methods

Abstract DNA extraction methods were evaluated to reduce PCR inhibitors and quantify Helicobacter pylori directly from water samples using real-time PCR. Three nucleic acid extraction methods were evaluated for different types of water samples. While the QIAamp DNA mini kit for tissue was suitable for DNA extraction from treated water, the QIAamp DNA stool mini kit was still efficient in analyzing samples from river water after heavy rain and with high concentration of PCR inhibitors. The FastDNA SPIN Kit for Soil could extract DNA effectively from microbes in river and stream waters without heavy rain. Immunomagnetic separation (IMS) was used prior to DNA extraction and was a useful tool for reducing PCR inhibitors in influent and stream samples. H. pylori in various waters could be quantified directly by real-time PCR while minimizing the effect of PCR inhibitors by an appropriate method through the evaluation of DNA extraction methods considering the characteristics of the matrix water. The findings of the present study suggest that the types or characteristics of water sample by source and precipitation are an important factor in detecting H. pylori and they can be applied when detecting and monitoring of other pathogens in water.

A comparative study of DNA extraction methods to be used in real-time PCR based quantification of ochratoxin A-producing molds in food products

Food Control ◽  
2012 ◽  
Vol 25 (2) ◽  
pp. 666-672 ◽  
Author(s):  
Alicia Rodríguez ◽  
Mar Rodríguez ◽  
M. Isabel Luque ◽  
Annemarie F. Justesen ◽  
Juan J. Córdoba
Keyword(s):  
Comparative Study ◽  
Real Time ◽  
Ochratoxin A ◽  
Dna Extraction ◽  
Real Time Pcr ◽  
Food Products ◽  
Extraction Methods ◽  
Dna Extraction Methods

scholarly journals Comparison of DNA extraction methods for Aspergillus fumigatus using real-time PCR

2006 ◽  
Vol 55 (9) ◽  
pp. 1187-1191 ◽  
Author(s):  
Lisa J. Griffiths ◽  
Martin Anyim ◽  
Sarah R. Doffman ◽  
Mark Wilks ◽  
Michael R. Millar ◽  
...  
Keyword(s):  
Real Time ◽  
Dna Extraction ◽  
Real Time Pcr ◽  
Diagnostic Technique ◽  
Extraction Methods ◽  
Simple Method ◽  
Bead Beating ◽  
Fungal Dna ◽  
Dna Extraction Methods ◽  
High Level

Newer methods such as PCR are being investigated in order to improve the diagnosis of invasive aspergillosis. One of the major obstacles to using PCR to diagnose aspergillosis is a reliable, simple method for extraction of the fungal DNA. The presence of a complex, sturdy cell wall that is resistant to lysis impairs extraction of the DNA by conventional methods employed for bacteria. Numerous fungal DNA extraction protocols have been described in the literature. However, these methods are time-consuming, require a high level of skill and may not be suitable for use as a routine diagnostic technique. Here, a number of extraction methods were compared: a freeze–thaw method, a freeze–boil method, enzyme extraction and a bead-beating method using Mini-BeadBeater-8. The quality and quantity of the DNA extracted was compared using real-time PCR. It was found that the use of a bead-beating method followed by extraction with AL buffer (Qiagen) was the most successful extraction technique, giving the greatest yield of DNA, and was also the least time-consuming method assessed.

Comparison of DNA extraction methods used to detect bacterial and yeast DNA from spiked whole blood by real-time PCR

2017 ◽  
Vol 140 ◽  
pp. 61-66 ◽  
Author(s):  
Libera M. Dalla-Costa ◽  
Luis G. Morello ◽  
Danieli Conte ◽  
Luciane A. Pereira ◽  
Jussara K. Palmeiro ◽  
...  
Keyword(s):  
Real Time ◽  
Dna Extraction ◽  
Real Time Pcr ◽  
Whole Blood ◽  
Extraction Methods ◽  
Dna Extraction Methods

Comparison of DNA extraction methods from muscle of canned tuna for species identification

Food Control ◽  
2007 ◽  
Vol 18 (10) ◽  
pp. 1211-1215 ◽  
Author(s):  
María José Chapela ◽  
Carmen G. Sotelo ◽  
Ricardo I. Pérez-Martín ◽  
Miguel Ángel Pardo ◽  
Begoña Pérez-Villareal ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Species Identification ◽  
Extraction Methods ◽  
Canned Tuna ◽  
Dna Extraction Methods

scholarly journals Development of a Magnetic Capture Hybridization Real-Time PCR Assay for Detection of Tumorigenic Agrobacterium vitis in Grapevines

2013 ◽  
Vol 103 (6) ◽  
pp. 633-640 ◽  
Author(s):  
Kameka L. Johnson ◽  
Desen Zheng ◽  
Supaporn Kaewnum ◽  
Cheryl Lynn Reid ◽  
Thomas Burr
Keyword(s):  
Real Time ◽  
Dna Extraction ◽  
Real Time Pcr ◽  
Detection Threshold ◽  
Pcr Primers ◽  
Immunomagnetic Separation ◽  
Extraction Methods ◽  
Economic Effects ◽  
Agrobacterium Vitis ◽  
Magnetic Capture

Agrobacterium vitis, the causal agent of grape crown gall, can have severe economic effects on grape production. The bacterium survives systemically in vines and, therefore, is disseminated in propagation material. We developed an assay for use in indexing programs that is efficient and sensitive for detecting A. vitis in grape tissue. Initially, real-time polymerase chain reaction (PCR) primers specific for diverse tumorigenic strains of A. vitis were developed using the virD2 gene sequence. To overcome the effects of PCR inhibitors present in plant tissue, DNA extraction methods that included magnetic capture hybridization (MCH), immunomagnetic separation (IMS), and extraction with the Mo Bio Powerfood kit were compared. The assays incorporating MCH or IMS followed by real-time PCR were 10,000-fold more sensitive than direct real-time PCR when tested using boiled bacterial cell suspensions, with detection thresholds of 101 CFU/ml compared with 105 CFU/ml. DNA extraction with the Powerfood DNA extraction kit was 10-fold more sensitive than direct real-time PCR, with a detection threshold of 104 CFU/ml. All three assays were able to detect A. vitis in healthy-appearing grapevine cuttings taken from infected vines.

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