A comparison of DNA extraction and purification methods to detect Escherichia coli O157:H7 in cattle manure

2003 ◽  
Vol 54 (2) ◽  
pp. 165-175 ◽  
Author(s):  
Teegan Trochimchuk ◽  
John Fotheringham ◽  
Edward Topp ◽  
Heidi Schraft ◽  
Kam Tin Leung
Keyword(s):  
Escherichia Coli ◽  
Dna Extraction ◽  
Cattle Manure ◽  
Escherichia Coli O157 ◽  
Extraction And Purification ◽  
Purification Methods

Comparison of Four Commercial DNA Extraction Kits for PCR Detection of Listeria monocytogenes, Salmonella, Escherichia coli O157:H7, and Staphylococcus aureus in Fresh, Minimally Processed Vegetables

2008 ◽  
Vol 71 (10) ◽  
pp. 2110-2114 ◽  
Author(s):  
P. ELIZAQUÍVEL ◽  
R. AZNAR
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Listeria Monocytogenes ◽  
Dna Extraction ◽  
Pcr Detection ◽  
Escherichia Coli O157 ◽  
Minimally Processed ◽  
Green Pepper ◽  
Minimally Processed Vegetables ◽  
And Staphylococcus Aureus

Four commercial DNA extraction methods, PrepMan Ultra (Applied Biosystems), InstaGene Matrix (BioRad), DNeasy Tissue kit (Qiagen), and UltraClean (MoBio), were tested for PCR detection of Listeria monocytogenes, Escherichia coli O157: H7, Salmonella, and Staphylococcus aureus in fresh, minimally processed vegetables. For comparative purposes, sensitivity assays with specific PCRs were carried out after DNA extraction with the four methods in green pepper, broccoli, and onion artificially inoculated with the four pathogens separately. As confirmed by statistical analysis, the DNeasy Tissue kit rendered the highest sensitivity values in the three matrices assayed for Salmonella, L. monocytogenes, and E. coli O157:H7 and in onion for S. aureus. Despite being the most expensive of the methods compared, the DNeasy Tissue Kit can be successfully applied for any of the four most commonly studied pathogens, thus saving time and overall reducing the cost of the analysis.

scholarly journals Effects of DNA Extraction and Purification Methods on Real-Time Quantitative PCR Analysis of Roundup Ready Soybean

2009 ◽  
Vol 92 (4) ◽  
pp. 1136-1144 ◽  
Author(s):  
Tigst Demeke ◽  
Indira Ratnayaka ◽  
Anh Phan
Keyword(s):  
Real Time ◽  
Dna Extraction ◽  
Quantitative Pcr ◽  
Real Time Pcr ◽  
Roundup Ready ◽  
Dna Recovery ◽  
Real Time Quantitative Pcr ◽  
Extraction And Purification ◽  
Purification Methods ◽  
Accuracy And Repeatability

Abstract The quality of DNA affects the accuracy and repeatability of quantitative PCR results. Different DNA extraction and purification methods were compared for quantification of Roundup Ready (RR) soybean (event 40-3-2) by real-time PCR. DNA was extracted using cetylmethylammonium bromide (CTAB), DNeasy Plant Mini Kit, and Wizard Magnetic DNA purification system for food. CTAB-extracted DNA was also purified using the Zymo (DNA Clean & Concentrator 25 kit), Qtip 100 (Qiagen Genomic-Tip 100/G), and QIAEX II Gel Extraction Kit. The CTAB extraction method provided the largest amount of DNA, and the Zymo purification kit resulted in the highest percentage of DNA recovery. The Abs260/280 and Abs260/230 ratios were less than the expected values for some of the DNA extraction and purification methods used, indicating the presence of substances that could inhibit PCR reactions. Real-time quantitative PCR results were affected by the DNA extraction and purification methods used. Further purification or dilution of the CTAB DNA was required for successful quantification of RR soybean. Less variability of quantitative PCR results was observed among experiments and replications for DNA extracted and/or purified by CTAB, CTAB+Zymo, CTAB+Qtip 100, and DNeasy methods. Correct and repeatable results for real-time PCR quantification of RR soybean were achieved using CTAB DNA purified with Zymo and Qtip 100 methods.

scholarly journals Soil survival of Escherichia coli O157:H7 acquired by a child from garden soil recently fertilized with cattle manure

2006 ◽  
Vol 101 (2) ◽  
pp. 429-436 ◽  
Author(s):  
A. Mukherjee ◽  
S. Cho ◽  
J. Scheftel ◽  
S. Jawahir ◽  
K. Smith ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cattle Manure ◽  
Garden Soil ◽  
Escherichia Coli O157

scholarly journals A PCR Specific for Escherichia coli O157 Based on the rfb Locus Encoding O157 Lipopolysaccharide

1998 ◽  
Vol 36 (6) ◽  
pp. 1801-1804 ◽  
Author(s):  
Patricia M. Desmarchelier ◽  
Sima S. Bilge ◽  
Narelle Fegan ◽  
Leanne Mills ◽  
James C. Vary ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Dna Extraction ◽  
Raw Milk ◽  
Escherichia Coli O157 ◽  
E Coli ◽  
Cell Lysates ◽  
O Antigen ◽  
Pcr Product

A PCR was developed for the detection of Escherichia coli O157 based on the rfbE O-antigen synthesis genes. A 479-bp PCR product was amplified specifically from E. coli O157 in cell lysates containing 200 or 2 CFU following crude DNA extraction. The PCR detected <1 CFU ofE. coli O157 per ml in raw milk following enrichment.

scholarly journals Lyophilization Prior to Direct DNA Extraction from Bovine Feces Improves the Quantification of Escherichia coli O157:H7 and Campylobacter jejuni

2009 ◽  
Vol 76 (5) ◽  
pp. 1686-1688 ◽  
Author(s):  
Delphine Rapp ◽  
John Waller ◽  
Gale Brightwell ◽  
Richard W. Muirhead
Keyword(s):  
Escherichia Coli ◽  
Real Time ◽  
Dna Extraction ◽  
Campylobacter Jejuni ◽  
Real Time Pcr ◽  
Escherichia Coli O157 ◽  
Fresh Feces ◽  
Direct Dna Extraction ◽  
Reliable Quantification ◽  
Bovine Feces

ABSTRACT Lyophilization was used to concentrate bovine feces prior to DNA extraction and analysis using real-time PCR. Lyophilization significantly improved the sensitivity of detection compared to that in fresh feces and was associated with reliable quantification of both Escherichia coli O157:H7 and Campylobacter jejuni bacteria present in feces at concentrations ranging between 2 log10 and 6 log10 CFU g− 1.

scholarly journals Rapid and sensitive detection of Escherichia coli O157:H7 using coaxial channel-based DNA extraction and microfluidic PCR

2018 ◽  
Vol 101 (11) ◽  
pp. 9736-9746 ◽  
Author(s):  
Huilin Zhang ◽  
Fengchun Huang ◽  
Gaozhe Cai ◽  
Yuntao Li ◽  
Jianhan Lin
Keyword(s):  
Escherichia Coli ◽  
Dna Extraction ◽  
Sensitive Detection ◽  
Escherichia Coli O157 ◽  
Coaxial Channel
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