scholarly journals Multicenter Comparative Study of Six Cryptosporidium parvum DNA Extraction Protocols Including Mechanical Pretreatment from Stool Samples

2020 ◽  
Vol 8 (9) ◽  
pp. 1450
Author(s):  
Nicolas Valeix ◽  
Damien Costa ◽  
Louise Basmaciyan ◽  
Stéphane Valot ◽  
Anne Vincent ◽  
...  
Keyword(s):  
Comparative Study ◽  
Real Time ◽  
Dna Extraction ◽  
Cryptosporidium Parvum ◽  
Real Time Pcr ◽  
Sample Pretreatment ◽  
Dna Amplification ◽  
Extraction Methods ◽  
Mechanical Pretreatment ◽  
Stool Samples

Background: Nowadays, many commercial kits allow the detection of Cryptosporidium sp. in stool samples after deoxyribonucleic acid (DNA) extraction. Protocols of stool pretreatment have been proposed to optimize oocysts’ DNA extraction. Among them, mechanical grinding was reported to improve the performance of Cryptosporidium oocysts’ DNA extraction. Methods: A multicenter comparative study was conducted within the framework of the French National Reference Center-Expert Laboratory for Cryptosporidiosis. Six extraction systems (i.e., manual or automated) associated with various mechanical pretreatment protocols, were compared for the Cryptosporidium parvum oocyst’ DNA extraction, before amplification using the same real-time PCR method targeting. Results: The sensitivity of real-time PCR assay was unequally impacted by the pretreatment/extraction protocol. We observed significant differences for the lowest concentrations of C. parvum oocysts (i.e., 0–94.4% and 33.3–100% respectively for 10 and 50 oocysts/mL). All in all, the protocol using Quick DNA Fecal/Soil Microbe-Miniprep® manual kit showed the best performances. In addition, optimal performances of mechanical pretreatment were obtained by combining a grinding duration of 60 s with a speed of 4 m/s using Fastprep24® with Lysing Matrix E®. Conclusions: Sample pretreatment, as well as the extraction method, needs to be properly adapted to improve the diagnostic performances of the C. parvum DNA amplification methods.

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 Assessment of real-time PCR for Helicobacter pylori DNA detection in stool with co-infection of intestinal parasites: a comparative study of DNA extraction methods

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Martina Leonardi ◽  
Giulia La Marca ◽  
Barbara Pajola ◽  
Francesca Perandin ◽  
Marco Ligozzi ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Comparative Study ◽  
Real Time ◽  
Dna Extraction ◽  
Real Time Pcr ◽  
Dna Detection ◽  
Intestinal Parasites ◽  
Extraction Methods ◽  
Dna Extraction Methods

scholarly journals Introducing automation to the molecular diagnosis of Trypanosoma cruzi infection: A comparative study of sample treatments, DNA extraction methods and real-time PCR assays

PLoS ONE ◽  
2018 ◽  
Vol 13 (4) ◽  
pp. e0195738 ◽  
Author(s):  
Alba Abras ◽  
Cristina Ballart ◽  
Teresa Llovet ◽  
Carme Roig ◽  
Cristina Gutiérrez ◽  
...  
Keyword(s):  
Trypanosoma Cruzi ◽  
Comparative Study ◽  
Real Time ◽  
Dna Extraction ◽  
Molecular Diagnosis ◽  
Real Time Pcr ◽  
Extraction Methods ◽  
Pcr Assays ◽  
Dna Extraction Methods ◽  
Cruzi Infection

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.

scholarly journals Evaluation of Two DNA Extraction Methods for Detection of Strongyloides stercoralis Infection

2018 ◽  
Vol 56 (4) ◽  
Author(s):  
Beatrice Barda ◽  
Rahel Wampfler ◽  
Somphou Sayasone ◽  
Khampheng Phongluxa ◽  
Syda Xayavong ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Strongyloides Stercoralis ◽  
Extraction Methods ◽  
Molecular Techniques ◽  
Diagnostic Methods ◽  
Content Type ◽  
Stool Samples ◽  
Dna Extraction Methods ◽  
Baermann Method

ABSTRACT Strongyloides stercoralis is present worldwide, but its prevalence is still uncertain, mainly due to the lack of sensitivity of diagnostic methods. Molecular techniques are under development, but a standardized protocol is still unavailable. We compared the sensitivity of real-time PCR, using two extraction protocols, with that of the Baermann technique. Samples were collected in the framework of the baseline screening of a randomized clinical trial evaluating moxidectin against S. stercoralis in Lao People's Democratic Republic. Two stool samples from each participant were processed by the Baermann method, and one subsample was processed by PCR. DNA was extracted using the QIAamp DNA stool minikit based on the standard protocol for the QIAamp DNA minikit (QIA) and using a modification of the QIA procedure (POL). Subsequently, all extracted samples were analyzed by real-time PCR. Overall, 95 samples were analyzed by the three diagnostic methods. Sixty-nine (72.6%) samples were positive according to the Baermann method, 25 (26.3%) by the QIA method, and 62 (65.3%) by the POL method. The sensitivities were 86% (95% confidence interval [CI], 76.7 to 92.9), 31.0% (95% CI, 21.3 to 42.6), and 78.0% (95% CI, 66.8 to 86.1) for the Baermann, QIA, and POL methods, respectively. The sensitivities calculated for each day of the Baermann method separately were 60% (48.4 to 70.8%) and 64% (52.2 to 74.2%) for days 1 and 2, respectively. In conclusion, the POL method revealed a good performance and was comparable to the Baermann test performed on two stool samples and superior to the Baermann method performed on one stool sample. Additional studies are needed to standardize a PCR protocol for S. stercoralis diagnosis.

scholarly journals Evaluation of PCR inhibitory effect of enrichment broths and comparison of DNA extraction methods for detection of Salmonella Enteritidis using real-time PCR assay

2010 ◽  
Vol 11 (2) ◽  
pp. 143 ◽  
Author(s):  
Ji-Yeon Hyeon ◽  
In-Gyun Hwang ◽  
Hyo-Sun Kwak ◽  
Chankyu Park ◽  
In-Soo Choi ◽  
...  
Keyword(s):  
Real Time ◽  
Dna Extraction ◽  
Real Time Pcr ◽  
Salmonella Enteritidis ◽  
Extraction Methods ◽  
Inhibitory Effect ◽  
Pcr Assay ◽  
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.

Evaluation of alternative DNA extraction processes and real-time PCR for detecting Cryptosporidium parvum in drinking water

2015 ◽  
Vol 15 (6) ◽  
pp. 1295-1303
Author(s):  
Gina H. Kimble ◽  
Vincent R. Hill ◽  
James E. Amburgey
Keyword(s):  
Drinking Water ◽  
Real Time ◽  
Dna Extraction ◽  
Cryptosporidium Parvum ◽  
Water Samples ◽  
Real Time Pcr ◽  
Extraction Procedure ◽  
The United States ◽  
Detection Sensitivity ◽  
Spin Column

USEPA Method 1623 is the standard method in the United States for the detection of Cryptosporidium in water samples, but quantitative real-time polymerase chain reaction (qPCR) is an alternative technique that has been successfully used to detect Cryptosporidium in aqueous matrices. This study examined various modifications to a commercial nucleic acid extraction procedure in order to enhance PCR detection sensitivity for Cryptosporidium. An alternative DNA extraction buffer allowed for qPCR detection at lower seed levels than a commercial extraction kit buffer. In addition, the use of a second spin column cycle produced significantly better detection (P = 0.031), and the volume of Tris–EDTA buffer significantly affected crossing threshold values (P = 0.001). The improved extraction procedure was evaluated using 10 L of tap water samples processed by ultrafiltration, centrifugation and immunomagnetic separation. Mean recovery for the sample processing method was determined to be 41% using microscopy and 49% by real-time PCR (P = 0.013). The results of this study demonstrate that real-time PCR can be an effective alternative for detecting and quantifying Cryptosporidium parvum in drinking water samples.

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