scholarly journals Comparison of different conditions for DNA extraction in sputum - a pilot study

2019 ◽  
Vol 14 ◽  
Author(s):  
Martina Oriano ◽  
Leonardo Terranova ◽  
Antonio Teri ◽  
Samantha Sottotetti ◽  
Luca Ruggiero ◽  
...  
Keyword(s):  
Real Time ◽  
16S Rdna ◽  
Dna Extraction ◽  
Real Time Pcr ◽  
Alpha Diversity ◽  
Diversity Indices ◽  
Extraction Yield ◽  
Chronic Respiratory Diseases ◽  
Dna Yield ◽  
Selection Of

Background: The analysis of microbiome in respiratory samples is a topic of great interest in chronic respiratory diseases. The method used to prepare sputum samples for microbiome analysis is very heterogeneous. The selection of the most suitable methodology for DNA extraction is fundamental to have the most representative data. The objective of this study was to compare different conditions for DNA extraction from sputum in adult patients with bronchiectasis. Methods: Five sputum samples from bronchiectasis patients were collected at the Policlinico Hospital in Milan, Italy. Eighteen conditions for DNA extraction were compared, including two enzyme-based (Roche and Zymo) and one beads-based (Mobio) technique. These techniques were tested with/without Dithiothreitol (DTT) and with/without lysostaphin (0.18 and 0.36 mg/mL) step. DNA was quantified, tested using Real-time PCR for 16S rDNA and S. aureus and, then, microbiome was evaluated. Results: Although 16S rDNA was similarly detected across all the different techniques, Roche kit gave the highest DNA yield. The lowest Ct values for Real-time PCR for S. aureus was identified when lysostaphin was added. Considering genera from microbiome, alpha diversity indices did not show any significant differences between techniques, while relative abundances were more similar in presence of DTT. Conclusions: None of the conditions emerged to be superior to the others even if enzyme-based kits seem to be needed in order to have a higher extraction yield.

scholarly journals Rnase A Enzyme Modification of Optimized SDS Protocol for DNA Extraction Suitable for Real-Time PCR Screening of GMOs

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Arita Sabriu-Haxhijaha ◽  
Velimir Stojkovski ◽  
Gordana Ilievska ◽  
Dean Jankuloski ◽  
Katerina Blagoevska
Keyword(s):  
Real Time ◽  
Dna Extraction ◽  
Real Time Pcr ◽  
Genetically Modified Crops ◽  
Dna Amplification ◽  
Rnase A ◽  
High Quality ◽  
Pcr Screening ◽  
Dna Yield ◽  
Corn Kernels

Abstract As the number of genetically modified crops increases rapidly, their accurate detection is significant for labelling and safety assessment. Currently, real-time PCR is the “golden standard” method for GMO detection. Hence, extraction of high quality DNA represents a crucial step for accurate and efficient DNA amplification. For GMO presence evaluation in the extracted DNA from raw corn kernels and roasted soybean, we used real-time PCR method, in consistent with the ISO17025 accreditation standards. As for DNA extraction, modified basic SDS protocol by adding RNase A enzyme in different steps of the protocol, with different time and temperature of incubation was used. The results showed as most suitable, the protocol where 10 µl of RNase A enzyme was added together with the lysis buffer at 65 °C for 30 minutes. Data for DNA yield and purity for roasted soybean was 469.6±3.3 µg/ml with A260/280 absorbance ratio 1.78±0.01. Suitability of DNA extracts for GMO analysis was assessed by screening for the presence of 35S promotor and Tnos terminator. Diluted extracts in concentrations 10, 1, 0.1, 0.01 and 0.0027 ng/µl, were tested in six replicates. Positive signal of amplification (LOD) was detected in all concentrations for both genetic elements in both matrices. The LOQ for 35S and Tnos for both matrices was 0.1 ng, while for Tnos in raw corn kernels was 0.01 ng. This in-house developed DNA extraction method is simple and obtains high-quality DNA suitable for GMO screening of 35S promotor and Tnos terminator in both raw and processed matrices.

scholarly journals How to Process Sputum Samples and Extract Bacterial DNA for Microbiota Analysis

2018 ◽  
Vol 19 (10) ◽  
pp. 3256 ◽  
Author(s):  
Leonardo Terranova ◽  
Martina Oriano ◽  
Antonio Teri ◽  
Luca Ruggiero ◽  
Camilla Tafuro ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Real Time ◽  
Dna Extraction ◽  
Real Time Pcr ◽  
Coefficient Of Variation ◽  
Extraction Yield ◽  
Rrna Gene ◽  
Microbiota Analysis ◽  
The 16S Rrna Gene

Different steps and conditions for DNA extraction for microbiota analysis in sputum have been reported in the literature. We aimed at testing both dithiothreitol (DTT) and enzymatic treatments of sputum samples and identifying the most suitable DNA extraction technique for the microbiota analysis of sputum. Sputum treatments with and without DTT were compared in terms of their median levels and the coefficient of variation between replicates of both DNA extraction yield and real-time PCR for the 16S rRNA gene. Treatments with and without lysozyme and lysostaphin were compared in terms of their median levels of real-time PCR for S. aureus. Two enzyme-based and three beads-based techniques for DNA extraction were compared in terms of their DNA extraction yield, real-time PCR for the 16S rRNA gene and microbiota analysis. DTT treatment decreased the coefficient of variation between replicates of both DNA extraction yield and real-time PCR. Lysostaphin (either 0.18 or 0.36 mg/mL) and lysozyme treatments increased S. aureus detection. One enzyme-based kit offered the highest DNA yield and 16S rRNA gene real-time PCR with no significant differences in terms of alpha-diversity indexes. A condition using both DTT and lysostaphin/lysozyme treatments along with an enzymatic kit seems to be preferred for the microbiota analysis of sputum samples.

scholarly journals Endocervical swabs transported in first void urine as combined specimens in the detection of Mycoplasma genitalium by real-time PCR

2009 ◽  
Vol 58 (1) ◽  
pp. 117-120 ◽  
Author(s):  
Andreas Edberg ◽  
Fredrik Aronsson ◽  
Eva Johansson ◽  
Elisabeth Wikander ◽  
Thomas Ahlqvist ◽  
...  
Keyword(s):  
Real Time ◽  
Dna Extraction ◽  
Real Time Pcr ◽  
Mycoplasma Genitalium ◽  
Extraction Methods ◽  
Pcr Inhibition ◽  
Dna Yield ◽  
Extraction Study ◽  
Dna Extraction Methods ◽  
Swab Specimen

The aim of this study was to determine whether a patient's endocervical swab specimen can be transported in first void urine (FVU) as combined specimens for the detection of Mycoplasma genitalium by real-time PCR. The study also compared two different DNA extraction methods for observation of possible PCR inhibition. Three specimens, one endocervical swab specimen transported in 2-SP medium, one endocervical swab specimen transported in FVU and a FVU specimen, were collected from 329 women. All sample types underwent manual DNA extraction whereas in the DNA extraction study, 329 endocervical swab specimens transported in FVU were subjected to both manual Chelex and automated BioRobot M48 DNA extraction. A total of 100 endocervical swab specimens transported in FVU from patients PCR-negative for M. genitalium in the study were used in the PCR inhibition analysis. M. genitalium was detected in 25/329 (7.6 %) women. The endocervical swab specimens transported in 2-SP medium and transported in FVU were positive for M. genitalium in 17/25 (68 %) and 24/25 (96 %) women, respectively. The FVU specimens alone were positive for M. genitalium in 22/25 (88 %) women. In the DNA extraction study, M. genitalium DNA was detected in 24/329 (7.3 %) and 28/329 (8.5 %) of endocervical swab specimens transported in FVU subjected to manual Chelex extraction and automated BioRobot M48 extraction, respectively. Partial PCR inhibition was detected in 6 % of samples subjected to manual Chelex extraction whereas no inhibition was detected with the automated BioRobot M48 extraction. Thus endocervical swab specimens transported in FVU demonstrate higher sensitivity than FVU specimens only and have considerably increased sensitivity compared with endocervical swab specimens transported in 2-SP medium for detection of M. genitalium DNA. Moreover, automated BioRobot M48 extraction was shown to be superior to a crude manual Chelex extraction, leaving no PCR inhibition and giving a slightly higher DNA yield and/or better sensitivity.

Real-time PCR-assisted selection of wheat plants transformed with HMW glutenin subunit genes

2005 ◽  
Vol 41 (1) ◽  
pp. 133-136 ◽  
Author(s):  
Valeria Terzi ◽  
Gabriela Pastori ◽  
Peter R. Shewry ◽  
Natale Di Fonzo ◽  
A. Michele Stanca ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Glutenin Subunit ◽  
Hmw Glutenin Subunit ◽  
Hmw Glutenin ◽  
Selection Of

scholarly journals Methods for optimizing DNA extraction before quantifying oral bacterial numbers by real-time PCR

2009 ◽  
Vol 296 (1) ◽  
pp. 45-51 ◽  
Author(s):  
Mangala A. Nadkarni ◽  
F. Elizabeth Martin ◽  
Neil Hunter ◽  
Nicholas A. Jacques
Keyword(s):  
Real Time ◽  
Dna Extraction ◽  
Real Time Pcr

scholarly journals Toxoplasma gondii and pre-treatment protocols for polymerase chain reaction analysis of milk samples: a field trial in sheep from Southern Italy

2017 ◽  
Vol 6 (1) ◽  
Author(s):  
Alice Vismarra ◽  
Elena Barilli ◽  
Maura Miceli ◽  
Carlo Mangia ◽  
Cristina Bacci ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Real Time ◽  
Dna Extraction ◽  
Real Time Pcr ◽  
Southern Italy ◽  
Raw Milk ◽  
Type I ◽  
Treatment Protocols ◽  
Milk Samples ◽  
Pre Treatment

Toxoplasmosis is a zoonotic disease caused by the protozoan <em>Toxoplasma gondii</em>. Ingestion of raw milk has been suggested as a risk for transmission to humans. Here the authors evaluated pre-treatment protocols for DNA extraction on <em>T. gondii</em> tachyzoite-spiked sheep milk with the aim of identifying the method that resulted in the most rapid and reliable PCR positivity. This protocol was then used to analyze milk samples form sheep from three different farms in southern Italy, including Real Time PCR for DNA quantification and PCR-RFLP for genotyping. The pre-treatment protocol using EDTA and Tris-HCl to remove casein gave the best results in the least amount of time compared to the others on spiked milk samples. One sample of 21 collected from sheep farms was positive on one-step PCR, Real Time PCR and resulted in a Type I genotype at one locus (SAG3). Milk usually contains a low number of tachyzoites and this could be a limiting factor for molecular identification. Our preliminary data has evaluated a rapid, cost-effective and sensitive protocol to treat milk before DNA extraction. The results of the present study also confirm the possibility of <em>T. gondii</em> transmission through consumption of raw milk and its unpasteurized derivatives.

A GeNorm algorithm-based selection of reference genes for quantitative real-time PCR in skin biopsies of healthy dogs and dogs with atopic dermatitis

2009 ◽  
Vol 129 (1-2) ◽  
pp. 115-118 ◽  
Author(s):  
Yvette M. Schlotter ◽  
Eveline Z. Veenhof ◽  
Bas Brinkhof ◽  
Victor P.M.G. Rutten ◽  
Bart Spee ◽  
...  
Keyword(s):  
Atopic Dermatitis ◽  
Real Time ◽  
Real Time Pcr ◽  
Reference Genes ◽  
Quantitative Real Time Pcr ◽  
Skin Biopsies ◽  
Healthy Dogs ◽  
Selection Of

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 ORAL HIV-ASSOCIATED KAPOSI SARCOMA: A COMPARISON BETWEEN IMMUNOHISTOCHEMISTRY AND qPCR TECHNIQUES FOR DETECTION OF HHV8

2015 ◽  
Vol 21 (1) ◽  
pp. 29
Author(s):  
Priscila Lie Tobouti ◽  
Juliana Seo ◽  
Michella Bezerra Lima ◽  
Bruno Tavares Sedassari ◽  
Norberto Nobuo Sugaya ◽  
...  
Keyword(s):  
Real Time ◽  
Dna Extraction ◽  
Real Time Pcr ◽  
Kaposi Sarcoma ◽  
Positive Staining ◽  
Extraction Protocol ◽  
Simple Phenol ◽  
Two Samples ◽  
Hematoxylin And Eosin ◽  
The Mean

<p><strong>Objective: </strong>To compare the diagnostic accuracy of immunohistochemistry compared to real-time PCR (using a simple phenol-chloroform DNA extraction protocol) in the detection of HHV8 in small oral biopsies of Kaposi sarcoma. Also to validate the use of this DNA extraction protocol to extract HHV8 DNA.</p><p><strong>Material and methods:</strong> Seventeen cases of oral KS were examined. Data including gender, age, and anatomic location were obtained from patient´s records and histological sections stained with hematoxylin and eosin (H&amp;E) were reviewed. Immunohistochemistry was used to detect HHV8 in lesions of interest, as well as real-time PCR.</p><p><strong>Results: </strong>All the patients were HIV positive, the mean age was 35.5 years old, and the affected oral sites were palate (47%), gingiva (29.4%), tongue (11.8%), lip (5.9%), and oral floor (5.9%). Fifteen samples showed positive staining for HHV8 and only two samples were negative. The same results were observed using real-time PCR HHV8-DNA detection.</p><p><strong>Relevance: </strong>Our findings suggest that immunohistochemistry is faster and cheaper to perform than real-time PCR and was shown to have similar levels of sensitivity and accuracy for detection of HHV8 even in small biopsies. Additionally DNA extraction using a non-commercial kit, as done in this study can further reduce the costs to a pathology service.</p>

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