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

Toxoplasmosis is a zoonotic disease caused by the protozoan Toxoplasma gondii. 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 T. gondii 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 T. gondii transmission through consumption of raw milk and its unpasteurized derivatives.

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

Multidisciplinary Respiratory Medicine ◽

10.4081/mrm.2019.4 ◽

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.

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

Water Science & Technology Water Supply ◽

10.2166/ws.2021.197 ◽

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.

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

Clinical and Laboratorial Research in Dentistry ◽

10.11606/issn.2357-8041.clrd.2015.97224 ◽

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

Objective: 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.Material and methods: 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&E) were reviewed. Immunohistochemistry was used to detect HHV8 in lesions of interest, as well as real-time PCR.Results: 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.Relevance: 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.

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Tendency for Interlaboratory Precision in the GMO Analysis Method Based on Real-Time PCR

Journal of AOAC International ◽

10.1093/jaoac/93.2.734 ◽

2010 ◽

Vol 93 (2) ◽

pp. 734-749 ◽

Cited By ~ 6

Author(s):

Takashi Kodama ◽

Yasunori Kurosawa ◽

Kazumi Kitta ◽

Shigehiro Naito

Keyword(s):

Standard Deviation ◽

Real Time ◽

Dna Extraction ◽

Real Time Pcr ◽

Quantitative Methods ◽

Genetically Modified ◽

Performance Criterion ◽

Gm Crops ◽

Evaluation Procedure ◽

Method Performance

Abstract The Horwitz curve estimates interlaboratory precision as a function only of concentration, and is frequently used as a method performance criterion in food analysis with chemical methods. The quantitative biochemical methods based on real-time PCR require an analogous criterion to progressively promote method validation. We analyzed the tendency of precision using a simplex real-time PCR technique in 53 collaborative studies of seven genetically modified (GM) crops. Reproducibility standard deviation (SR) and repeatability standard deviation (Sr) of the genetically modified organism (GMO) amount () was more or less independent of GM crops (i.e., maize, soybean, cotton, oilseed rape, potato, sugar beet, and rice) and evaluation procedure steps. Some studies evaluated whole steps consisting of DNA extraction and PCR quantitation, whereas others focused only on the PCR quantitation step by using DNA extraction solutions. Therefore, SR and Sr for GMO amount () are functions only of concentration similar to the Horwitz curve. We proposed SR 0.1971C0.8685 and Sr 0.1478C0.8424, where C is the GMO amount (). We also proposed a method performance index in GMO quantitative methods that is analogous to the Horwitz Ratio.

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

Journal of AOAC International ◽

10.1093/jaoac/92.4.1136 ◽

2009 ◽

Vol 92 (4) ◽

pp. 1136-1144 ◽

Cited By ~ 18

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.

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Comparison of six methods of DNA extraction for the diagnosis of bovine brucellosis by real-time PCR

Archives of Microbiology ◽

10.1007/s00203-019-01675-3 ◽

2019 ◽

Vol 201 (8) ◽

pp. 1025-1028

Author(s):

Ingred Sales Preis ◽

André Moura ◽

Patrícia Gomes de Souza ◽

Paulo Martins Soares Filho ◽

Antônio Augusto Fonseca Júnior

Keyword(s):

Real Time ◽

Dna Extraction ◽

Real Time Pcr ◽

Bovine Brucellosis

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Quantitative Detection of Campylobacter jejuni on Fresh Chicken Carcasses by Real-Time PCR

Journal of Food Protection ◽

10.4315/0362-028x-70.6.1373 ◽

2007 ◽

Vol 70 (6) ◽

pp. 1373-1378 ◽

Cited By ~ 30

Author(s):

ANNA-CLARA RÖNNER ◽

HANS LINDMARK

Keyword(s):

Detection Limit ◽

Real Time ◽

Dna Extraction ◽

Campylobacter Jejuni ◽

Real Time Pcr ◽

Close Correlation ◽

Quantitative Detection ◽

Quantitative Real Time Pcr ◽

Direct Plating ◽

Pcr Method

Campylobacter jejuni infection is a significant cause of foodborne gastroenteritis worldwide. Consumption and handling of poultry products is believed to be the primary risk factor for campylobacteriosis. Risk assessments require quantitative data, and C. jejuni is enumerated usually by direct plating, which sometimes allows growth of non-Campylobacter bacteria. The objective of the present study was to develop a quantitative real-time PCR method (q-PCR) for enumerating C. jejuni in chicken rinse without a culturing step. The procedure to obtain the template for the PCR assay involved (i) filtration of 10 ml of chicken rinse, (ii) centrifugation of the sample, and (iii) total DNA extraction from the pellet obtained using a commercial DNA extraction kit. The detection limit of the method was comparable to that for plating 100 μl of chicken rinse on modified charcoal cefoperazone deoxycholate agar, and the detection limit could be further improved 10-fold by concentrating the DNA eluate by ethanol precipitation. A close correlation for spiked chicken rinse was obtained for the results of the quantitative real-time PCR method and direct plating (r = 0.99). The coefficient of correlation for the methods was 0.87 when samples from chicken carcasses on the slaughter line were analyzed, whereas a lower correlation (r = 0.76) was obtained when samples from retail carcasses were analyzed. Greater variation in the proportion of dead and/or viable but not culturable Campylobacter types in the retail samples may explain the decreased correlation between the methods. Overall, the new method is simple and fast and the results obtained are closely correlated with those for direct plating for samples containing a low proportion of dead Campylobacter cells.

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