Development of a direct DNA extraction protocol for real-time PCR detection of Giardia lamblia from surface water

Ecotoxicology ◽  
2009 ◽  
Vol 18 (6) ◽  
pp. 661-668 ◽  
Author(s):  
Xin Yu ◽  
Michele I. Van Dyke ◽  
Andrea Portt ◽  
Peter M. Huck
Keyword(s):  
Surface Water ◽  
Real Time ◽  
Dna Extraction ◽  
Real Time Pcr ◽  
Giardia Lamblia ◽  
Pcr Detection ◽  
Extraction Protocol ◽  
Direct Dna Extraction

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>

scholarly journals Persistence of a Stx-Encoding Bacteriophage in Minced Meat Investigated by Application of an Improved DNA Extraction Method and Digital Droplet PCR

2021 ◽  
Vol 11 ◽  
Author(s):  
B. Spilsberg ◽  
C. Sekse ◽  
Anne M. Urdahl ◽  
Live L. Nesse ◽  
Gro S. Johannessen
Keyword(s):  
Real Time ◽  
Dna Extraction ◽  
Shiga Toxin ◽  
Real Time Pcr ◽  
Food Storage ◽  
Shiga Toxins ◽  
Digital Droplet Pcr ◽  
Extraction Protocol ◽  
Droplet Pcr ◽  
Minced Meat

Shiga toxin-producing Escherichia coli (STEC) are important food-borne pathogens with Shiga toxins as the main virulence factor. Shiga toxins are encoded on Shiga toxin-encoding bacteriophages (Stx phages). Stx phages may exist as free bacteriophages in the environment or in foods or as prophages integrated into the host genome. From a food safety perspective, it is important to have knowledge on the survival and persistence of Stx phages in food products since these may integrate into the bacterial hosts through transduction if conditions are right. Here, we present the results from a study investigating the survival of a Stx phage in minced meat from beef stored at a suboptimal temperature (8°C) for food storage along with modifications and optimizations of the methods applied. Minced meat from beef was inoculated with known levels of a labeled Stx phage prior to storage. Phage filtrates were used for plaque assays and DNA extraction, followed by real-time PCR and digital droplet PCR (ddPCR). The results from the pilot study suggested that the initial DNA extraction protocol was not optimal, and several modifications were tested before a final protocol was defined. The final DNA extraction protocol comprised ultra-centrifugation of the entire phage filtrate for concentrating phages and two times phenol–chloroform extraction. The protocol was used for two spiking experiments. The DNA extraction protocol resulted in flexibility in the amount of DNA available for use in PCR analyses, ultimately increasing the sensitivity of the method used for quantification of phages in a sample. All three quantification methods employed (i.e., plaque assays, real-time PCR, and ddPCR) showed similar trends in the development of the phages during storage, where ddPCR has the benefit of giving absolute quantification of DNA copies in a simple experimental setup. The results indicate that the Stx phages persist and remain infective for at least 20 days under the storage conditions used in the present study. Stx phages in foods might represent a potential risk for humans. Although it can be speculated that transduction may take place at 8°C with subsequent forming of STEC, it can be expected to be a rare event. However, such an event may possibly take place under more optimal conditions, such as an increase in storage temperature of foods or in the gastrointestinal tract of humans.

scholarly journals A simple and rapid protocol of crude DNA extraction from apple trees for PCR and real-time PCR detection of ‘Candidatus Phytoplasma mali’

2009 ◽  
Vol 156 (1-2) ◽  
pp. 96-101 ◽  
Author(s):  
M. Aldaghi ◽  
S. Massart ◽  
O. Dutrecq ◽  
A. Bertaccini ◽  
M.H. Jijakli ◽  
...  
Keyword(s):  
Real Time ◽  
Dna Extraction ◽  
Real Time Pcr ◽  
Pcr Detection ◽  
Apple Trees ◽  
Candidatus Phytoplasma Mali

Real-time PCR detection of Toxoplasma gondii in surface water samples in São Paulo, Brazil

2019 ◽  
Vol 118 (2) ◽  
pp. 631-640 ◽  
Author(s):  
Ana Tereza Galvani ◽  
Ana Paula Guarnieri Christ ◽  
José Antonio Padula ◽  
Mikaela Renata Funada Barbosa ◽  
Ronalda Silva de Araújo ◽  
...  
Keyword(s):  
Surface Water ◽  
Toxoplasma Gondii ◽  
Real Time ◽  
Water Samples ◽  
Real Time Pcr ◽  
Sao Paulo ◽  
Pcr Detection ◽  
São Paulo ◽  
Surface Water Samples

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.

Evaluation of a shortened QIAsymphony DNA extraction protocol for stool samples using a multiplex real-time PCR for the detection of enteric pathogens

2011 ◽  
Vol 84 (2) ◽  
pp. 365-367 ◽  
Author(s):  
E. van Zanten ◽  
G.J. Wisselink ◽  
S. Stoll ◽  
R. Alvarez ◽  
A.M.D. Kooistra-Smid
Keyword(s):  
Real Time ◽  
Dna Extraction ◽  
Real Time Pcr ◽  
Enteric Pathogens ◽  
Extraction Protocol ◽  
Stool Samples
Sign in / Sign up

Export Citation Format

Share Document