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.

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.

Analysis of Polymorphisms in Genes Differentially Expressed in the Enamel of Mice with Different Genetic Susceptibilities to Dental Fluorosis

2018 ◽  
Vol 53 (2) ◽  
pp. 228-233 ◽  
Author(s):  
Senda Charone ◽  
Erika Calvano Küchler ◽  
Aline de Lima Leite ◽  
Mileni Silva Fernandes ◽  
Vinicius Taioqui Pelá ◽  
...  
Keyword(s):  
Drinking Water ◽  
Real Time ◽  
Dna Extraction ◽  
Genetic Polymorphisms ◽  
Clinical Evaluation ◽  
Real Time Pcr ◽  
Genomic Dna ◽  
Dental Fluorosis ◽  
Clinical Aspects ◽  
Genomic Dna Extraction

Genes expressed during amelogenesis are candidates to increase the risk of dental fluorosis (DF). Thus, this study aimed to evaluate the association between polymorphisms in enamel development genes and susceptibility to DF in mice. Mice of both sexes, representing strains 129P3/J (n = 20; resistant to DF) and A/J (n = 20; susceptible to DF), were divided into 2 groups. Each strain received a diet with a low concentration of fluoride (F) and drinking water containing 0 or 50 mg/L of F for 6 weeks. Clinical evaluation and analysis of Vickers enamel microhardness of the incisors were performed. Livers were collected for genomic DNA extraction. Seventeen genetic polymorphisms in Amelx, Ambn, Ambn, Col14a1, Col1a1, Col5a2, Enam, Fam20a, Fam83h, Foxo1, Klk4, Mmp20, Serpinf1, Serpinh1, Smad3, Tuft1, and Wdr72 were genotyped by real-time PCR using Taqman chemistry. Overrepresentation of alleles and genotypes in DF was evaluated using the χ2 test with an alpha of 5%. The clinical aspects of the enamel and the surface enamel microhardness confirmed the DF condition. In the polymorphisms rs29569969, rs13482592, and rs13480057 in Ambn, Col14a1, and Mmp20, respectively, genotype and allele distributions were statistically significantly different between A/J and 129P3/J strains (p < 0.05). In conclusion, polymorphisms in Ambn, Col14a1, and Mmp20 are associated with the susceptibility to DF.

scholarly journals Detection of Elm Yellows Phytoplasma in Elms and Insects Using Real-Time PCR

Plant Disease ◽  
2010 ◽  
Vol 94 (11) ◽  
pp. 1355-1360 ◽  
Author(s):  
Padmini Herath ◽  
Gregory A. Hoover ◽  
Elisa Angelini ◽  
Gary W. Moorman
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Extraction Procedure ◽  
Accurate Method ◽  
The United States ◽  
State University ◽  
Phylogenetic Groups ◽  
The Real ◽  
Elm Yellows ◽  
Taqman Minor Groove Binder

A rapid and accurate method to detect the common strain of elm yellows (EY) phytoplasma in elm and insect samples was developed using a real-time polymerase chain reaction (PCR) procedure based on the TaqMan minor-groove-binder probe. Primers and probe were designed based on the EY phytoplasma-specific translocation protein secY gene DNA sequence. Success of the DNA extraction procedure was evaluated by amplifying the chloroplast trnL gene of Ulmus americana. The real-time PCR assay reacted positively with EY and EY phytoplasma strain ULW DNA, an isolate which occurs in Europe. It did not cross-react with Illinois EY or aster yellows phytoplasma DNA, both of which are known to occur in elm trees in the United States, nor did it amplify several other phytoplasmas belonging to the 16SrV and other phylogenetic groups. The real-time PCR protocol was used to identify 30 EY-positive elm trees on The Pennsylvania State University, University Park campus. Threshold cycle (CT) values obtained from the EY phytoplasma-infected elm trees ranged from 15 to 37. EY phytoplasma was detected in several leafhopper taxa. This real-time PCR method can be used for the diagnostic screening of elm trees and for the identification of possible insect vectors of EY phytoplasma.

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 new large scale soil DNA extraction procedure and real-time PCR assay for the detection of Sclerotium cepivorum in soil

2012 ◽  
Vol 134 (3) ◽  
pp. 467-473 ◽  
Author(s):  
James W. Woodhall ◽  
Kathryn M. Webb ◽  
Patricia M. Giltrap ◽  
Ian P. Adams ◽  
Jeff C. Peters ◽  
...  
Keyword(s):  
Real Time ◽  
Dna Extraction ◽  
Real Time Pcr ◽  
Large Scale ◽  
Extraction Procedure ◽  
Pcr Assay ◽  
Soil Dna ◽  
Sclerotium Cepivorum ◽  
Soil Dna Extraction

scholarly journals Real-Time PCR for Detection and Quantification of the Protistan Parasite Perkinsus marinus in Environmental Waters

2004 ◽  
Vol 70 (11) ◽  
pp. 6611-6618 ◽  
Author(s):  
Corinne Audemard ◽  
Kimberly S. Reece ◽  
Eugene M. Burreson
Keyword(s):  
Real Time ◽  
Water Samples ◽  
Real Time Pcr ◽  
Environmental Water ◽  
The United States ◽  
Environmental Water Samples ◽  
Perkinsus Marinus ◽  
Environmental Waters ◽  
Specific Primers ◽  
Dna Recovery

ABSTRACT The protistan parasite Perkinsus marinus is a severe pathogen of the oyster Crassostrea virginica along the east coast of the United States. Very few data have been collected, however, on the abundance of the parasite in environmental waters, limiting our understanding of P. marinus transmission dynamics. Real-time PCR assays with SybrGreen I as a label for detection were developed in this study for quantification of P. marinus in environmental waters with P. marinus species-specific primers and of Perkinsus spp. with Perkinsus genus-specific primers. Detection of DNA concentrations as low as the equivalent of 3.3 � 10−2 cell per 10-μl reaction mixture was obtained by targeting the multicopy internal transcribed spacer region of the genome. To obtain reliable target quantification from environmental water samples, removal of PCR inhibitors and efficient DNA recovery were two major concerns. A DNA extraction kit designed for tissues and another designed for stool samples were tested on environmental and artificial seawater (ASW) samples spiked with P. marinus cultured cells. The stool kit was significantly more efficient than the tissue kit at removing inhibitors from environmental water samples. With the stool kit, no significant difference in the quantified target concentrations was observed between the environmental and ASW samples. However, with the spiked ASW samples, the tissue kit demonstrated more efficient DNA recovery. Finally, by performing three elutions of DNA from the spin columns, which were combined prior to target quantification, variability of DNA recovery from different samples was minimized and more reliable real-time PCR quantification was accomplished.

scholarly journals On-Site DNA Extraction and Real-Time PCR for Detection of Phytophthora ramorum in the Field

2005 ◽  
Vol 71 (11) ◽  
pp. 6702-6710 ◽  
Author(s):  
J. A. Tomlinson ◽  
N. Boonham ◽  
K. J. D. Hughes ◽  
R. L. Griffin ◽  
I. Barker
Keyword(s):  
Real Time ◽  
Dna Extraction ◽  
Real Time Pcr ◽  
Cost Effective ◽  
The United States ◽  
Phytophthora Ramorum ◽  
Taqman Assay ◽  
Molecular Testing ◽  
Field Independent ◽  
Cost Effective Method

ABSTRACT Phytophthora ramorum is a recently described pathogen causing oak mortality (sudden oak death) in forests in coastal areas of California and southern Oregon and dieback and leaf blight in a range of tree, shrub, and herbaceous species in the United States and Europe. Due to the threat posed by this organism, stringent quarantine regulations are in place, which restrict the movement of a number of hosts. Fast and accurate diagnostic tests are required in order to characterize the distribution of P. ramorum, prevent its introduction into pathogen-free areas, and minimize its spread within affected areas. However, sending samples to a laboratory for testing can cause a substantial delay between sampling and diagnosis. A rapid and simple DNA extraction method was developed for use at the point of sampling and used to extract DNAs from symptomatic foliage and stems in the field. A sensitive and specific single-round real-time PCR (TaqMan) assay for P. ramorum was performed using a portable real-time PCR platform (Cepheid SmartCycler II), and a cost-effective method for stabilizing PCR reagents was developed to allow their storage and transportation at room temperature. To our knowledge, this is the first description of a method for DNA extraction and molecular testing for a plant pathogen carried out entirely in the field, independent of any laboratory facilities.

scholarly journals Direct detection of Mycobacterium avium SUBSP. paratuberculosis in bovine milk by multiplex real-time PCR

2013 ◽  
Vol 29 (3) ◽  
pp. 513-525 ◽  
Author(s):  
A. Selim ◽  
M. El-Haig ◽  
E.S. Galila
Keyword(s):  
Real Time ◽  
Dna Extraction ◽  
Mycobacterium Avium ◽  
Real Time Pcr ◽  
Direct Detection ◽  
Detection Sensitivity ◽  
Mycobacterium Avium Subsp ◽  
Pcr Assay ◽  
Mycobacterium Avium Subsp Paratuberculosis ◽  
Pcr Targeting

This study aimed to direct detection of Mycobacterium avium subsp. paratuberculosis (MAP) in milk by evaluating a multiplex real-time PCR assay targeting IS900 and ISMAV2 sequences including the amplification of PUC19-plasmid as internal control. The sensitivity of the assays was evaluated by testing MAP isolates in broad linear range of DNA (50 ng - 5 fg/?l). For the validation of the specificity, 6 MAP isolates and 22 isolates of genus Mycobacteriacea were tested. Results revealed that reproducible detection limit for real-time PCR targeting IS900 and ISMAV2 was 5 fg/?l and 50 fg/?l respectively. By targeting ISMAV2 sequence, 100% specificity was detected. However, a cross reaction with 5 ng/?l of genome of 3 M. avian subspecies avium strains was detected by targeting IS900 and negative in lower genome quantity (5pg/?l). To maximize the assay?s detection sensitivity, an efficient strategy for MAP-DNA extraction from spiked milk was assessed. Targeting of IS900 was sensitive and targeting ISMAV2 was very specific. Therefore, a multiplex real-time PCR assay targeting IS900 and ISMAV2 in combination with two commercial DNA extraction kits could be an ideal sensitive and specific protocol for routine large scale analysis of milk samples and other clinical specimens from man and animals.

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