scholarly journals Real-Time PCR Assays for Quantification ofqnrGenes in Environmental Water Samples and Chicken Feces

2012 ◽  
Vol 79 (5) ◽  
pp. 1743-1745 ◽  
Author(s):  
Elisabet Marti ◽  
José Luis Balcázar
Keyword(s):  
Real Time ◽  
Water Samples ◽  
Real Time Pcr ◽  
Environmental Water ◽  
Quinolone Resistance ◽  
Environmental Water Samples ◽  
Complex Samples ◽  
Chicken Feces ◽  
Pcr Assays

ABSTRACTReal-time PCR assays were developed for the enumeration of plasmid-mediated quinolone resistance (PMQR) determinants, such as theqnrA,qnrB, andqnrSgenes, in different water samples and chicken feces. The results indicate that the developed assays are specific and sensitive for the quantification ofqnrgenes in complex samples.

scholarly journals Detection and enumeration of Cryptosporidium oocysts in environmental water samples by Real-time PCR assay

2017 ◽  
Vol 4 (3) ◽  
pp. 42-47
Author(s):  
Mohammad-Reza Mahmoudi ◽  
Mojgan Bandepour ◽  
Bahram Kazemi ◽  
Asad Mirzaei ◽  
◽  
...  
Keyword(s):  
Real Time ◽  
Water Samples ◽  
Real Time Pcr ◽  
Environmental Water ◽  
Environmental Water Samples ◽  
Pcr Assay ◽  
Cryptosporidium Oocysts

scholarly journals Molecular-Beacon Multiplex Real-Time PCR Assay for Detection of Vibrio cholerae

2006 ◽  
Vol 72 (9) ◽  
pp. 6424-6428 ◽  
Author(s):  
Aneta J. Gubala ◽  
David F. Proll
Keyword(s):  
Vibrio Cholerae ◽  
Real Time ◽  
Water Samples ◽  
Real Time Pcr ◽  
Molecular Beacon ◽  
Environmental Water ◽  
Molecular Beacons ◽  
Pcr Assay ◽  
Specificity And Sensitivity ◽  
Pcr Assays

ABSTRACT A multiplex real-time PCR assay was developed using molecular beacons for the detection of Vibrio cholerae by targeting four important virulence and regulatory genes. The specificity and sensitivity of this assay, when tested with pure culture and spiked environmental water samples, were high, surpassing those of currently published PCR assays for the detection of this organism.

scholarly journals Real-Time PCR for Quantification of Giardia and Cryptosporidium in Environmental Water Samples and Sewage

2003 ◽  
Vol 69 (9) ◽  
pp. 5178-5185 ◽  
Author(s):  
Rebecca A. Guy ◽  
Pierre Payment ◽  
Ulrich J. Krull ◽  
Paul A. Horgen
Keyword(s):  
Real Time ◽  
Water Samples ◽  
Real Time Pcr ◽  
Simultaneous Detection ◽  
Environmental Water ◽  
Qpcr Assay ◽  
Detection Methods ◽  
Environmental Water Samples ◽  
Dna Yield ◽  
Chelex 100

ABSTRACT The protozoan pathogens Giardia lamblia and Cryptosporidium parvum are major causes of waterborne enteric disease throughout the world. Improved detection methods that are very sensitive and rapid are urgently needed. This is especially the case for analysis of environmental water samples in which the densities of Giardia and Cryptosporidium are very low. Primers and TaqMan probes based on the β-giardin gene of G. lamblia and the COWP gene of C. parvum were developed and used to detect DNA concentrations over a range of 7 orders of magnitude. It was possible to detect DNA to the equivalent of a single cyst of G. lamblia and one oocyst of C. parvum. A multiplex real-time PCR (qPCR) assay for simultaneous detection of G. lamblia and C. parvum resulted in comparable levels of detection. Comparison of DNA extraction methodologies to maximize DNA yield from cysts and oocysts determined that a combination of freeze-thaw, sonication, and purification using the DNeasy kit (Qiagen) provided a highly efficient method. Sampling of four environmental water bodies revealed variation in qPCR inhibitors in 2-liter concentrates. A methodology for dealing with qPCR inhibitors that involved the use of Chelex 100 and PVP 360 was developed. It was possible to detect and quantify G. lamblia in sewage using qPCR when applying the procedure for extraction of DNA from 1-liter sewage samples. Numbers obtained from the qPCR assay were comparable to those obtained with immunofluorescence microscopy. The qPCR analysis revealed both assemblage A and assemblage B genotypes of G. lamblia in the sewage. No Cryptosporidium was detected in these samples by either method.

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.

Sign in / Sign up

Export Citation Format

Share Document