scholarly journals Evaluation of real-time PCR for quantitative detection of Escherichia coli in beach water

2013 ◽  
Vol 12 (1) ◽  
pp. 51-56 ◽  
Author(s):  
Jason Tszhin Lam ◽  
Edwin Lui ◽  
Simon Chau ◽  
Cathie Show Wu Kueh ◽  
Ying-kit Yung ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Real Time ◽  
Real Time Pcr ◽  
Culture Method ◽  
Quantitative Detection ◽  
Chain Reaction ◽  
Limit Of Quantification ◽  
E Coli ◽  
Polymerase Chain ◽  
Beach Water

The current investigation evaluated the use of real-time polymerase chain reaction (PCR) for quantitative detection of Escherichia coli in marine beach water. Densities of E. coli in 263 beach water samples collected from 13 bathing beaches in Hong Kong between November 2008 and December 2009 were determined using both real-time PCR and culture-based methods. Regression analysis showed that these two methods had a significant positive linear relationship with a correlation coefficient (r) of 0.64. Serial dilution of spiked samples indicated that the real-time PCR had a limit of quantification of 25 E. coli colonies in 100 mL water sample. This study showed that the rapid real-time PCR has potential to complement the traditional culture method of assessing fecal pollution in marine beach water.

Quantitative Real-Time Polymerase Chain Reaction Detection of BK Virus Using Labeled Primers

2010 ◽  
Vol 134 (3) ◽  
pp. 444-448 ◽  
Author(s):  
Zhengming Gu ◽  
Jianmin Pan ◽  
Matthew J. Bankowski ◽  
Randall T. Hayden
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Real Time Pcr ◽  
Quantitative Polymerase Chain Reaction ◽  
Bk Virus ◽  
Clinical Samples ◽  
Quantitative Detection ◽  
Chain Reaction ◽  
Pcr Method ◽  
Polymerase Chain

Abstract Context.—BK virus infections among immunocompromised patients are associated with disease of the kidney or urinary bladder. High viral loads, determined by quantitative polymerase chain reaction (PCR), have been correlated with clinical disease. Objective.—To develop and evaluate a novel method for real-time PCR detection and quantification of BK virus using labeled primers. Design.—Patient specimens (n = 54) included 17 plasma, 12 whole blood, and 25 urine samples. DNA was extracted using the MagNA Pure LC Total Nucleic Acid Isolation Kit (Roche Applied Science, Indianapolis, Indiana); sample eluate was PCR-amplified using the labeled primer PCR method. Results were compared with those of a user-developed quantitative real-time PCR method (fluorescence resonance energy transfer probe hybridization). Results.—Labeled primer PCR detected less than 10 copies per reaction and showed quantitative linearity from 101 to 107 copies per reaction. Analytical specificity of labeled primer PCR was 100%. With clinical samples, labeled primer PCR demonstrated a trend toward improved sensitivity compared with the reference method. Quantitative assay comparison showed an R2 value of 0.96 between the 2 assays. Conclusions.—Real-time PCR using labeled primers is highly sensitive and specific for the quantitative detection of BK virus from a variety of clinical specimens. These data demonstrate the applicability of labeled primer PCR for quantitative viral detection and offer a simplified method that removes the need for separate oligonucleotide probes.

Quantitative detection of human enteric adenoviruses in river water by microfluidic digital polymerase chain reaction

2014 ◽  
Vol 70 (3) ◽  
pp. 555-560 ◽  
Author(s):  
Naohiro Kishida ◽  
Naohiro Noda ◽  
Eiji Haramoto ◽  
Mamoru Kawaharasaki ◽  
Michihiro Akiba ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
River Water ◽  
Water Samples ◽  
Real Time Pcr ◽  
Quantitative Detection ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Digital Polymerase Chain Reaction ◽  
Enteric Adenoviruses

We describe an assay for simple and accurate quantification of human enteric adenoviruses (EAdVs) in water samples using a recently developed quantification method named microfluidic digital polymerase chain reaction (dPCR). The assay is based on automatic distribution of reaction mixture into a large number of nanolitre-volume reaction chambers and absolute copy number quantification from the number of chambers containing amplification products on the basis of Poisson statistics. This assay allows absolute quantification of target genes without the use of standard DNA. Concentrations of EAdVs in Japanese river water samples were successfully quantified by the developed dPCR assay. The EAdVs were detected in seven of the 10 samples (1 L each), and the concentration ranged from 420 to 2,700 copies/L. The quantified values closely resemble those by most probable number (MPN)-PCR and real-time PCR when standard DNA was validated by dPCR whereas they varied substantially when the standard was not validated. Accuracy and sensitivity of the dPCR was higher than those of real-time PCR and MPN-PCR. To our knowledge, this is the first study that has successfully quantified enteric viruses in river water using dPCR. This method will contribute to better understanding of existence of viruses in water.

Comparison of Real-Time Polymerase Chain Reaction and Enzyme-Linked Immunosorbent Assay for Sensitive and Quantitative Detection of Hazelnuts in Nut Pastes

2018 ◽  
Vol 101 (6) ◽  
pp. 1864-1867 ◽  
Author(s):  
Ľubica Piknová ◽  
Veronika Janská ◽  
Tomáš Kuchta ◽  
Peter Siekel
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Sandwich Elisa ◽  
Pcr Analysis ◽  
Quantitative Detection ◽  
Enzyme Linked Immunosorbent Assay ◽  
Chain Reaction ◽  
Wide Range ◽  
Order Of Magnitude ◽  
Polymerase Chain

Abstract Background: Hazelnuts, being a frequent agent of allergenic reactions, need to be detected in food products. Thus, it is necessary to develop and further investigate appropriate methods for detection. Objective: The aim of the study was to compare the analysis of nut pastes (peanut paste spiked with different amounts of hazelnut paste) as a model of contamination of confectionery. Methods: Real-time PCR and sandwich ELISA (RidaScreen Hazelnut Fast Kit) were used. Results: For real-time PCR, LOQ of 2 mg/kg and a quantification range from 2 to 10 000 mg/kg were determined. For ELISA, LOQ of 1 mg/kg and a quantification range from 1 to 100 mg/kg were determined. Conclusions: The comparison shows that the methods had comparable sensitivity with LOQs in the same order of magnitude. Although ELISA was slightly more sensitive, it required dilution of samples at higher concentrations of the analyte because of its narrow quantification range. Results of this study suggest that real-time PCR and ELISA are both suitable methods for the analysis of nut pastes over a wide range of concentrations. Achieved results could be useful for control as well as for technological purposes. Highlights: Real-time PCR analysis of peanut paste spiked with different amounts of hazelnut paste as a model is proposed. Sandwich ELISA analysis of peanut paste spiked with different amounts of hazelnut paste as a model is proposed. The analytical parameters of real-time PCR and ELISA methods are compared.

Rapid and Simultaneous Quantitation of Escherichia coli O157:H7, Salmonella, and Shigella in Ground Beef by Multiplex Real-Time PCR and Immunomagnetic Separation†

2007 ◽  
Vol 70 (6) ◽  
pp. 1366-1372 ◽  
Author(s):  
LUXIN WANG ◽  
YONG LI ◽  
AZLIN MUSTAPHA
Keyword(s):  
Escherichia Coli ◽  
Detection Limit ◽  
Real Time ◽  
Real Time Pcr ◽  
Ground Beef ◽  
Immunomagnetic Separation ◽  
Quantitative Detection ◽  
Escherichia Coli O157 ◽  
The Real ◽  
E Coli

The objective of this study was to establish a multiplex real-time PCR for the simultaneous quantitation of Escherichia coli O157:H7, Salmonella, and Shigella. Genomic DNA for the real-time PCR was extracted by the boiling method. Three sets of primers and corresponding TaqMan probes were designed to target these three pathogenic bacteria. Multiplex real-time PCR was performed with TaqMan Universal PCR Master Mix in an ABI Prism 7700 Sequence Detection System. Final standard curves were calculated for each pathogen by plotting the threshold cycle value against the bacterial number (log CFU per milliliter) via linear regression. With optimized conditions, the quantitative detection range of the real-time multiplex PCR for pure cultures was 102 to 109 CFU/ml for E. coli O157:H7, 103 to 109 CFU/ml for Salmonella, and 101 to 108 CFU/ml for Shigella. When the established multiplex real-time PCR system was applied to artificially contaminated ground beef, the detection limit was 105 CFU/g for E. coli O157:H7, 103 CFU/g for Salmonella, and 104 CFU/g for Shigella. Immunomagnetic separation (IMS) was further used to separate E. coli O157:H7 and Salmonella from the beef samples. With the additional use of IMS, the detection limit was 103 CFU/g for both pathogens. Results from this study showed that TaqMan real-time PCR, combined with IMS, is potentially an effective method for the rapid and reliable quantitation of E. coli O157:H7, Salmonella, and Shigella in food.

scholarly journals Development of Real-Time Polymerase Chain Reaction Assay with TaqMan Probe for the Quantitative Detection of Infectious Hypodermal and Hematopoietic Necrosis Virus from Shrimp

2006 ◽  
Vol 89 (1) ◽  
pp. 240-244 ◽  
Author(s):  
Zhi-Qin Yue ◽  
Hong Liu ◽  
Wei-Ji Wang ◽  
Zhi-Wen Lei ◽  
Cheng-Zhu Liang ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Real Time Pcr ◽  
Taqman Probe ◽  
Quantitative Detection ◽  
Necrosis Virus ◽  
Chain Reaction ◽  
Pcr Assay ◽  
The Real ◽  
Polymerase Chain

Abstract An assay was developed for the detection of infectious hypodermal and hematopoietic necrosis virus (IHHNV) based on real-time quantitative polymerase chain reaction (PCR). A pair of primers and a TaqMan probe were designed that are specific for the recognition of a conservative region in the IHHNV genome. The IHHNV real-time PCR assay had a detection limit of 9 DNA copies,with a dynamic range of detection between 9 106 and 9 DNA copies. The primer pairs and probe were specific to IHHNV and did not cross-reactwith shrimp genomic DNAor other shrimp viruses such as White Spot Syndrome Virus (WSSV), Monodon Baculovirus (MBV), and hepatopancreatic parvovirus (HPV). This assay has a broad application for basic and clinical investigations. For clinical samples, the real-time PCR assay detected all the positive samples screened by conventional PCR, which indicated the sensitivity of the real-time assay. The IHHNV real-time PCR assay with high sensitivity, specificity, wide range of detection ability, and simplicity is particularly useful for screening large numbers of specimens and measuring viral loads to monitor the broodstock.

A rapid (one day), sensitive real-time polymerase chain reaction assay for detecting Escherichia coli O157:H7 in ground beef

2006 ◽  
Vol 52 (10) ◽  
pp. 992-998 ◽  
Author(s):  
Jane Holicka ◽  
Rebecca A Guy ◽  
Anita Kapoor ◽  
David Shepherd ◽  
Paul A Horgen
Keyword(s):  
Escherichia Coli ◽  
Polymerase Chain Reaction ◽  
Real Time ◽  
Polymerase Chain Reaction Assay ◽  
Ground Beef ◽  
Automatic Process ◽  
Escherichia Coli O157 ◽  
Chain Reaction ◽  
E Coli ◽  
Polymerase Chain

The purpose of this study was to apply our rapid, integrated double enrichment 5′ nuclease real-time polymerase chain reaction assay for the detection of Escherichia coli O157:H7 and evaluate its efficacy. The assay targeted ground beef, an important vehicle in disease epidemiology. The assay reliably determined in 8 h the presence of E. coli O157:H7 in ground beef at the level of 1 colony-forming unit (CFU)/g. The sensitivity and specificity of the assay were compared with that of standard enrichment diagnostic techniques. A correlation of 100% in detection was achieved to the limit of 1 CFU/g. This assay can be used as a rapid, automatic process for identification of E. coli O157:H7 in ground beef or can be integrated with standard culture procedures, resulting in considerable cost and time savings.Key words: real-time PCR, E. coli O157:H7, ground beef, molecular diagnostics, rapid O157:H7 assay.

Screening Bovine Carcass Sponge Samples for Escherichia coli O157 Using a Short Enrichment Coupled with Immunomagnetic Separation and a Polymerase Chain Reaction–Based (BAX) Detection Step†

2001 ◽  
Vol 64 (10) ◽  
pp. 1610-1612 ◽  
Author(s):  
DONG-HYUN KANG ◽  
GENEVIEVE A. BARKOCY-GALLAGHER ◽  
MOHAMMAD KOOHMARAIE ◽  
GREGORY R. SIRAGUSA
Keyword(s):  
Escherichia Coli ◽  
Culture Method ◽  
Immunomagnetic Separation ◽  
Escherichia Coli O157 ◽  
Chain Reaction ◽  
Selective Enrichment ◽  
E Coli ◽  
Screening Protocol ◽  
Polymerase Chain ◽  
Culture Positive

A bovine carcass sponge sample screening protocol for detecting Escherichia coli O157:H7 was composed of a short selective enrichment followed by an immunomagnetic separation (IMS) and target detection using the BAX E. coli O157 polymerase chain reaction assay. This screening protocol was compared to a culture-based method for detection of the organism in carcass sponge samples. Enriched samples were subjected to IMS; the bead suspension was divided and plated on selected media or stored at −20°C, then subjected to BAX analysis. The results showed a high degree of agreement between the plating method and the BAX system. Fifty-two of the 59 culture-positive samples were also positive using the BAX system (88.1% sensitivity). Of the 76 samples that appeared negative for the presence of E. coli O157:H7 by the culture method, 66 were determined as negative using the BAX system (86.8% specificity). Four of the 10 samples found negative by the initial culture method and positive by the BAX method were subsequently found to be culture positive upon reanalysis. Based on these data, the BAX system combined with a short, selective enrichment and IMS may be a rapid, reliable, and simple method to screen for E. coli O157:H7 in carcass sponge samples. Our data indicate that optimization and subsequent testing of this protocol for use as a carcass screening tool are warranted.

Sign in / Sign up

Export Citation Format

Share Document