NDLI: Rapid screening of waterborne pathogens using phage-mediated separation coupled with real-time PCR detection

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Rapid screening of waterborne pathogens using phage-mediated separation coupled with real-time PCR detection

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Analytical and Bioanalytical Chemistry : Volume 408, Issue 13, May 2016

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Rapid screening of waterborne pathogens using phage-mediated separation coupled with real-time PCR detection

Content Provider	Springer Nature Link
Author	Wang, Ziyuan Wang, Danhui Kinchla, Amanda J. Sela, David A. Nugen, Sam R.
Copyright Year	2016
Abstract	Escherichia coli O157:H7 is a ubiquitous pathogen which can be linked to foodborne outbreaks worldwide. In addition to the significant illnesses, hospitalizations, and deaths resulting from the outbreaks, there can be severe economic consequences to farmers, food manufacturers, and municipalities. A rapid detection assay which can validate sanitation and water quality would prove beneficial to these situations. Here, we report a novel bacteriophage-mediated detection of E. coli O157:H7 which utilizes the specific recognition between phages and their host cell as well as the natural lysis component of the infection cycle for DNA release. Carboxylic acid-functionalized magnetic beads were conjugated with bacteriophage and used to separate and concentrate E. coli O157:H7. The effects of bead incubation time, salinity, pH, and temperature on the bio-magnetic separation were investigated and compared to an antibody-based counterpart. The conditions of 0.01 M PBS, pH 7.0, and 20 min of reaction at 37 °C were found to be optimal. The capture efficiency of the coupled assay was approximately 20 % higher than that of antibody-based separation under extreme conditions. The resulting bead-phage-bacteria complexes were quantitatively detected by real-time PCR (qPCR). Our results demonstrated that the use of phage-based magnetic separation coupled with qPCR improved the sensitivity of detection by 2 orders of magnitude compared that without phage-based pre-concentration. Specificity and selectivity of the assay system was evaluated, and no cross-reactivity occurred when Salmonella typhimurium, Staphylococcus aureus, and Pseudomonas aeruginosa were tested. The total assay time was less than 2 h.
Starting Page	4169
Ending Page	4178
Page Count	10
File Format	PDF
ISSN	16182642
Journal	Analytical and Bioanalytical Chemistry
Volume Number	408
Issue Number	15
e-ISSN	16182650
Language	English
Publisher	Springer Berlin Heidelberg
Publisher Date	2016-04-12
Publisher Place	Berlin, Heidelberg
Access Restriction	One Nation One Subscription (ONOS)
Subject Keyword	Escherichia coli O157:H7 Bacteriophage Magnetic separation Real-time PCR Water testing E. coli Analytical Chemistry Biochemistry Laboratory Medicine Characterization and Evaluation of Materials Food Science Environmental Monitoring/Analysis
Content Type	Text
Resource Type	Article
Subject	Analytical Chemistry Biochemistry

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Detection of Escherichia coli O157:H7 in Food Using Real-Time Multiplex PCR Assays Targeting the stx $_{1}$, stx $_{2}$, wzy $_{O157}$, and the fliC $_{h7}$ or eae Genes

Detection of E. coli O157:H7 in Food Using Automated Immunomagnetic Separation Combined with Real-Time PCR

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Evaluation of Two Immunomagnetic Separation Techniques for the Detection and Recovery of Escherichia coli O157:H7 from Finished Composts

An evaluation of immunomagnetic separation-real-time PCR (IMS-RTiPCR) combined assay for rapid and specific detection of Escherichia coli O157:H7 in raw milk and ground beef

Characterization and Food Application of the Novel Lytic Phage BECP10: Specifically Recognizes the O-polysaccharide of Escherichia coli O157:H7

Rapid detection of Escherichia coli O157:H7 with multiplex real-time PCR assays.

Real-time PCR for the detection of Escherichia coli O157:H7 in dairy and cattle wastewater

Türkiye’de baharatlarda Escherichia coli O157:H7’nin belirlenmesinde real-time PCR ve konvansiyonel kültürel yöntemin karşılaştırılması

Rapid screening of waterborne pathogens using phage-mediated separation coupled with real-time PCR detection

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Detection of Escherichia coli O157:H7 in Food Using Real-Time Multiplex PCR Assays Targeting the stx $_{1}$, stx $_{2}$, wzy $_{O157}$, and the fliC $_{h7}$ or eae Genes

Detection of E. coli O157:H7 in Food Using Automated Immunomagnetic Separation Combined with Real-Time PCR

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Real-time PCR for the detection of Escherichia coli O157:H7 in dairy and cattle wastewater

Türkiye’de baharatlarda Escherichia coli O157:H7’nin belirlenmesinde real-time PCR ve konvansiyonel kültürel yöntemin karşılaştırılması

Rapid screening of waterborne pathogens using phage-mediated separation coupled with real-time PCR detection