Grantee Research Project Results
2007 Progress Report: Rapid and Quantitative Detection of Helicobacter Pylori and E. Coli O157 in Well Water Using a Nano-Wired Biosensor and QPCR
EPA Grant Number: R833005Title: Rapid and Quantitative Detection of Helicobacter Pylori and E. Coli O157 in Well Water Using a Nano-Wired Biosensor and QPCR
Investigators: Alocilja, Evangelyn C. , Rose, Joan B. , Dreelin, Erin
Institution: Michigan State University
EPA Project Officer: Packard, Benjamin H
Project Period: November 1, 2006 through October 31, 2009 (Extended to October 31, 2011)
Project Period Covered by this Report: November 1, 2006 through October 31,2007
Project Amount: $600,000
RFA: Development and Evaluation of Innovative Approaches for the Quantitative Assessment of Pathogens in Drinking Water (2005) RFA Text | Recipients Lists
Research Category: Drinking Water , Water
Objective:
The specific objectives of this research are to: (1) develop a protocol for processing water samples for the biosensor and QPCR; (2) assess the performance of the biosensor and QPCR for sensitivity, specificity, recovery, and false positives/negatives of detection and enumeration for E. coli O157:H7 and H. pylori in groundwater samples from the field; (3) develop a method for detecting and enumerating E. coli O157:H7 and H. pylori by QPCR using bacteria isolated and screened by the biosensor system; and (4) validate a method for testing viability of E. coli O157:H7.
Progress Summary:
qPCR Method: qPCR primers and methods for E. coli O157:H7, E.coli and Enterococci were reviewed. Some of the genes that have been targeted for detection include stx-1, stx-2, eae and tir, the virulence genes, and rfbE, a structural gene that codes for the enzymes required for the biosynthesis of the O157 antigen. Our goal is to use the rfbE gene for the detection of E. coli O157 from the groundwater samples. This is one gene that can specifically target the O157 strains of E. coli and could be potentially used in different environmental samples containing different strains of E. coli even though detection of this gene does not provide evidence of their virulence. If this is positive, other markers then may be tested for. Previously, we had developed a qPCR method for Helicobacter. We will now be starting on groundwater assessments; however we will need to investigate antibodies in regard to detection and specificity.
Field sampling to date: Concentration of a water sample was performed by filtering 4 L onto a membrane filter. This filter was then eluted and the eluate concentrated to a pellet by centrifugation. Extraction was performed using MOBIO ultraclean Megasoil DNA kit. The human and bovine fecal marker genes were amplified by polymerase chain reaction (PCR) using published primer sets. Visualization of PCR products was performed with gel electrophoresis. The gel was examined for a PCR product of the appropriate molecular weight as determined by a DNA molecular weight standard ladder and/or a positive control PCR product.
Biosensor: There have been four modifications made to the biosensor: (1) Magnetic nanoparticles coated in polyaniline and monoclonal antibodies are now being combined with the sample in order to concentrate it before it is applied to the test strip; (2) The development of a probe station for the measurement of I-V characteristics; (3) The electrodes are now being applied using a shadow mask and/or screen printing method rather than being manually drawn on in order to increase consistency from strip to strip; and (4) A new platform has been designed to better control the environment around the test strip.
Future Activities:
qPCR: The goal for this year will be to examine animal manure and human wastewater for the following markers, examining the stability, sensitivity and reproducibility of the target and assay. This will also necessitate further refinement of the water sampling and processing method. Manure and wastewater samples will be shared with the Biosensors Laboratory.
Biosensor: The next steps for this project will be to validate the new platform structure and to compare it to the old platform that was being used in order to ensure that it is improving the performance of the sensor. We also plan on testing the biosensor with higher sample volumes to see if that improves the sensitivity of the biosensor. In addition, we are planning to test the effect of a new method for the assembly of the biosensor involving the application of the electrodes before the functionalization of the capture pad. Once validations have been completed on these improved procedures, sensitivity testing for E. coli O157:H7 will be conducted and extended to groundwater testing.
Validation: Additional steps for viability assay optimization include the introduction of Mueller Hinton Broth II (BD Cat# 297963) as growth media to minimize background luminescence as demonstrated with TSB, employment of a multi-well plate luminometer instead of a single tube luminometer to increase quality control throughout experimentation, and reagent lot comparison to ensure the reliability of the current reagents in use.
Journal Articles on this Report : 1 Displayed | Download in RIS Format
Other project views: | All 11 publications | 4 publications in selected types | All 3 journal articles |
---|
Type | Citation | ||
---|---|---|---|
|
Nayak AK, Rose JB. Detection of Helicobacter pylori in sewage and water using a new quantitative PCR method with SYBR green. Journal of Applied Microbiology 2007;103(5):1931-1941. |
R833005 (2007) R833005 (2008) |
|
Supplemental Keywords:
magnetic polyaniline, nanoparticles, multi-well luminometer,, RFA, Scientific Discipline, INTERNATIONAL COOPERATION, Water, TREATMENT/CONTROL, POLLUTANTS/TOXICS, Sustainable Industry/Business, Sustainable Environment, Technology, Technology for Sustainable Environment, Environmental Monitoring, Drinking Water, Microorganisms, Environmental Engineering, pathogens, bacteria, nanotechnology, drinking water monitoring, E. Coli, biotechnology, biosensors, biosensorProgress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.