You are here: home » Oceans and Human Health » Pathogen Source Tracking » Adapting Luminex
Adapting Luminex to Microbial Source Tracking
Project Goal
This project provides an innovative approach to monitoring coastal water quality for microbial contamination. It harnesses the power of biotechnology to rapidly detect multiple source tracking markers and indicator organisms simultaneously. In contrast, current source tracking methods either use a library-based approach (which has a variety of drawbacks) or utilize a series of separate analyses to detect host-specific markers. Fecal indicator detection is performed in additional separate steps using slow methods that take longer than 24 hours and are focused on a single organism. This approach will rapidly detect a matrix of source tracking markers and indicator organisms, giving environmental managers comprehensive information on which decisions can be based.
Click here for a full project report
Abstract
Microbial contamination and toxic algal blooms impact coastal water quality. As the nation’s coastal areas become more urbanized, poor water quality has increasingly negative economic, health, and environmental impacts. Proper assessment and understanding of the factors that effect coastal water quality requires the ability to rapidly and simultaneously detect multiple microbial contaminants and determine their sources with regard to location and type of contamination. Such data informs mitigation strategies and health risk assessments. Environmental managers need quick and accurate measures of water quality so that they can restrict human access to contaminated marine waters and products. This project aims to harness biotechnology advances in the realm of clinical science and apply them to coastal water quality applications. We are adapting a DNA hybridization assay technology, the Luminex ® 100™ to achieve this goal. The system is essentially a flow cytometer equipped with two lasers, one that identifies a color-coded bead (to which a molecular probe is attached) and the other that registers whether or not the probe has captured a target (DNA amplified by PCR, in this case). The system incorporates a suspension array that assays multiple analytes rapidly in a single well of a microtiter plate. The system works in a 96-well plate format. In each well, detection of multiple targets can be achieved, and each well is read rapidly – in about 45 seconds. Therefore, this system has the ability to provide rapid, multiplexed, high-throughput detection of biological organisms. We designed and field tested a variety of primer/probe sets to identify the DNA signatures of various bacteria that indicate fecal contamination and expanded our efforts to include molecular markers that indicate whether contamination is from animal or human sources. The L uminex response to cultures indicated that the system was specific and sensitive. The Luminex response to environmental samples was consistent with the number of bacterial cells available to extract and results were consistent with DNA sequencing. The current array utilizes probes to simultaneously detect the following: the genus Enterococcus, Bacteroides distasonis, Escherichia coli/Shigella spp., the human specific HF8 cluster of Bacteroides, and the human-specific esp gene of Enterococcus spp. Overall, the data suggest that this technology has the potential to simultaneously detect multiple targets for coastal water quality applications, particularly as progress is made to efficiently extract DNA from water and sediment matrices.
Objectives
The short-term objectives are to:
- Adapt source tracking markers for use in Luminex xMAPTM assays.
- Adapt currently available source tracking markers to Luminex beads.
- Design appropriate group-specific primers to provide appropriate amplicons in which the source tracking probes will bind.
- Multiplex the technique to provide simultaneous detection of source tracking markers and fecal indicators.
- Determine the sensitivity of analysis, and examine options for upstream processing of nucleic acids to improve sensitivity.
- Field testing of the source tracking markers and comparison to other methods for verification and quality control.
Expected Results
The versatility of the Luminex platform is attracting the industrial sector to develop kits based on xMAP technology. The Luminex market analysis conducted (funded by CICEET, RTI International, 2005) identified several companies that were interested in discussions regarding Luminex kits for coastal water quality applications. After completion of the work outlined here, Luminex Corp. is a willing partner for the next steps in technology transfer—the design and commercialization of source tracking kits.
Accomplishments:
- The following manuscript has been completed has been submitted: “Luminex Detection of Fecal Indicators in River Samples, Marine Recreational Water, and Beach Sand.” I.B. Baums, K.D. Goodwin, T. Kiesling, D. Wanless, and J.W. Fell.
- The Luminex assay as described in the manuscript above has been substantially redesigned in order to increase sensitivity and specificity. Changes include more specific priming (removal of the universal reverse primer), optimization of the multiplex PCR amplification to increase sensitivity (optimization of primer concentrations and annealing conditions, designing amplicons to be of more similar length), and switching of amplification targets to increase specificity of priming with environmental samples (e.g., 23S of Enterococcus spp. rather than 16S).
- The following source tracking markers were incorporated into Luminex analysis: the human-associated esp gene of enterococci, the human-specific HF8 marker of Bacteroides spp., and a dog-specific marker designed for the primers DF475F/Bac708R. Detection of HF8 has been successfully achieved in environmental samples. The requirement of a preculture step to achieve detection of the esp gene was confirmed. Adequate sensitivity was not achieved with the dog-specific marker, and that marker has been redesigned. Further testing is funding-dependent.
- DNA extraction efficiency experiments revealed poor recovery with standard DNA spin kits (0-2%). Extraction protocols have switched to bead beating to generate crude lysate. However, extraction efficiency remains unacceptably low (0-39%), and work continues in this area.
- Water processing protocols now incorporate a culture step on selective media in order to increase sensitivity of the assay to source tracking markers.
- The FileMaker Pro database has been completed. Initial code has been refined and simplified to make it more robust and more easily adaptable to HML needs. The database is now being beta-tested, particularly with regard to report generation.
- AOML and HML personnel have worked successfully as a team, despite physical distance, in order to maximize the work that can be accomplished under an unfavorable funding climate.
Transfer of Results
Potential end-users of this technology are coastal resource managers, environmental researchers, and aquaculture professionals. This project provides an innovative approach to monitoring coastal water quality for microbial contamination. The approach described here will rapidly detect a matrix of source tracking markers and indicator organisms, giving environmental managers comprehensive information on which decisions can be based.
As the technology is ready to transfer to environmental and public health managers, activities will be developed using methods such as workshops, symposia and conferences advertised through websites, listserves, and forums developed by the University of Miami’s Center for Oceans and Human Health , the University of Miami’ National Institute of Environmental Health, the Alliance for Coastal Technologies , and the Cooperative Institute of Coastal and Estuarine Technology (CICEET).
Publications/Presentations:
K.D. Goodwin Garcia, C., Bonilla, J.A., Bonilla, T.D., Wanless, D., Abdelzaher, A. LaGier, M.J., Solo-Gabriele, H., Concentration and Extraction: Efforts to Overcome Issues with Processing Coastal Water Samples for Downstream Molecular Analysis. American Society of Microbiology, Florida Branch Meeting, Cocoa Beach, FL, March 31- April 1, 2006.
K.D. Goodwin. Next Generation Sensors for the Integrated Ocean Observing System. Public Health Risks: Coastal Observations for Decision Making, St. Petersburg, FL, Jan. 23-25, 2006.
K.D. Goodwin. Adapting the Luminex System for Water Quality Analysis. Oceans & Human Health Initiative Principal Investigators Meeting, Charleston, SC, Jan. 18-20, 2006.
J. Gooch, K. Goodwin, J. Gregory, J. Jacobs., J. Lewis, W. Litaker, B. Robinson, J. Stewart. Concentration, Extraction and Detection: Efforts to Overcome Common Issues with Isolating Microbes from Environmental Samples. Oceans & Human Health Initiative Principal Investigators Meeting, Charleston, SC, Jan. 18-20, 2006.
K.D. Goodwin. Adaptation of the Luminex 100 System to Use in Recreational Waters, Sustainable Beaches Conference ’05, St. Petersburg, FL, Oct. 31-Nov. 2, 2005.
M.E. Durbin, A.M. Zaher, N.F. Heybeck, H.M. Solo-Gabriele, S. Elmir, K.D. Goodwin, C. Sinigalliano. The Inter-Tidal Zone is the Source of Enterococci to a Subtropical Recreational Beach. ASM, Atlanta, GA, June 5-9 2005.
K.D. Goodwin. Development of Molecular Biological Tools for Monitoring Coastal Water Quality Monitoring, Sea Tech, Dania, FL, March 17, 2005.
Public Information and Outreach:
Our goal is to interact with the HML Education and Outreach program on technology transfer issues including lessons learned, contacts made, and ideas of how the program can provide information to developers, end-users, and stakeholders regarding technology transfer. In addition, this project will interface with the University of Miami NSF/NIEH Center for Oceans and Human Health: Dr. Goodwin is a member of the Internal Advisory Committee for the University of Miami RSMAS OHH Center. This project is integrated with on-going projects at that center and utilizes the outreach vehicles (website, science symposiums) enlisted by the RSMAS OHH Center .
For More Information
Contact: Kelly Goodwin, (305)361.4384
Email: Kelly.Goodwin@noaa.gov
Literature Cited
Barnes B, Gordon DM. Coliform dynamics and the implications for source tracking. Environ Microbiol 2004; 6: 501-509.
Bellisario R, Colinas RJ, Pass KA. Simultaneous measurement of antibodies to three HIV-1 antigens in newborn dried blood-spot specimens using a multiplexed microsphere-based immunoassay. E Hum Develop 2001; 64: 21-25.
Bellisario R, Colinas RJ, Pass KA. Simultaneous measurement of thyroxine (T4) and thyrotropin (TSH) from newborn dried blood-spot specimens using a multiplexed fluorescent microsphere immunoassay. Clin Chem 2000; 46: 1422-1424.
Bernhard, AE, Field, KG. Identification of nonpoint sources of fecal pollution in coastal waters by using host-specific 16S ribosomal DNA genetic markers from fecal anaerobes. Appl Environ Microbiol 2000; 66: 1587-1594.
Bernhard, AE, Field, KG. PCR Assay to Discriminate Human and ruminant Feces on the Basis of Host Differences in Bacteroides-Prevotella Genes Encoding 16S rRNA. Appl Environ Microbiol 2000; 66: 4571-4574.
Boehm AB, Grant SB, Kim JH, Mowbray SLO, Mcgee CD, Clark CD, Foley DM, Wellman DE. Decadal and shorter period variability of surf zone water quality at Huntington Beach, California. Environ Sci Technol 2002; 36: 3885-3892.
Cabelli VJ, Dufour AP, Levin MA, McCabe J, Haberman PW. Relationship of microbial indicators to health effects at bathing beaches. Amer Jour Public Health 1979; 69: 690-696.
Cheng S, McCleskey FK, Gress MJ, Petroziello JM, Liu R, Namdari H, Beninga K, Salmen A, DelVecchio VG. A PCR assay for identification of Enterococcus faecium. J Clin Microbiol 1997; 35: 1248-1250.
Deasy BM, Rea MC, Fitzgerald GF, Cogan TM, Beresford RP. A rapid PCR based method to distinguish between Lactococcus and Enterococcus. System Appl Microbiol 2000; 23: 510-522.
Desmarais TR, Solo-Gabriele HM, Palmer CJ. Influence of Soil on Fecal Indicator Organisms in a Tidally Influenced Subtropical Environment. Appl Environ Microbiol 2002; 68: 1165-1172.
Diaz MR, Fell JW. High through-put detection of pathogenic yeasts in the genus Trichosporon. J Clin Microbiol 2004; 42: 3696-3706.
Dufour, A. P. Health effects criteria for fresh recreational waters. EPA-600/1-84/004. 1984. Office of Research and Development, USEPA.
Dunbar SA, Vander Zee CA, Oliver KG, Karem KL, Jacoboson JW. Quantitative, multiplexed detection of bacterial pathogens: DNA and protein application of the Luminex LabMAPTM system. J Microbiol Methods 2003; 53: 245-252.
EPA. Guidelines establishing test procedures for the analysis of pollutants; Analytical methods for biological pollutants in ambient water; Final Rule. Federal Register V68, No. 139 40 CFR Part 136, 43272-43283. 2003.
Fulton R, McDade R, Smith P, Kienker L, Kettman J. Advanced multiplexed analysis with the FlowMetrix system. Clin Chem 1997; 43: 1749-1756.
Hammerum AM, Jensen LB. Prevalence of esp, encoding the enterococcal surface protein, in Enterococcus faecalis and Enterococcus faecium isolates from hospital patients, poultry, and pigs in Denmark. J Clin Microbiol 2002; 40: 4396.
Khatib L, Tsai YL, Olson BH. A biomarker for the identification of cattle fecal pollution in water using the LTIIa toxin gene from Enterotoxigenic E. coli. Appl Microbiol Biotechnol 2002; 59: 97-104.
Khatib L, Tsai YL, Olson BH. A biomarker for the identification of swine fecal pollution in water using the STII toxin gene from Enterotoxigenic E. coli. Appl Microbiol Biotechnol 2003; 63: 231-238.
Leclerc H, Schwarzbrod L, Dei-Cas E. Microbial agents associated with waterborne diseases. Critical Reviews in Microbiology 2002; 28: 371-409.
Oliver, K, Patel, L, Kemp, J., Daves, J., Bell, L., and Zivin, R. The Luminex LabMAP system: a rapid, homogeneous, multianalyte platform. Society for Biomolecular Screening Meeting, Edinbugh, UK. 1999.
RTI International. Technology transfer of two assays to detect microbial contaminants in coastal waters. Phase I report and recommendations for phase II. 2005.
Scott TM, Jenkins TM, Lukasik J, Roase JB. Potential use of a host associated molecular marker in Enterococcus faecium as an index of human fecal pollution. Environ Sci Technol 2005; 39: 283-287.
Scott TM, Rose JB, Jenkins TM, Farrah SR, Lukasik J. Microbial source tracking: current methodology and future directions. Appl Environ Microbiol 2002b; 68: 5796-5803.
Scott TM, Rose JB, Jenkins TM, Farrah SR, Lukasik J. Microbial source tracking: current methodology and future directions. Appl Environ Microbiol 2002a; 68: 5796-5803.
Ye F, Li MS, Taylor JD, Nguyen Q, Colton HM, Casey WM, Wagner M, Weiner MP, Chen J. Fluorescent microsphere-based readout technology for multiplexed human single nucleotide polymorphism analysis and bacterial identification. Hum Mutat 2001; 17: 305-316.