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Harmful Algal Bloom (HAB) Identification Tools
Project Goal
While great strides have been made in the ability to detect and quantify HAB-forming organisms, methodologies need to be updated to increase throughput and accessibility to other researchers. The goal of this project is to develop molecular-based assays for the rapid, parallel detection of HAB species common to South Carolina waters, especially species that are abundant in holding ponds. Learn more about the background information for this project »
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Abstract
The goal of the harmful algal identification tools project is to create a single DNA-based test that can screen for the presence of the most problematic algal species on the eastern seaboard. Harmful algal blooms (HABs) can cause extensive damage to fisheries, recreational waterways, and human health. Knowing exactly which harmful algal species (if any) are present during a bloom event (noticeable discoloration of the water) is paramount to effective decision making by resource managers. Current technologies require a trained expert, expensive and time consuming analyses, or both. We are creating a single tool that can be implemented by a variety of users to rapidly and accurately identify multiple harmful algal bloom (HAB) forming species. Since currently available technologies are either too cumbersome or cost prohibitive to meet our needs, we have developed an entirely new technology. Species Identification via Chimeric Amplification (SIVCA) uses a combination of short DNA primers to selectively amplify specific genes from different harmful algal species. We have successfully developed and implemented probe sets targeted to 12 different species. In addition, we have used this novel method of gene amplification to generate two detection methods, one that is easily accessible and low cost; the other being more expensive but with dramatically higher throughput. We plan to bring the total number of species in the assay up to 18 and to transfer the reactions over to robotic equipment, further increasing our automation and throughput. Even at its current developmental stage, SIVCA has the potential to revolutionize the way multiple species are detected and can have broad implications in not only environmental health, but also in ecology and even bioterrorism.
Objectives
- Create a single reaction that can simultaneously amplify marker genes from all 18 species of microalgae;
- Design primers for assay development;
- Utilize robotic equipment to automate the entire amplification-to-detection pathway; and
- Field validate assay.
Expected Results
This project will utilize the advances made in multiplex PCR technology along with the automated PCR and sequence detection machinery at the HML to create a high-throughput, cost-effective, transferable technology that will enable the simultaneous detection of 18 different harmful algal species.
Accomplishments to date:
- Development of SIVCA (species identification via chimeric amplification), a novel two-stage Multiplex Polymerase Chain Reaction (PCR) protocol. The polymerase chain reaction (PCR) is a common tool used by molecular biologists to selectively amplify specific genes (or pieces of genes) from a mixed pool of DNA. This new method of PCR involves identification and labeling of multiple targets in the first step and amplification and addition of a fluorescent dye in the second. This new technique for multiplexing and fluorescently labeling target DNA provides two major benefits: 1) by varying the amount of each first stage primer, differences in reaction efficiency are compensated for, and 2) a single fluorescent label can be used to label all products resulting in dramatic cost savings. This new technique will be useful not only to OHHI and NOAA scientists, but will also provide a new paradigm for simultaneous multiplexing and fluorescent labeling of PCR products by other scientists. One of the difficulties in multiplex PCR is differences in efficiency from one target to another. SIVCA allows us to compensate for those differences and amplify many target species in a single sample. Current PCR assays for algal identification target a single species, but our two-stage multiplex can identify 12 species in only 2 reactions! Already we are increasing the potential throughput by ten fold.
- Development of a low-cost version of the assay. A key corollary to the success of the project is the ability for users with varying levels of technology and funds to have an assay that will meet their needs. We designed the assay such that all of the products are distinguishable from one another by size (molecular weight) alone. This means that a small lab with minimal resources can perform these analyses quickly and reliably, albeit with lower throughput. Using relatively inexpensive (and reusable) high-density agarose we can distinguish all products in each of the reactions. This requires neither specialized equipment (apart from what is normally present in a molecular biology lab) nor the expensive fluorescent primer, resulting in a very low-cost assay. Increased accuracy can be accomplished with a minimal increase in price by use of acrylamide electrophoresis.
- Development of an automated high-throughput detection assay. Using SIVCA we have been able to attach a fluorescent dye to all products amplified in our reactions. Due to the difference in size of each product, we can detect and identify each product using a commercially available capillary electrophoresis system. This automation allows us to screen up to 96 samples at a time, all in a matter of hours. To screen that many samples for 12 species would require at least eleven days with currently available real-time PCR protocols.
Transfer of Results
The technology we are developing for this project can be used by a variety of laboratories and agencies. Because of the use of standard PCR and electrophoresis equipment in the early stages of development, this assay will be usable in a lower-throughput form that will also be drastically lower in cost and widely available. We envision a two-fold approach to utilization of this technology, including small natural resource agency, private, and academic scientists; and large academic, private, and government institutions.
The smaller academic and natural-resources facilities who are monitoring a limited number of sites or with limited frequency will have limited resources and facilities. With the equipment that they likely already have on hand they can simply order the primers specified in our final report and carry out the assay in their laboratories. Even without a capillary sequencer, the PCR products can be separated using simple acrylamide electrophoresis. This eliminates the need for the costly fluorophore addition to one of the primers, making the assays even more attractive to smaller laboratories with a limited budget.
Large academic and governmental institutions as well as contract laboratories may have already invested in the automation equipment needed for the high-throughput applications we develop for application at the HML. In addition, environmental monitoring contract laboratories may wish to add this assay to those they offer on a fee-per-sample basis. The versatility of this approach and its adaptability to various levels of equipment availability should make its transfer to users straightforward. Finally, we will disseminate our results and methodologies as well as a summary of their strengths and limitations to environmental and public health officials as well as municipal planners through conference presentations and local government meetings, and the South Carolina Task Group on Harmful Algae. This will ensure that agencies and municipalities are aware of its existence and potential application.
Publications:
Brown, P.J.P, Lewitus, A.J., Wilde, S.B. and Chapman, R. 2007 Species Identification via Chimeric Amplification: A novel technique for simultaneous detection of multiple harmful algal species. Manuscript in preparation for submission to Nature: Methods in early 2007.
Public Information and Outreach:
One of the major benefits of our HAB identification tool is its usefulness to natural resource and land management officials. The decision to close a beach or a shell fish bed due to an algal bloom can have exceptional financial implications. A resource manager doesn’t want to make that kind of decision without all the facts. There are several algal species that are similar to HAB species but are not themselves toxic. Our tool rapidly and accurately identifies many potentially harmful species while excluding these non-toxic look-alikes. While it would be ideal to only base these decisions on whether or not a bloom was toxic, the toxic mechanisms for many of these organisms have not been determined. Additionally, until more is known about the environmental triggers of algal toxicity, prudence demands that decisions be made based on the presence of the organism, not whether or not its toxic at the moment. This enables decision makers to make rapid and informed decisions regarding closure of commercial and recreational resources, thus protecting human health without endangering commerce. Due to the scalability of this tool, the low-cost option will be available to state agencies and many local municipalities, enabling local officials to make public health decision regarding HABs quickly and for the common good.
For More Information
Contact: Patrick Brown, (843)762.8868
Email: BrownP@dnr.sc.gov