Environmental Marine Biotechnology: Development of DNA Microarrays as Sensors for Diverse Marine Pathogens in the Environment

Objectives:Pathogen monitoring and early detection systems in coastal environments are currently facing a monumental task: the need to survey an increasing number of organisms and an alarming rise in the numbers of vulnerable sites. This necessitates new technologies that allow the assessment of the global distribution as well as the local variation of a variety for pathogenic populations in an efficient and cost-effective manner.Methodology:We propose to develop and tests a powerful pathogen detection system based on DNA microarrays (“gene chips”). This new technology would allow the simultaneous detection and quantification of hundreds to thousands of microbial species based on differentiating DNA sequence variation. A single array can be customized to identify organisms ranging from pathogenic bacteria, protists and viruses to toxin producing algae. This avoids lengthy cultivation based tests, problems with viable but non-culturable pathogens, and inaccuracies associated with ‘indicator species.’ The entire process of array construction to detection and data analysis will be automated, and is especially amenable for use in centralized analytical facilities where samples can be sent to and treated in a standardized manner. Rationale:The goals of the proposed research are to (1) design a microarray containing oligonucleotide probes targeting representatives from key genera of marine pathogens, (2) test issues relating to the accuracy and detection limits of the probe array using cultures as well as environmental samples, (3) validate array data by quantitative PCR, a sensitive method for enumerating gene copy number as a proxy for cell abundance, and (4) formulate protocols from sample collection to hybridization for future routine assa- of pathogens. In building and designing the chips, we will take advantage of the instrumentation and expertise that is available to us at no cost in the newly established microarray facility at MIT and draw upon our experience in developing quantification tests with quantitative PCR. Ultimately, microarrays as a technique of the future will allow simple, cost-effective and rapid testing of coastal waters enabling us to determine global pathogen distributions and elucidate host-pathogen interactions.