Waterborne Pathogen Prevention and Detection Using Traditional Methods and Microarray Probe Detection Claribel Orellana CE /5/07

Biotechnology Priorities: Agriculture and Medicine Priorities: Agriculture and Medicine Malaria Vaccine Malaria Vaccine Stem cell research Stem cell research Completion of the human genome map Completion of the human genome map ‘Golden Rice’ modified to make vitamin A ‘Golden Rice’ modified to make vitamin A GCSF for increasing white blood count in chemotherapy patients GCSF for increasing white blood count in chemotherapy patients

World Concern: Water Quality Half the world affected by contaminated water Half the world affected by contaminated water Unsanitary conditions and lack of resources Unsanitary conditions and lack of resources Cause: waterborne pathogens Cause: waterborne pathogens

Waterborne Pathogens Three different types: bacteria, viruses, and bacteria Three different types: bacteria, viruses, and bacteria Name of micro-organismsMajor diseasesMajor reservoirs and primary sources Bacteria Salmonella typhiTyphoid feverHuman feces Salmonella paratyphiParatyphoid feverHuman feces Vibrio choleraCholeraHuman feces and freshwater zooplankton Enteropathogenic E. coliGastroenteritisHuman feces Yersinia enterocoliticaGastroenteritisHuman and animal feces Legionella pneumophila and related bacteria Acute respiratory illness (legionellosis) Thermally enriched water Protozoa Giardia lambliaGiardiasis (gastroenteritis)Water and animal feces Helminths Ascaris lumbricoidesascariosisAnimal and human feces

Typical Contamination Unprotected water source Unprotected water source Inadequate sanitation Inadequate sanitation Animal and fecal matter reaching water source Animal and fecal matter reaching water source Surface runoff through the ground, water pipes and wells Surface runoff through the ground, water pipes and wells Cattle pond Surface drain Well Cattle pond

Typical Contamination Animal and fecal matter reaching water source Animal and fecal matter reaching water source Surface runoff through the ground, water pipes and wells Surface runoff through the ground, water pipes and wells

Traditional Practices for Safe Water Point-of-use disinfection Point-of-use disinfection Use of sodium and calcium hypochlorite Use of sodium and calcium hypochlorite Safer and easier to use and distributeSafer and easier to use and distribute Destroys most pathogensDestroys most pathogens Electrolysis Electrolysis System run on solar powerSystem run on solar power Generators can generate enough disinfectant for 10,000 people. Generators can generate enough disinfectant for 10,000 people. InexpensiveInexpensive

Traditional Practices for Safe Water Safe Storage Safe Storage Stored water vs. municipal tap Stored water vs. municipal tap Container comparison Container comparison CDC containerCDC container Cantero Cantero

DNA Microarrays DNA microarrays: reverse dot-blots for which sequence- specific “probes” are attached to substrate in a lattice pattern DNA microarrays: reverse dot-blots for which sequence- specific “probes” are attached to substrate in a lattice pattern spots are usually micrometers and micrometers away from each other and they represent specific probe sequences spots are usually micrometers and micrometers away from each other and they represent specific probe sequences Simultaneous detection vs. cultivation Simultaneous detection vs. cultivation

Polymerase Chain Reaction (PRC) PCR amplifies DNA sequences PCR amplifies DNA sequences

Microarray Process Sequences are hybridized Sequences are hybridized Specific bacterial targets are detected Specific bacterial targets are detected

Precision Factors Assay sensitivity Assay sensitivity Sample size Sample size Efficiency of pathogen isolation Efficiency of pathogen isolation Efficiency of nucleic acid extraction Efficiency of nucleic acid extraction Effect of co-precipitating factors that inhibit PCR Effect of co-precipitating factors that inhibit PCR

Benefits and Limitations Benefits: Benefits: Simultaneously detects pathogens Simultaneously detects pathogens Not limited to identification by product length Not limited to identification by product length Limitations: Limitations: Requires pathogens to be identified before configuring array Requires pathogens to be identified before configuring array Needs to be validated Needs to be validated Currently more expensive than traditional methods Currently more expensive than traditional methods

Microarray Flow Process

Application EPA looking into feasibility EPA looking into feasibility Is being monitored Is being monitored Will most likely become a standard Will most likely become a standard Soon be more cost-effective Soon be more cost-effective Priority is quick pathogenic detection Priority is quick pathogenic detection