Waterborne Pathogen Prevention Waterborne Pathogen Prevention and Detection Using Traditional and Detection Using Traditional Methods and Microarray Probe Methods and Microarray Probe

DetectionDetection

Claribel OrellanaClaribel Orellana

CE 421CE 421

12/5/0712/5/07

BiotechnologyBiotechnology

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

World Concern: Water QualityWorld Concern: Water Quality

• Half the world affected by contaminated waterHalf the world affected by contaminated water• Unsanitary conditions and lack of resourcesUnsanitary conditions and lack of resources• Cause: waterborne pathogensCause: waterborne pathogens

Waterborne PathogensWaterborne Pathogens

• Three different types: bacteria, viruses, and bacteriaThree different types: bacteria, viruses, and bacteria

Name of micro-organisms Major diseases Major reservoirs and primary sources

Bacteria

Salmonella typhi Typhoid fever Human feces

Salmonella paratyphi Paratyphoid fever Human feces

Vibrio cholera Cholera Human feces and freshwater zooplankton

Enteropathogenic E. coli Gastroenteritis Human feces

Yersinia enterocolitica Gastroenteritis Human and animal feces

Legionella pneumophila and related bacteria

Acute respiratory illness (legionellosis)

Thermally enriched water

Protozoa

 Giardia lamblia Giardiasis (gastroenteritis) Water and animal feces

Helminths

 Ascaris lumbricoides ascariosis Animal and human feces

Typical ContaminationTypical Contamination

• Unprotected water sourceUnprotected water source• Inadequate sanitationInadequate sanitation• Animal and fecal matter reaching water sourceAnimal and fecal matter reaching water source• Surface runoff through the ground, water pipes and wellsSurface runoff through the ground, water pipes and wells

Cattle pond

Surface drain

Well

Well

Cattle pond

Typical ContaminationTypical Contamination

• Animal and fecal matter reaching water sourceAnimal and fecal matter reaching water source• Surface runoff through the ground, water pipes and wellsSurface runoff through the ground, water pipes and wells

Traditional Practices for Safe WaterTraditional Practices for Safe Water

• Point-of-use disinfectionPoint-of-use disinfection• Use of sodium and calcium hypochloriteUse 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 WaterTraditional Practices for Safe Water

• Safe StorageSafe Storage• Stored water vs. municipal tapStored water vs. municipal tap• Container comparisonContainer comparison

• CDC containerCDC container

• CanteroCantero

DNA MicroarraysDNA Microarrays

• DNA microarrays: reverse dot-blots for which sequence-DNA microarrays: reverse dot-blots for which sequence-specific “probes” are attached to substrate in a lattice specific “probes” are attached to substrate in a lattice pattern pattern

• spots are usually 100-200 micrometers and 200-500 spots are usually 100-200 micrometers and 200-500 micrometers away from each other and they represent micrometers away from each other and they represent specific probe sequences specific probe sequences

• Simultaneous detection vs. cultivationSimultaneous detection vs. cultivation

Polymerase Chain Reaction (PRC)Polymerase Chain Reaction (PRC)

• PCR amplifies DNA sequencesPCR amplifies DNA sequences

Microarray ProcessMicroarray Process

• Sequences are hybridizedSequences are hybridized• Specific bacterial targets are detectedSpecific bacterial targets are detected

Precision FactorsPrecision Factors

• Assay sensitivityAssay sensitivity• Sample sizeSample size• Efficiency of pathogen isolationEfficiency of pathogen isolation• Efficiency of nucleic acid extractionEfficiency of nucleic acid extraction• Effect of co-precipitating factors that inhibit PCREffect of co-precipitating factors that inhibit PCR

Benefits and LimitationsBenefits and Limitations

• Benefits:Benefits:• Simultaneously detects pathogensSimultaneously detects pathogens• Not limited to identification by product lengthNot limited to identification by product length

• Limitations:Limitations:• Requires pathogens to be identified before configuring arrayRequires pathogens to be identified before configuring array• Needs to be validatedNeeds to be validated• Currently more expensive than traditional methodsCurrently more expensive than traditional methods

Microarray Flow ProcessMicroarray Flow Process

ApplicationApplication

• EPA looking into feasibilityEPA looking into feasibility• Is being monitoredIs being monitored• Will most likely become a standardWill most likely become a standard• Soon be more cost-effectiveSoon be more cost-effective• Priority is quick pathogenic detectionPriority is quick pathogenic detection