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  1. 1. Bacteriophage Based Bioluminescence Biosensor For Pathogen Detection In Food N.A.G. Sandaruwan
  2. 2. Why detection of micro organisms in food is important? “2.2 million people die annually due to food and water borne illnesses, and 1.9 millions among them are children” -WHO
  3. 3.  Conventional methods used for this are microbiological and biochemical analysis, ELISA, PCR etc.  They are overly time consuming, not amendable to on site detection and need a extensive training.  Biosensors have been identified as attractive alternatives to the existing conventional pathogen detection methods.
  4. 4. What is a Biosensor? A biosensor is an analytical device, used for the detection of an analyte, that combines a biological component with a physicochemical detector.
  5. 5. Classification of Biosensors
  6. 6. Bioluminescence
  7. 7. History of Bioluminescence Technique  In 1667 Robert Boyle, English chemist discovered bioluminescence.  He observed few spot of bluish-green light on a chicken carcasses which was purchased the week prior.  Dubois confirmed oxygen dependency and demonstrated the necessity of an enzyme (luciferase) and a luciferase substrate for light emission.
  8. 8. Bioluminescence  Need luciferase enzyme.  Substrate is catalytic oxidated by the enzyme to produce light in the presence of molecular oxygen. FMNH2 + RCHO + O2 FMN + RCOOH + H2O + Light
  9. 9. Bioluminescence in a microorganism Membrane synthesis Myristol ACP Myristic acid Myristic Aldehyde Light (490nm) FMNH2 Luciferase Luciferas e
  10. 10. Genetic Background  Luciferase can be found in bacteria ( lux ) and eukaryotes ( luc ).  It’s encoded by lux A and B genes.  In addition light generation requires lux C,D and E genes.
  11. 11. How to use a Bacteriophage? Select a specific bacteriophage Change the DNA component Introduce to the medium Multification
  12. 12. Reporter phage with lux gene Introducing to the sample Contamination with targeted MO Reporter phage attack to MO Inject lux gene Luciferase enzyme is produced Light emmission
  13. 13. Industrial applications  A511::lux AB recombinant phage is used to identify Listeria bacterium in Spikked ricotta cheese, chocolate pudding etc.  A511::lux AB is used to check the quality of minced meat and soft cheese.  P22::lux AB is used to identify Salmonella Spp. ( ex:- in eggs )
  14. 14. Advantages of bacteriophage based biosensers  Very sensitive.  Less time consuming  Minimal sample preparation.  Specificity of detection can distinguish between live and dead MO s.
  15. 15. Limitations  Presence of specific phage inhibition.  Antiviral bacterial immunity system.
  16. 16. Thank you!