Rapid methods for detection of Food-borne Pathogens.


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A presentation on some Rapid and efficient methods for detection of pathogens which are carried by food.

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Rapid methods for detection of Food-borne Pathogens.

  1. 1. Rapid Methodsfor Detection ofFood-bornePathogens.KALEEM IQBAL0309-MPHIL-BIO-T-12INSTITUTE OF INDUSTRIAL BIOTECHNOLOGY, GCU LAHORE.
  2. 2. Need for detection Food-borne diseases cost Billions of dollars to theworld annually. Pathogens may enter the food through manydifferent ways. Uneven distribution of bacteria in the food, presenceof indigenous microbes. To minimize time and human error.
  3. 3. Method For a method to be effective it should meet the followingrequirements: Detection method must be specific. The method should detect the desired specific pathogens. Method must be sensitive to detect small no. of pathogens. Should produce a quantitative analysis to determine the severity. Method should be multiplex.
  4. 4. Biosensors Devices that convert a biological responseto electric signals. Consists of a bioreceptor element and atransducer. The transducer converts the signal toelectrical current and passes it to theamplifier.
  5. 5. Principle of BiosensorsTarget analyte
  6. 6. Bioluminescence Biosensor Measures change in luminescence emitted byliving micro-organisms. There are two types of bioluminescence found inmicrobes. ATP Bioluminescence: Bacterial Bioluminescence.
  7. 7. ATP Bioluminescence Used to measure the effectiveness of cleaning surfaces and utensils. Take a swab sample and combine it with a mixture of luciferase. Following reaction takes place:Luciferin + ATP  luciferyl adenylate + PPiLuciferyl adenylate + O2  oxyluciferin + AMP + light
  8. 8. Bacterial Luminescence The gene responsible for luminescencein bacteria is called lux gene. The gene is introduced into the hostspecific bacteria. Detectors are then used to measurethe emitted light. Capable of detecting 100 cells/hr.
  9. 9. Impedimentary (Electrical impedance) Microbial growth causes change in impedance and can be detectedusing Electrochemical Impedance Spectroscopy (EIS). Monitor large number of samples simultaneously. Relatively short detection time 6-24 hours.
  10. 10. Some Biosensors and Kits.Table 1. Miniaturized biochemical kits and automated systems for identifying foodborne bacteriaSystem Format Manufacturer OrganismsCobas IDA biochemical Hoffmann LaRoche EnterobacteriaceaeMicro-IDb biochemical REMEL Enterobacteriaceae,ListeriaMISb Fatty acida Microbial-ID Enterobacteriaceae,Listeria, Bacillus,Staphylococcus,CampylobacterWalk/Away biochemicala MicroScan Enterobacteriaceae,Listeria,Bacillus, Staphylococcus,CampylobacterRiboprinter nucleic acida Qualicon Salmonella,Staphylococcus,Listeria, Escherichia coliMalthusb conductancea Malthus Salmonella, Listeria,Campylobacter, E. coli,Pseudomonas, coliformsBactometer impedancea bioMerieux Salmonella
  11. 11. Antibody Based Assays Highly specific interactions of antigen-antibody used for detection ofpathogens. Latex Agglutination. Reverse Phase Latex Agglutination. Immunodiffusion. ELISA. Immunoprecipitation:
  12. 12. DNA Based Assays These include the methods based on the use of nucleic acids fordetection of pathogens. There are three main nucleotide based assays: DNA hybridization. Polymerase Chain Reaction. DNA microarray.
  13. 13. DNA Hybridization DNA probes, sequences of known segments are hybridized with the targetgenomes. If the relevant sequence is there in the sample, the probe binds with thatportion and gives fluorescence. Target DNA is denatured at a high temperature. Probes are labelled with radioactive labels for detection. Autoradiography is then used to detect the probe-target complexes.
  14. 14. Polymerase Chain Reaction (PCR) Primers designed againstpathogenic genes are usedto amplify the samplesequences. If the amplified product isobtained this means thatthe pathogenic DNA isthere in the sample. This method is more specificand rapid thanconventional methods.
  15. 15. Real Time PCR Just like PCR but it monitors the changes in the reaction as they occur bycontinuous collection of fluorescent signals. Fluorescent dyes such as SYBR green are used in Rt. PCR. As the dye binds to dsDNA it undergoes change in shape and increasesthe fluorescence. Real time PCR is being used for: Viral quantification. Drug efficacy. Pathogen detection. Genotyping.
  16. 16. DNA Microarray. The target sequences are bound to achip which is normally glass slide ornylon membranes called arrays. mRNA is extracted from control andexperimental cells and labelled withspecific dyes e.g. Cy3 and Cy5. They are then hybridized with the targetprobes attached to the arrays. This is a rapid and efficient method andcan detect thousands of specificsequences.
  17. 17. THANKS