Rapid methods for detection of Food-borne Pathogens.
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The document discusses rapid methods for detecting food-borne pathogens, emphasizing the economic impact of food-borne diseases and the need for effective detection techniques. It outlines various biosensor technologies and assays, including bioluminescence, impedance-based detection, and molecular techniques such as PCR and DNA microarray, highlighting their specificity and sensitivity. Additionally, it lists examples of automated systems and kits available for identifying foodborne bacteria.
Rapid methods for detection of Food-borne Pathogens.
- 1. Rapid Methods for Detection of Food-borne Pathogens. KALEEM IQBAL 0309-MPHIL-BIO-T-12 INSTITUTE OF INDUSTRIAL BIOTECHNOLOGY, GCU LAHORE.
- 2. Need for detection Food-borne diseases cost Billions of dollars to the world annually. Pathogens may enter the food through many different ways. Uneven distribution of bacteria in the food, presence of indigenous microbes. To minimize time and human error.
- 3. Method For a method to be effective it should meet the following requirements: 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. Biosensors Devices that convert a biological response to electric signals. Consists of a bioreceptor element and a transducer. The transducer converts the signal to electrical current and passes it to the amplifier.
- 5. Principle of Biosensors Target analyte
- 6. Bioluminescence Biosensor Measures change in luminescence emitted by living micro-organisms. There are two types of bioluminescence found in microbes. ATP Bioluminescence: Bacterial Bioluminescence.
- 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 + PPi Luciferyl adenylate + O2 oxyluciferin + AMP + light
- 8. Bacterial Luminescence The gene responsible for luminescence in bacteria is called lux gene. The gene is introduced into the host specific bacteria. Detectors are then used to measure the emitted light. Capable of detecting 100 cells/hr.
- 9. Impedimentary (Electrical impedance) Microbial growth causes change in impedance and can be detected using Electrochemical Impedance Spectroscopy (EIS). Monitor large number of samples simultaneously. Relatively short detection time 6-24 hours.
- 10. Some Biosensors and Kits. Table 1. Miniaturized biochemical kits and automated systems for identifying foodborne bacteria System Format Manufacturer Organisms Cobas IDA biochemical Hoffmann LaRoche Enterobacteriaceae Micro-IDb biochemical REMEL Enterobacteriaceae, Listeria MISb Fatty acida Microbial-ID Enterobacteriaceae, Listeria, Bacillus, Staphylococcus, Campylobacter Walk/Away biochemicala MicroScan Enterobacteriaceae, Listeria, Bacillus, Staphylococcus, Campylobacter Riboprinter nucleic acida Qualicon Salmonella, Staphylococcus, Listeria, Escherichia coli Malthusb conductancea Malthus Salmonella, Listeria, Campylobacter, E. coli, Pseudomonas, coliforms Bactometer impedancea bioMerieux Salmonella
- 11. Antibody Based Assays Highly specific interactions of antigen-antibody used for detection of pathogens. Latex Agglutination. Reverse Phase Latex Agglutination. Immunodiffusion. ELISA. Immunoprecipitation:
- 12. DNA Based Assays These include the methods based on the use of nucleic acids for detection of pathogens. There are three main nucleotide based assays: DNA hybridization. Polymerase Chain Reaction. DNA microarray.
- 13. DNA Hybridization DNA probes, sequences of known segments are hybridized with the target genomes. If the relevant sequence is there in the sample, the probe binds with that portion 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. Polymerase Chain Reaction (PCR) Primers designed against pathogenic genes are used to amplify the sample sequences. If the amplified product is obtained this means that the pathogenic DNA is there in the sample. This method is more specific and rapid than conventional methods.
- 15. Real Time PCR Just like PCR but it monitors the changes in the reaction as they occur by continuous 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 increases the fluorescence. Real time PCR is being used for: Viral quantification. Drug efficacy. Pathogen detection. Genotyping.
- 16. DNA Microarray. The target sequences are bound to a chip which is normally glass slide or nylon membranes called arrays. mRNA is extracted from control and experimental cells and labelled with specific dyes e.g. Cy3 and Cy5. They are then hybridized with the target probes attached to the arrays. This is a rapid and efficient method and can detect thousands of specific sequences.
- 17. THANKS