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.
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.
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.
Bioluminescence Biosensor Measures change in luminescence emitted byliving micro-organisms. There are two types of bioluminescence found inmicrobes. ATP Bioluminescence: Bacterial Bioluminescence.
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
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.
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.
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
Antibody Based Assays Highly specific interactions of antigen-antibody used for detection ofpathogens. Latex Agglutination. Reverse Phase Latex Agglutination. Immunodiffusion. ELISA. Immunoprecipitation:
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.
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.
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.
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.
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.