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

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