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Food poisoning is an ever present health issue that is believed to exceed £1.5 billion a year to the UK economy. Microbial contamination of meat is the potential source of infection commonly associated with Campylobacter spp., Salmonella spp., Escherichia
Immunological techniques include:
Detection of bacteria is based on the ability of antibodies to recognise specific macromolecules, such as proteins or polysaccharides. Monoclonal or polyclonal antibodies are used for observation of a quantitative reaction of an antigen. The use of well selected antibodies is important for sensitivity and specificity of immuno-assays.
Polymerase chain reaction (PCR) has rapidly become one of the most widely used techniques in the process of identification of micro organisms. This is the fastest way to screen bacterial colonies. Most nucleic-acid-based methods cannot distinguish between live and dead cells. However, some infectious diseases caused by a small amount of pathogen and it may be more complicated to establish causative agent by other methods. In case of Salmonella food poisoning cases, the presence of live bacterial cells is significant because only the presence of live cells may be considered significant.
PCR is used to amplify the target DNA about 100-1000 base pairs long. A DNA or RNA target sequence should be unique in order to detect, characterize and identify micro organisms. Artificially synthesized pair of single stranded oligonucleotide primers complementary to the flanking regions of the target sequence (Figure 8) along with a DNA polymerase is used for selective and repeated amplification of target DNA. The primers are complementary to either end of the target sequence but lie on opposite strands. The primers are usually 20-30 nucleotides long and bind to complementary flanking region at 3' end (Rao, 2011)
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Flaking region--------II--------------
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Figure 8: Target
DNA for amplification by PCR (Rao, 2011)
Polymerase Chain Reaction is performed in the thermal cycler, which can regulate temperature during amplification and be programmed for the duration of process.
The process involves the repetition of three steps (Vierstraete, 1999) (Figure 9):
| Figure
9: The different steps in PCR.
Image downloaded from http://purpleopurple.com/ |
Primer extension, in which nucleotides
are added to the primers at a temperature of 60-75oC
– in the 5' to 3' direction – to form a double-stranded copy of
the target DNA.
|
Figure 10: Verification of the PCR product on gel. (Vierstraet, 1999) Image downloaded from http://users.ugent.be/~ |
The amplification product
can be detected using gel electrophoresis (Figure 10). The presence
of a band containing DNA fragments of a particular size indicates the
presence of the target sequence. The absence of a band indicates the
absence of the target sequence. In this case, other techniques can be
used in combination with PCR to detect specific target sequences.
DESIGN STUDY
INTRODUCTION
Microbial induced food poisoning continues to be a significant and on-going health problem in both developed and developing countries. For example, in the UK there are > 80,000 reported cases per year, costing the UK economy over £1.5 billion per annum (Ref?). The most common food-poisoning causing bacteria include Campylobacter spp., Salmonella spp., E. coli O157 and Staphylococcus aureus with consumption of infected meat products is the usual mode of transmission (Ref?).
Accurate and definitive bacterial identification and pathogen detection is essential for correct disease diagnosis, determination of appropriate treatment and to establish the origin of disease outbreaks associated with microbial infections. Correct identification is also required to allow the screening of food products prior to their consumption. Methods for the detection and identification of micro-organisms can be culture-based methods using selective growth media, immunological techniques to detect specific microbial antigens and DNA-based testing, using PCR, are all used for the purpose of identification of pathogenic micro-organisms.
The aim of this project is to develop
a PCR – based method for the detection of common food-poisoning causing
bacteria in meat products.
FLOW CHART
Design of oligonucleotde primers
Species-specific oligonucleotide primers
will be directed against the non-conserved regions of the 16S ribosomal
RNA gene from the targeted organism(s) (Campylobacter jejuni,
Salmonella spp., Escherichia coli O157 and Staphylococcus
aureus). Gene sequences from a number of micro-organisms will be
download from the DNA sequence database, Genbank (www.ncbi.nlm.nih.gov/Genbank)
Once the targeted regions have identified, oligonucleotide primers will be designed to bind to these regions taking into account the following “rules” for designing primers:
The specificity of the designed primers will be confirmed using BLAST (www.ncbi.nlm.nih.gov/BLAST).
Optimisation of PCR conditions for amplification of targeted gene sequences
Optimal primer sequences and appropriate primer concentrations are essential for maximal specificity and efficiency in PCR as is optimisation of amplification conditions. For example, the number of cycles performed in the PCR reaction is important for amplification of sufficient amount of the targeted gene for further analysis such as agarose gel electrophoresis / DNA sequencing. This will require a series of trial experiments to optimise amplification conditions.
Factors that will be examined include
MgCl2 concentration, annealing temperature (Tm),
number of PCR cycles, etc.
Detection of microbial DNA in meat samples
Once PCR conditions have been optimised
using purified genomic DNA (see above), meat samples from commercial
sources will tested for the presence of microbial DNA. This will first
involve optimising conditions for the extraction of DNA from meat samples
that have been artificially “spiked” with purified genomic DNA.
Once this has been optimised a variety of meat samples will be tested
for the presence of the micro-organisms of interest.
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