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| | | | A closer look at rapid detection of micro-organisms by 'real-time' PCR | | | | | | 19 February 2007
Michael Wood – Technical Manager, Bodycote Norpath, part of Bodycote Health Sciences.
Michael graduated from the University of Newcastle upon Tyne with a degree in Microbiology and Biochemistry. He worked in the National Health Service and Public Health Laboratory Service as a Microbiologist before moving into the food sector.
In 1987 Michael, with a small group of scientists founded Norpath Laboratories. Michael has extensive knowledge of microbial detection and identification procedures, validation of methods and techniques as well as being involved in the research of new technologies.
Since the acquisition of Norpath Laboratories by Bodycote in the summer of 2006, Michael has continued his involvement with the research and evaluation of new and innovative technologies in microbial detection.
The rapid detection of pathogens and other microbiological contaminants in food is critical for ensuring the safety of consumers. Traditional methods to detect food borne bacteria often rely on growth in culture media, followed by isolation, biochemical and sometimes serological identification, which can be time consuming; up to 5 days in some cases. Recent advances in technology make detection and identification faster, more sensitive and more specific than traditional method.
For food manufacturers whether under pressure to reduce pre-release storage times or where shelf life is critical and for responding quickly to food crisis issues such as food poisoning, rapid detection methods in the laboratory are an attractive option. These methods can give results in 24-30 hours.
The latest technologies identify microorganisms in foods by detecting specific areas of their DNA known as genes. The systems have been developed in such a way that the genes detected are highly specific to the individual organisms.
Careful selection of the detection elements, called DNA ‘primers’ can allow specific identification of pathogens such as Salmonella, Listeria and E. coli 0157. The detection mechanism is so specific that it can eliminate the need for further confirmation steps. Final (‘confirmed’) results can be available in 30 hours.
The method is based firstly on the detection of the target gene using a specific DNA ‘primer’ followed by its amplification. In the amplification process many copies of the gene are made using a technique known as the Polymerase Chain Reaction or PCR.
Polymerase Chain Reaction is a technique for multiplying a specific target DNA sequence which is chosen to be unique to the microorganism under test. The DNA is first released from the organism by disrupting the cell wall and the relevant Primers. Polymerase, an enzyme that copies DNA is also added together with a fluorescent probe.
The DNA molecule is made up of two complimentary strands which are first separated from each other or ‘denatured’ by heating to 95C. On cooling to 55C the Primers bind or ‘anneal’ to the target DNA sequence. Further heating to 72C enables the Polymerase to begin synthesis of new strands of DNA.
This three-stage cycle of ‘denaturation, annealing and synthesis’ is repeated 30 to 60 times. The quantity of target DNA is doubled at each new PCR cycle. The fluorescent probe carries two markers at either end: a fluorophore and a quencher. In the reaction mixture the probe is stable and emits no fluorescence as long as the fluorophore and the quencher remain in close proximity. However during PCR amplification the Polymerase splits the probe, liberating the fluorophore and allowing the fluorescent signal intensity to be measured.
Each copy of the gene is chemically linked to a fluorescent dye. As the copy numbers mount the increased fluorescence is monitored and recorded in ‘real-time’ – as it happens – allowing an early indication of the presence of the target and in some cases an additional indication of the numbers of the microorganism in the original sample.
At present Bodycote F & CP Group offers clients PCR based detection systems for Listeria and Salmonella in foods. Work in development includes detection of E. coli 0157 and Campylobacter in foodstuffs and detection and enumeration of Legionnella spp in water.
In all these systems, target DNA extraction, amplification and detection procedures have been optimized for all food and environmental samples. Extending the technique to additional micro-organisms requires only the characterization of specific DNA ‘primers’. Research into detection systems for Yeasts and Enterobacter sakazakii is currently in progress. Similar techniques can be applied to the detection of GM food ingredients and meat speciation.
The detection of pathogens and other microbiological contaminants in food is critical for ensuring the safety of consumers. Microbiological monitoring is essential at all stages of the manufacturing process to support HACCP planning. In crisis situations confidence in results is critical. There are however a number of factors that should be borne in mind when comparing the merits of traditional versus rapid methodologies.
Traditional microbiological methods require the sample to be cultured and the identity of isolates confirmed. Culture may require several stages to allow for the recovery of stressed cells and confirmation may require additional growth steps. Overall these methods are labour intensive and times consuming.
In general rapid methods eliminate many of the time consuming cultural steps. Used as screening tests they allow product or ingredients found to be ‘negative’ to be released in a much shorter timescale. In some cases, positive results will require confirmation by traditional techniques that can negate any benefits. Real-time PCR however, can have the specificity to eliminate these final confirmation steps.
All rapid techniques have an additional cost implication, however this should be set against the benefits of faster throughput and increased shelf life.
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