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An Investigation Into The Use Of PCR-RFLP Profiling For The Identification Of Fruit Species In Fruit Juices.
Project Code: Q01111
The Food Standards Agency (FSA) has supported the development of PCR-based approaches for food analysis. Recently the FSA has been focussing on methods that utilise lab-on-a-chip capillary electrophoresis, which is a low-cost, easy-to-use system that enables accurate sizing and quantification of DNA fragments. This system is ideally suited to authenticity applications that are based on DNA profiling. Successful methods are already being used by local Government laboratories to uphold food labelling requirements.
A lab-on-a-chip, PCR-RFLP profiling approach suitable for the identification of six fruit species (apple, blueberry, elderberry, grape, pear and pomegranate) used in the production of fruit juices was developed. Blueberry and pomegranate juices are recognised premium grade juices, while juices such as apple and pear are less commercially valuable. The FSA was keen to develop methods that would allow authorities to detect adulteration or contamination of premium juices with cheaper juices. The profiling approach reported here used PCR and restriction enzyme digestion of a chloroplast DNA target, the psbA-trnH intergenic spacer region. This sequence is a recommended taxonomic target and has been adopted for plant barcoding purposes by several groups involved with the Barcode of Life initiative.
Our findings, using PCR amplicons produced from DNA extracted from whole plant materials (leaf or fruit), showed that it was possible to differentiate some of the six species on the basis of their PCR amplicon sizes; elderberry (560bp) could be differentiated from the other five species, while apple (392bp) and pear (411bp) could also be distinguished from the other species, but not from each other. Blueberry (465bp), grape (459bp) and pomegranate (463bp) could be differentiated from the other three species, but not from each other.
Using leaf and fruit materials, PCR-RFLP profiles were produced using the restriction enzymes ApoI and DdeI and the Agilent 2100 Bioanalyzer. Differentiation of all six species was readily achieved using either of these two enzymes; however, the use of both enzymes provided more substantiation to the results. A database of PCR amplicon sizes and PCR-RFLP profiles was produced for the six species investigated in this study. It is envisaged that this database could be further expanded using experimental data or information from the Barcode of Life database.
A number of methods were used to extract DNA from fruit juices produced from concentrate. Although DNA amplification was achieved using the DNA extracted from these juices, the amplicons produced did not match those in the database. In general, the amplicons produced from the juices were smaller than those in the database and in many cases the juices produced several PCR products rather than a single product as expected. Although these results indicated that DNA was being extracted from the juices, it was not possible to confirm the origin of the juice DNA. It is suggested that the DNA extracted from juice concentrate could have been derived from the yeasts and moulds naturally found on fruits that the process of producing juice concentrates caused degradation of fruit DNA, or that genetic differences between fruit varieties could account for the differences between the database and juice results.
The database of PCR amplicon sizes and PCR-RFLP profiles that was produced during this study has a wide application for the investigation of authenticity of fruit based food products such as fruit puree or conserves that are used as ingredients of food products, e.g. yoghurts and jams. This PCR-RFLP based approach is a quick and easy method. If a suitable DNA extraction protocol for use with juices derived from concentrates can be developed, this method should offer analysts a reliable approach for fruit juice authentication.
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