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Adaptation of DNA analysis techniques for the identification of illegally imported bushmeat for use on the Agilent 2100 bioanalyser
Project Code: Q01109
Food DNA Services Limited
Ogden, R ; McEwing, R
This report details the results of work to develop and transfer a method for the genetic identification of bushmeat species onto the Agilent 2100 BioAnalyzer analysis platform. The method employed is based on a technique known as PCR-RFLP that previously has been demonstrated to allow discrimination between species using a system of species-specific restriction profiles.
The system is designed to identify the following species: chimpanzee, gorilla, bush buck, African sheep, dwarf zebu, zebu, duiker (black-backed duiker, Peter’s duiker, black-fronted duiker, blue duiker), bush pig, pouched rat, cane rat and porcupine, all of which are known to be traded internationally as bushmeat. These species are associated with disease risks to humans and livestock and may also be endangered in their country of origin.
Following the collection of reference samples, the experimental procedures consisted of designing and validating the PCR method, DNA sequencing of the target species, selection of suitable restriction enzymes, RFLP profile construction, preparation of a working SOP and finally validation of the total system.
The results indicated that samples from all eleven target species groups could be reliably amplified using standardized experimental conditions. Validation studies further demonstrated the ability to perform the PCR assay with a wide range of DNA concentrations using a variety of tissue samples prepared in a number of ways. This provides the basis for applying the assay to the types of sample typically seized during investigations.
Selection and testing of the restriction enzymes resulted in two enzymes, HinfI and MseI, being chosen for subsequent assay development. The combination of products from these enzymes yields fragment profiles that are species-specific within the target set. On the basis of these results, a profile construction method was designed that allowed fragments to be easily scored and converted into a species identification.
Validation of the assay showed variability in the size of fragments within a single run (one Labchip), between runs (between Labchips) and between laboratories (between operators, machines and Labchips). The exact cause of the variation is unknown, but at least some component is associated with the BioAnlyzer technology.
The impact of sizing variation has led to a redesign of the fragment profile method which now minimizes the impact of the observed variability. The assay is therefore capable of identifying the target species with a sufficient level of confidence for survey work. Legal applications would be presumptive rather than definitive due to the nature of PCR-RFLP; a secondary test such as DNA sequencing would subsequently be required to confirm the results.
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