Species identification by DNA sequencing

eFOOD-Lab_International_02_2014

Species identification by DNA sequencing 2/14 eFOOD-Lab international 7 Application of Next Generation Sequencing on Food and Feed Analysis Our Author: Ilka Haase, Eurofins Genomics, Anzinger Str. 7a, Ebersberg, Phone: +49 8092 8289-296, Email: ilkahaase@eurofins.com, DNA regions with high sequence variability (= differences in the nucleotides). Public databases like NCBI (National Center for Biotechnology Information), BOLD (Barcode of Life Data Systems) or FISH-BOL (Fish Barcode of Life Initiative) provide such sequences as so-called barcodes for more and more animal, plant and microbial species. Commonly suggested and accepted barcodes are (i) for animals a sequence region of the mitochondrial cytochrome C oxidase subunit I (COI), (ii) for plants sequence regions of the plastid ribulose bisphosphate carboxylase (rbcL) and maturase K (matK) genes, (iii) for bacteria the 16 S sequence region and (iv) for fungi sequences of the internal transcribed spacer (ITS) region (www.boldsystems.org; 1). By using universal primer pairs for the barcodes given above, the analysis of any unknown sample of an organism group is possible. For medical, botanical and zoological studies, sample DNA is normally extracted from fresh tissue, the respective barcode (animal, plant, bacteria or fungi) is amplified by PCR (polymerase chain reaction) and the amplified DNA fragment is analysed by Sanger sequencing (see Fig. 1a). A comparison of the obtained sample sequence with the database’s entries allows the identification of the species in the sample. The analysis of food and feed samples by DNA barcoding might be challenging because of processing steps like e.g. milling, high temperatures, acidic conditions, that all have an influence on the DNA quality and lead to partial DNA degradation. Resulting DNA fragments are either too short to contain enough sequence information for a species identification or cause a complete failure of the barcode amplification. Additionally, in complex food and feed samples that contain e.g. two animal species, an overlay of the two barcoding sequences can be observed in the chromatogram of the Sanger sequencing The use of DNA-based methods for the analysis of plant, animal and also microbial species in food has become more and more popular the last years. Especially since the horse meat scandal in 2013, authenticity as well as quality control of food and feed by DNA is on everybody’s tongue. Species identification by DNA barcoding The identification of a certain species of an organism group (plants, animals, fungi, bacteria) can be achieved by the use of Fig. 1. Barcoding by Sanger sequencing. (a) Sequence chromatogram of a single species sample. (b) Two species mix with a major and a minor component. In the sequence chromatogram, the peaks of the minor species can only be observed when present > 10 %. Qualit y Mana gement


eFOOD-Lab_International_02_2014
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