Authentic Identification Techniques For Fish Fillets Or Fish Based On DNA Barcodes

In recent years, there weren’t authoritative identification techniques for fishproducts identification and perfect quality assurance system for aquatic productscirculation and processing in China. Food quality and safety issues had occurredfrequently due to mislabels or adulteration of seafood. In the market, it appeared thatlow-value fish posing as high-value fish, poisonous fish used as raw food materials orno fish in the fish products. This research is references of molecular biology andbioinformatics research. Then, DNA barcoding, PCR-RFLP and fluorescencequantitative PCR techniques were chosen to establish a technology system for tracingmeat source of fish products, applying to the different experimental conditions. It has animportant and practical significance for circulation and processing of fish products. Themain research results are as follows:1. The optimization of DNA barcodingCritical steps of DNA barcoding were optimized, such as DNA extraction,universal primers screening, PCR amplification, PCR product purification, DNAsequencing and analysis. It showed that DNA extraction by High Salt Method wassuitable for DNA extraction of most fish products，followed by Kit method and CTABmethod. It could improve the DNA extraction yield of fish products using n-hexane aspretreatment reagent of samples contained more oil. Primers F1/R1, R2/F1or F2/R2was suitable for most fish COI gene amplication by DNA barcoding. Primers F1/R1orHCF1/HCR1was also suitable for species identification of meat from Oysters, pork,chicken, duck and beef. The optimization results of PCR conditions showed that:detection limit of DNA was100ng for fish identification by barcoding. when initialtemplates amount of DNA was added about150ng, the polymerase enzyme additionamount was1U, the universal primer was0.6μL, annealing temperature was50℃,Nonspecific amplification bands of PCR products electrophorese were less and theaverage optical density of main band were the largest. Sequencing success rate could be improved by building PMD-18T vectors for samples that their PCR amplication weresuccessful but DNA Sequencing were failure. DNA barcoding was a simple andeffective molecular technique, suitable for ingredient identification of single animalorigin food that processed mildly.2. Application of DNA barcoding in fish or fish fillets species identification215samples were collected from different supermarkets in Qingdao, includingfrozen fishes（94）, frozen cod fillets（27）, salmon fillets(33), baked fish fillets(25), etc.Then, these samples were identified by DNA barcoding. The results showed that theDNA barcoding was a useful and accurate technique for species identification of mostfishes. Besides, the identification results of thirty different fishes were the same byDNA barcoding and morphology,“Sillago japonica” was mislabeled as "sardines" in thesupermarket. All frozen fish fillets were identified as “codfish”, mainly Gaduschalcogrammus (50%), but there was error label marking that toothfish was labeled assablefish.33samples of salmon fillits’ labels were all correct, there weren’t rainbowtrout posing atlantic salmon. Of the25baked fish fillets,68%labels were not inaccordance with the fish ingredients, and some Lagocephalus lunaris which containedtoxins may lead to food safety incidents after people consumption. Fish ball (with singlemeat ingredient) contained pork or catfish. DNA barcoding was not a useful techniquefor authenticity of commercial fish balls with complex meat components. Besides, DNAbarcode database of fishes was established from two aspects. For one hand,193mesured fish DNA barcoding sequences were converged in the EXCEL spreadsheet,indicating the sample detailed information, method of DNA extraction, primers,amplification conditions, purification of PCR product, DNA sequencing method, etc.For another hand, DNA barcode database of fishes, project name "Fish species ofChina" was established using data collection module of BOLD site.3. Rapid salmons’ species identification methods established based on DNA barcodesTwo pairs of universal primers CO I gene was designed based on the DNAbarcoding and the target fragment size were595bp and844bp, respectively. The resultsshowed that two pairs of primers can be used as universal primers for Salmonids. Then,844bp PCR products of five kinds of salmon were analysis by RFLP. It showed that fivekinds of salmon could be identified to species level. Using Msc I and Hind III restriction endonucleases could be identified the Norwegian Salmon quickly and accurately. Usingthe Msc I, Sal I, Dra I and Ade I (or the Msc I, Sal I, Dra I and Van91I) were able toidentify five kinds of salmons to the species level.In order to identify species of rainbow trout and atlantic salmon rapidly, a methodwas established based on SYBR Green I Real-time PCR combined with melting curveanalysis. In this experiment, CO I was taken as target gene and two pairs of universalprimers for salmons were designed. Then, the annealing temperatures and the ratio oftwo pairs of universal primers were optimized. Meanwhile, stability Test of this methodwas also evaluated. It showed that the salmon species identification was mostsignificantly affected by the ratio of two pairs of universal primers, followed byannealing temperatures. The commercial Atlantic salmon products could be easilydifferentiated from rainbow trout products by melting curve analysis when initialtemplates amount of salmons was added about100ng, the two salmons’ universalprimers concentration ratio was1:1, annealing temperature was50℃. In addition, Therepeatability experiments were well performed(p=0.772),86.29℃for Atlantic salmonand83.50℃for rainbow trout, respectively. Further studies suggested that theestablished method also could be used for identification of Atlantic salmon amongsockeye salmon, white salmon and silver salmon. The results showed that this methodwas simple, low cost, accurate and sensitive with a good reproducibility.