Research On Molecular Identification Methods Of Common Squid Species In Family Ommastrephidae

Squids are distributed worldwide,including many species of commercial importance.They are often made into varieties of flavor foods and sold out.The catches and consumptions of them increased in recent years.As different kinds of species have extremely different nutrition values,flavours,and market price,the food safety of fish products becomes the focus topic.While many countries have developed relative legislations concerning food adulteration and mislabeling,the judgement and implementation must be based on the rapid and accurate species identification.The rapid identification methods for squid species especially their processed products,however,have not been well developed.In our study,the Ommastrephidae species were the research object,and closely related specimens such as Octopus vulgaris and Sepia pharaonis were used as negative controls.DNA barcoding,quantitative real-time PCR(qPCR),and loop-mediated isothermal amplification(LAMP)technologies were used for identification and analysis of Ommastrephidae species.The developed methods would play an important role in fulfilling labeling regulations and squid fishery control.The main results were as follows:1.The universal primers set was used to amplify the mitochondrial COI fragment of Ommastrephes bartramii,Dosidicus gigas,Illex argentinus,and Todarodes pacificus,and their DNA barcodes(658 bp)were obtained.Aligning with the barcodes of 17 Ommastrephidae species downloaded from BOLD and GenBank database,it could be found that the 3 rd codon is the major region of sequence variation.As sequence variation is not saturated,these barcodes are suitable for phylogenetic analysis.The full-length barcode was divided into 9 sections for preliminary research of DNA mini-barcoding.The statistic analysis based on K2P model showed that average interspecific distances of all barcodes were 18-41 times of intraspecific distances,and there were distinct barcoding gaps in full-length barcode,mini-barcode-204-2 and 102-2.In cluster analysis,phylogenetic trees constructed based on these three barcodes showed that all species fell into reciprocally monophyletic with high bootstrap values.Therefore,DNA barcoding can identify squid species,and mini-barcoding can be used for species identification of highly processed squid products.2.Q-PCR(TaqMan)systems were developed based on mitochondrial gene:the Probe-12S rDNA system was targeting Ommastrephidae species and species-specific systems were targeting four squid species,respectively.After analyzing mitochondrial genes reported in GenBank,the Cytb gene was selected for O.bartramii detection,COI gene for D.gigas and T.pacificus detection,ATPase 6 gene for I.argentinus detection,and 12S rDNA gene for designing Ommastrephidae-specific primers and probe.The results showed that four species-specific TaqMan systems were.able to distinguish target species from others and Probe-12S rDNA system could differentiate Ommastrephidae species from other cephalopods.The amplification efficiencies were calculated to be 95.8-102.6%according to slopes of their standard curves.In DNA mixture samples,the target DNA was amplified efficiently without disturbance.Squid contents in processed products have also been detected,thus our methods meet the needs of rapid detection.3.Mitochondrial COI gene was selected for designing species-specific primers targeting D.gigas.Two sets of LAMP based assays,RealAmp and Visual-LAMP,were developed.As results showed,when incubated at 65 ? for 30 min,only D.gigas DNA could be detected by RealAmp or Visual-LAMP.The absolute detection limit was 10 pg per reaction,and relative detection limit was 0.01%.These methods can be applied to all kinds of processed squid and squid-containing products.Therefore,they provided technical support for labeling regulations and managing squid fishery.