Research On Three Molecular Methods For Identification Of Common Cod Species In Family Gadidae

Codfish usually refers to the Gadiformes,which contains many important species with high economic value,such as Gadus morhua,G.macrocephalus and Melanogrammus aeglefinus.The consumption of Codfish in China is increasing in recent years,and the prices of different Cod species vary greatly.However,most Codfish are processed into frozen fish fillets or other products without correct scientific name in packaging.So,it is difficult for consumers to distinguish Cod species through the appearance,which causes the adulteration phenomenon in Cod market.Therefore,it is urgent to establish rapid,accurate,simple and practical methods for the identification of commercial Cod products.In this study,the Gadidae species were choosed as the subject of research,and two non-Gadidae species(Dissostichus eleginoides and Lepidocybium flavobrunneum)were choosed as negative controls.Fluorescence quantitative polymerase chain reaction(qPCR)system,loop-mediated isothermal amplification(LAMP)technology and colorimetric detection based on gold nanoparticles(AuNPs)were used for the analysis and identification of Gadidae species.The mainly results were as follows:1.A qPCR assay was established for analysis and identification of Gadidae family and three Cod species.16 S rDNA gene was used to design primers to distinguish the Gadidae and non-Gadidae species.The mitochondrial Cytb gene was selected for discrimination of G.morhua,G.macrocephalus,M.aeglefinus,D.eleginoides and L.flavobrunneum.The detection result showed that 16 S rDNA qPCR system and five species-specific qPCR systems performed well with a high specificity,and could distinguish the target species from others.Using the standard curves constructed for each gene,the content of target Cod species could be quantitatively detected in single or mixed samples.In the species-specific qPCR systems,the absolute limit of detection of G.morhua,G.macrocephalus,M.aeglefinus,D.eleginoides and L.flavobrunneum were 3.8?4.7?3.45?4.65 and 4.35 pg/?L.And the relative detection limit(LODr)reached 0.01% in simulated mixture samples.By the detection of 20 commercial Cod products,the Cod species could be detected in raw materials and deep-processedproducts as well.The positive results detected by the species-specific qPCR systems were consistent with the results obtained by DNA sequencing.Thus,the qPCR systems developed in this study have strong practicability and could meet the needs of routine testing.2.Two LAMP assays(fluorescence LAMP and visual LAMP)were established for the identification of three Cod species in Gadidae(G.morhua,G.macrocephalus and M.aeglefinus).12 S rDNA gene was used to design primers to distinguish the Gadidae and non-Gadidae species,and the mitochondrial Cytb gene was selected for discrimination of three Cod species.The result showed that 12 S rDNA LAMP system could distinguish the Gadidae species form others easily.The species-specific LAMP systems of three Gadidae species also performed well.In the species-specific LAMP systems,the absolute limit of detection(LODa)of G.morhua,G.macrocephalus and M.aeglefinus were 285,37 and 197pg/?L,and the relative limit of detection(LODr)reached to 1%,0.1% and 1%,respectively.In the 12 S rDNA LAMP system,the LODa of three Cod species were 28.5,37 and 19.7 pg/?L,respectively,and the LODr reached to 0.1%.In the detection of commercial Cod products,the two LAMP assays established in this study were also applicable to the species detection of Cod and deep-processed Cod products,which provided a rapid and effective technology for Cod market management in future.3.A rapid colorimetric detection system based on AuNPs and specific probe was developed for detection of Gadidae species with nake eyes.12 S rDNA gene was used to design specific probe to distinguish the Gadidae and non-Gadidae species.Three Cod species from Gadidae family(G.morhua,G.macrocephalus and M.aeglefinus)were selected as the target species to optimize the reaction condition of the 12 S rDNA-AuNPs-probe system.The results indicated that the established system has a high specificity.The detection limits of G.morhua,G.macrocephalus and M.aeglefinus were 2.2,3.15 and 2.65 ng/?L,respectively.Further more,20 commercial fish samples were detected to verify the assays.The colorimetric results of all samples with AuNPs assays were coherent with the results obtained by conventional PCR,which indicated that the reliability of the AuNPs assays could meet the requirements of species identification in commercial products.In addition,the whole reaction process and results observation could be completed within 30 min,which could be adopted as a new method for on-site rapid detection of processed Cod and Cod-containing products.