Study On Application Of DNA Markers To Identify Components And Species Of Pufferfish

Fugu (puffer fish), with scientific name of swellfish, is Osteichthyes,Triggerfish Passeriformes, puffer suborder, puffer genus, is a warm water marinebenthic fish. By mistake ingestion or mixed into the processing of aquatic products,the incident of poisoning often happen because of puffer fish containing toxicsubstance, tetrodotoxin (TTX). DNA molecular marker techniques including PCRtechnology and DNA sequencing technology is based on amplification ofspecies-specific gene and its sequence analysis to distinguish the target organismsfrom other species, with so many advantages such as strong specificity, highsensitivity, simple and rapid, it can identify species at the molecular level. In thisstudy, DNA-PCR and sequencing technology were used to identify component andspecies of puffer fish, to provide scientific basis for the rapid identification of pufferfish, toxic and harmful substances mixed artificially into aquatic products.In this study, the DNA of11samples of puffer fish, had been extracted by fourmethods respectively. Four DNA extracting methods were compared by means of fourindexes, including DNA concentration, DNA purity, DNA agarose gel electrophoresis,and PCR amplification. The results proves that the best method is modified extractionmethod for mitochondrial DNA, which DNA extracted can meet the requirement ofPCR amplification.According to the cytochrome b of Lagocephalus gloveri and Lagocephaluswheeleri published in GenBank, seven pairs of pufferfish specificity primers had beendesigned by Primer Premier5.00version and then composed. After the screeningexperiment, the primer HT-1, which could detect target DNA fragments in the wholeeleven samples. And the optimum25μL volume was as follows：5×PCR buffer was5μL, MgCl₂2.0mmol/L, Taq DNA polymerase1.0U, dNTPs200μmol/L, premier0.5μmol/L,sample concentration300ng.The optimal PCR conditions were asfollows：predenaturation at94℃for5min, denaturation at94℃for30s, annealed at62.5℃for30s,extension at72℃for30s, for40cycles, final extension at72℃for5 min, conserved at4℃.Identification process of puffer fish can be described as follows: extract mtDNAof samples firstly, then PCR amplification, finally observe the stripe of PCR productsafter agarose gel electrophoresis. Meanwhile, the limit of detection (LOD) can reach0.1%. Furthermore, the detection rate of G.loveri, L.lagocephalus andT.oblonguscould reach100%,97.5%and100%respectively. And those of other twopuffer fish samples alsocan reach100%, with the LOD level of0.1%.Duplex PCR technology can not only verify the result of PCR test, but alsoeffectively avoid false-positive appearance and improve the accuracy of detection.Therefore, primed HT-1and primed FISH is on the basis of duplex PCR experiment.On the basis of the initial set of PCR amplification system and amplificationprocedures, the optimization experiments were carried out on two pairs of primerconcentration and annealing temperature. The Optimization system of duplex PCRamplification were as follows：5μL5×PCR buffer, MgCl22.0mmol/L, Taq DNApolymerase3.0U, dNTPs200μmol/L, premier final concentration0.4μmol/L, DNAtemplate400ng, the total volume25μL. The optimal PCR conditions were asfollows：predenaturation at94℃for5min, denaturation at94℃for30sec, annealedat59℃for30s,extension at72℃for30s, for40cycles, final extension at72℃for5min, conserved at4℃.In addition, the PCR products of all puffer samples were sequenced, and theanalysis the sequence alignment was carried out. The sequence homology of11samples was analysed by DNAMAN software, to obtain homology coefficient matrixand construct homology tree.11samples were divided into3groups,100%ofhomology rate was found among samples of each group,89%of homology rates wasfound between the first group and the second group, and85%of homology rate wasfound between both of two groups and the third group. The homologous rate analysiswas applied for determiming2unknown species of puffer fish samples belonging tothe taxonomic position.