| Puffer fish, with scientific name of swellfish, is a kind of toxic but delicious fish which related to our diet culture and history closely, it is welcome by the domestic and foreign consumers because of its delicious taste and high nutritional value. In our country, puffer fish industry has formed a certain scale since 1990, it’s rise and fall has a close relationship with the interests of the practitioners and the stability of the aquaculture industry. In recent years, this industry is facing the challenge of trade technical barriers at export sale and the opportunity of rapid development and prosperity at domestic sale. In this case, it is imminent to build an advanced and completedetection method. At present, the puffer fish detection is mainly through the detection of tetrodotoxin (TTX), but this methods have certain degree flaws, and it can not be obtained a wide range of application and development. Meanwhile, the PCR technology can be used to detect specificial DNA fragment as molecular marker for distinguishing the target organisms and other species, so species identification can be done at the molecular level by PCR. In this paper, the PCR used in the detection of puffer fish and will open a new way of thinking in direct detection of puffer fish.In this study,9 kinds of puffer fish were used as samples. Based on reference, 5 normal methods were used to extract DNA respectively. A better DNA extraction method was picked out based on the result ofDNA concentration, DNA purity and DNA agarose gel electrophoresis. A better DNA extraction method was optimized based on the 3 primary factors that influence DNA extraction process, and the optimum method was ascertained ultimately.Using optimization technology, the annealing temperature, primer concentration and DNA template concentration were inspected in order to improve the specificity and stability of the PCR detection, finally the amplification system and amplification process parameters of ordinary PCR and SYBR-PCR were determined, and the sensitivity and detection limit of the both methods were compared. The results show that they are all the same no matter ordinary PCR or SYBR-PCR. Comparedthe operation efficiency and relationship to operator’s health, SYBR-PCR is more suiTab for modern inspection and quarantine. The SYBR-PCR amplification system were as follows:SYBR Premix Ex TaqTM (2×) was 10 μL, primer (10 μM) was 0.4 μL, DNA template concentration was 150 ng, H2O was 7.2 μL. The optimal SYBR-PCR amplification conditions were as follows:predenaturation at 95 ℃ for 30 s, denaturation at 95 ℃for 5 s, extension at 66 ℃for 34 s, for 40 cycles, and melting curve analysis program were as follows:95℃ for 15 s,60℃ for 1 min,95 ℃for 15s.In addition, the PCR products of every puffer fish had been sequenced; analysis the sequence alignment and enzyme digestion sites by DNAMAN and NEBcutter 2.0; select out three kinds of restriction enzymes that can identify all amplification sequence of every puffer fish to do enzyme digestion, then NmeAⅢ was choosen to overcome the interruption of false positive cased byPCR products of Lateolabrax japonicas and Thunnus albacores. The enzyme maps and digested fragments have obvious difference with the puffer fish. At last NmeAⅢ was selected as validation test ofPCR to avoid false positive and false detection. The results of this parper not only provided technical support for establishing a detecting method and national standard of puffer fish, but also laid a foundation for future similar systems. |
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