Abstract:

According to the sequence of cytochrome b gene of puffer fish published in GenBank, seven pairs of puffer fishspecific primers were designed with Primer Premier 5.00 version. After PCR screening, the primer HT-1, which could detect the target DNA fragment in eight samples of puffer fish from different aquatic breeding farm in Fujian province, was selected to establish a PCR method for the detection of puffer fish in this work. Furthermore, six key factors affecting PCR including Taq enzyme and template DNA amounts as well as final magnesiumion (Mg2+), dNTPs and primer concentrations were optimized in order to establish optimal PCR system. And the optimal composition of a 20μL PCR system in water consisted of 2μL of 10 × PCR buffer, 1.5 mmol/L MgCl2, 1.0 U Taq DNA polymerase, 300μmol/L dNTPs, 0.2μmol/L primer and 400 ng of template DNA. The thermal program for PCR was as follows: predenaturation at 94 ℃ for 5 min, denaturation at 94 ℃ for 30 sec, annealing at 62 ℃ for 30 s, polymerization at 72 ℃ for 30 s, and after 40 cycles, final polymerization at 72 ℃ for 5 min. The proposed PCR method was found to be specific through the comparison of PCR results amplified from puffer fish DNA and non-puffer fish DNA. The detection limit was 0.1%, and PCR detection rate for 0.1% content of puffer fish samples was 97.5% or more.

Key words: puffer fish, primer screening, PCR detection, optimization