PCR 方法检测河豚鱼的引物筛选及反应体系优化

根据Genbank 公布的河豚鱼细胞色素b 基因序列，应用软件Primer Premier 5.00 版设计了7 对引物，经过PCR 筛选，确定可以在所有8 个供试河豚鱼样品中检出目的DNA 片段的引物HT-1，用于建立河豚鱼的PCR 检测方法。对该PCR 方法中6 个因素包括退火温度、Mg2+ 终浓度、Taq DNA 聚合酶用量、dNTPs 终浓度、引物终浓度和模板DNA 用量进行优化，确定优化的PCR 扩增体系：10 × PCR 缓冲液2μL，MgCl2 终浓度1.5mmol/L，Taq DNA 聚合酶1.0U，dNTPs 终浓度300μmol/L，引物终浓度0.2μmol/L，DNA 模板400ng，加纯水至总体积20μL。扩增程序定为94℃预变性5min，94℃变性30s，62℃退火30s，72℃延伸30s，40 个循环，72℃延伸5min。据此建立河豚鱼成分PCR 检测方法，并通过河豚鱼与非河豚鱼的PCR 检测结果比较，验证了该方法的河豚鱼特异性。研究结果还表明，该方法的检出限为0.1%，含量为0.1% 的河豚鱼样品PCR 检出率至少在97.5% 以上。

Primer Screening and Optimization of PCR System for the Detection of Puffer Fish

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.