食品科学 ›› 2010, Vol. 31 ›› Issue (20): 376-381.doi: 10.7506/spkx1002-6630-201020078

• 分析检测 • 上一篇    下一篇

PCR 方法检测河豚鱼的引物筛选及反应体系优化

陈文炳1,赵 晨1 ,2,邵碧英1,江树勋1,闫 诚1,李寿崧1,林河通2   

  1. 1.福建出入境检验检疫局 2.福建农林大学食品科学学院
  • 收稿日期:2010-06-09 修回日期:2010-09-10 出版日期:2010-10-25 发布日期:2010-12-29
  • 通讯作者: 陈文炳 E-mail:621213wbc@163.com
  • 基金资助:

    国家质量监督检验检疫总局科技项目(2008IK175)

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

CHEN Wen-bing1,ZHAO Chen1,2,SHAO Bi-ying1,JIANG Shu-xun1,YAN Cheng1,LI Shou-song1,LIN He-tong2   

  1. 1. Fujian Entry-exit Inspection and Quarantine Bureau, Fuzhou 350001, China ;
    2. College of Food Science, Fujian Agriculture and Forestry University, Fuzhou 350002, China
  • Received:2010-06-09 Revised:2010-09-10 Online:2010-10-25 Published:2010-12-29
  • Contact: CHEN Wen-bing1 E-mail:621213wbc@163.com

摘要:

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。

关键词: 河豚鱼, 引物筛选, PCR 检测, 优化

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

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