猪源性成分检测中3种DNA提取方法比较

王萍,乔勇升,韩芷玲

(江苏省泰州市食品药品检验所，江苏泰州225300)

摘要：为了达到对动物源性成分快速、准确的检定，对提取动物源性基因组DNA的方法进行了比较分析。考察酚-三氯甲烷法、聚乙烯吡咯烷酮法(PVP法)和试剂盒法这3种方法对DNA提取的浓度、纯度及实时荧光PCR扩增效果的影响。结果表明：酚-三氯甲烷法提取DNA浓度最高，试剂盒法纯度最高且实时荧光PCR扩增效果最好，但PVP法操作简单、安全、经济，提取的DNA用于实时荧光PCR扩增检出限可达1.98 pg/μL。使用PVP法对市场上10份不同类型的动物性产品进行了检测，检出两份产品中含有猪源性成分。PVP法应是3种方法中的首选方法，本文为实验室选择合适的DNA提取方法提供了依据。

关键词：猪源性；DNA提取；酚-三氯甲烷法；PVP法；试剂盒法；实时荧光PCR

doi:10.3969/j.issn.1672-3678.2015.06.011

收稿日期：2015-03-24

作者简介：王萍(1983—)，女，江苏泰州人，工程师，硕士，研究方向：食品检测，E-mail:wingping02091111@126.com

中图分类号：TS207.3

文献标志码：A

文章编号：1672-3678(2015)06-0061-04

Abstract：In order to detect animal-derived material rapidly and accurately,we compared and analyzed the DNA extraction methods from the animal-derived material. We investigated the effect of three methods (phenol-chloroform method,polyvinylpyrrolidone method(PVP method) and reagent kit method) on the concentration of the extracted DNA,the purity of the extracted DNA and the result of the real-time fluorescent PCR amplification. The DNA concentration of the phenol-chloroform DNA extraction method was the highest,the purity of reagent kit was the highest and the effect of the real-time fluorescent PCR amplification was the best,but the PVP method was convenient,safe and economical,the detection limit of the real-time fluorescent PCR amplification was 1.98 pg/μL. Ten different types of animal products in the market were tested with PVP method,two products were detected to contain the porcine-derived material. The PVP method should be the preferred of the three methods. The assay provided a basis for the laboratory to choose a suitable DNA extraction method.

Keywords：porcine-derived; DNA extraction; phenol-chloroform method; PVP method; reagent kit method; real-time fluorescent PCR

Comparison of three DNA extraction methods in detection of porcine-derived material

WANG Ping,QIAO Yongsheng,HAN Zhiling

(Jiangsu Taizhou Institute for Food and Drug Control,Taizhou 225300,China)

食品安全关系着人类身体健康，是全社会关注的热点问题。近年来肉制品掺假屡禁不止，肉制品以次充好，引起人们对食品安全的恐慌，这就要求食品监管部门能够对动物源性成分进行快速、准确的检验和鉴定。

用于动物源性成分的鉴定主要有物理、化学、免疫学和分子生物学等方法。其中，以检测DNA为基础的实时荧光定量PCR技术具有特异性强、灵敏度高、定量准确、自动化程度高等优点，被广泛用于分子生物学和医学研究等领域。DNA模板质量对荧光定量PCR很关键，在提取过程中若是处理不当，残留的一些有机物以及其他一些杂物就会抑制PCR的进行，从而造成假阴性，影响后续操作的进行；……