Developing PCR Primers Using a New Computer Program for Detection of Multiple Animal-Derived Materials in Feed

Authors: Shinoda, Naoki1; Kusama, Toyoko1; Yoshida, Tomotaro1; Sugiura, Tatsuki2; Kadowaki, Koh-Ichi3; Onodera, Takashi4; Sugiura, Katsuaki1

Source: Journal of Food Protection®, Number 11, November 2008, pp. 2168-2373 , pp. 2257-2262(6)

Publisher: International Association for Food Protection

Buy & download fulltext article:


Price: $37.00 plus tax (Refund Policy)


To reduce the risk of the bovine spongiform encephalopathy agent being recycled to cattle through animal feed, in October 2001 Japan introduced a feed ban prohibiting the use of animal proteins in feed. PCR identification of feed ingredients is part of the audit program to ensure the proper implementation of the feed ban. For efficient analysis, screening of feed products for materials from multiple species is essential. In our study, we developed a computer program GSPRIMER (http://www. famic. go. jp/ffis/feed/gsprimer/) that facilitates development of PCR primers specific to multiple species. The most important feature of GSPRIMER is its ability to estimate the specificity and homology of a potential primer in incremental steps from the 3 terminal. We analyzed all regions of mitochondrial DNA from the target and nontarget species using GSPRIMER. We designed species-specific primer sets for three animal species (sheep, goats, and swine) and group-specific primer sets for ruminants and animals susceptible to transmissible spongiform encephalopathy. The primers were efficiently screened by the PCR protocol using a mixture of mitochondrial DNA from nontarget species as a template. As a result, one primer set each for sheep and goats, two for swine, and three for a group of ruminant species were developed. The detection limit of one of the ruminant primer sets ranged from 0.05 to 0.01% bovine meat and bone meal and 0.1 pg of bovine DNA. We also successfully applied the primer set to 17 commercial feed samples that were known to be free from ruminant-derived materials. No false-positive results were found.

Document Type: Research Article

Affiliations: 1: Food and Agricultural Materials Inspection Center, 2-1 Shintoshin, Chuo-ku, Saitama-shi, Saitama 330-9731, Japan 2: OSDN Corp., Ebisu Business Tower 13F, 1-19-19 Ebisu, Shibuya-ku, Tokyo 150-0013, Japan 3: Ministry of Agriculture, Forestry and Fisheries, 1-2-1 Kasumigaseki, Chiyoda-ku, Tokyo 100-8950, Japan 4: Department of Molecular Immunology, School of Agricultural and Life Sciences, University of Tokyo, Bunkyo-ku Yayoi 1-1-1, Tokyo 113-8657, Japan

Publication date: November 1, 2008

More about this publication?
  • IAFP members must first sign in on the right to access full text articles of JFP

    First published in 1937, the Journal of Food Protection®, is a refereed monthly publication. Each issue contains scientific research and authoritative review articles reporting on a variety of topics in food science pertaining to food safety and quality. The Journal is internationally recognized as the leading publication in the field of food microbiology with a readership exceeding 11,000 scientists from 70 countries. The Journal of Food Protection® is indexed in Index Medicus, Current Contents, BIOSIS, PubMed, Medline, and many others.

    Print and online subscriptions are available to Members and Institutional subscribers. Online visitors who are not IAFP Members or journal subscribers will be charged on a pay-per-view basis. Information can be obtained by calling +1 800.369.6337; +1 515.276.3344; fax: +1 515.276.8655, E-mail: or Web site:
  • Information for Authors
  • Submit a Paper
  • Subscribe to this Title
  • Membership Information
  • Information for Advertisers
  • ingentaconnect is not responsible for the content or availability of external websites
Related content



Free Content
Free content
New Content
New content
Open Access Content
Open access content
Subscribed Content
Subscribed content
Free Trial Content
Free trial content

Text size:

A | A | A | A
Share this item with others: These icons link to social bookmarking sites where readers can share and discover new web pages. print icon Print this page