The safe use of cattle feed free from meat and bone meal is an important prerequisite to prevent further spread of bovine spongiform encephalopathy. We designed primers to detect very small amounts of meat and bone meal in ruminant feed. Mitochondrial subunit 8 of the ATP synthase gene was used as a target sequence. PCR-based assays revealed amplification of DNA from mammals, ruminants, and individual species using these primers. The method allowed detection of the presence of meat and bone meal in ruminant feed from 0.1 to 0.01%. Sensitivity and effectiveness of the method for detecting prohibited animal proteins in ruminant feed was evaluated.

Fisheries: 1ῌ2ῌ1 Kasumigaseki, Chiyoda-ku, Tokyo 100ῌ8950, Japan; ῍ Corresponding author In Japan, PCR identification of species-specific, animal group-specific and plant DNA is employed as part of the audit program to ensure compliance with the feed ban in place for the control of bovine spongiform encephalopathy (BSE). Since October 2001, animal proteins other than dairy proteins, egg proteins and gelatin have been prohibited to be used in feed for ruminants. Meat-and-bone meal (MBM) derived from poultry, pig and/or fish is allowed to be used in feed for poultry, pigs and fish. Porcine MBM is permitted in feed for domestic animals other than cattle since April 2005. Given the fact that pigs and cattle are the two major sources of MBM in Japan, the identification of porcine DNA with high specificity and sensitivity has become increasingly important to ensure that MBM products are free from ruminant materials. Two PCR primer sets (PPA8 and PPA6) were newly designed using mtATP8 and mtATP6 as the target sequences, with relatively short amplification sizes. PPA8 and PPA6 were able to specifically detect porcine DNA with the detection limits of 0.01ῌ and 0.001ῌ of porcine MBM in feed, respectively. PPA6 was superior to PPA8 in terms of detection of DNA damaged/fragmented during rendering procedures. The PCR method using these primer sets is registered as the o$cial analytical method for feed in Japan.

The Japanese Government has prohibited the use of seafood protein, as well as mammalian protein, in ruminant feed. There is an o$cial method to detect meat and bone meal, but no method is yet available to detect fishmeal in ruminant feed. We tried to develop a suitable method to detect fishmeal in ruminant feed, similar to the o$cial method "PCR detection of animal-derived DNA in feed". Our previously reported primers (fishcon5 and fishcon3-1) showed low sensitivity, so we designed new primers based on a DNA sequence from yellowfin tuna mitchondrial DNA. Among the primers, FM5 and FM3 specifically detected fish DNA (sardine, yellowfin tuna, skipjack tuna, chub mackerel, Pacific saury, salmon, rainbow trout, Japanese anchovy, codfish and Japanese horse mackerel) from fish meat, and did not amplify DNA from animals and plants. The sensitivity for detection of the presence of fishmeal in ruminant feed was 0.01ῌ0.001ΐ. Q´̯῝̰ῨῢῩῌ ῐῐῩQQῲ´̱ῐῠ̯̮̰ῢQ