Development of primers for detection of heat-treated cetacean materials in porcine meat and bone meal.

The feed ban introduced after the detection of the first case of bovine spongiform encephalopathy in 2001 in Japan has been modified to allow some of the previously prohibited animal materials to be used in animal feed. Recently, porcine materials were allowed to be used in feed for pigs, poultry, and fish. Materials from other mammals, including whales, remain prohibited. In the absence of a method to detect the prohibited whale materials in porcine materials, there is a possibility that the whale materials are being used for feed for pigs, poultry, and fish. To detect illegal use of whale materials mixed with porcine materials, we have developed PCR primers specific to a group of most cetacean species, using a computer-based method we developed previously. The primer sets were capable of detecting whale meat meal that had been autoclaved at 133 degrees C for up to 20 min. The detection limit of whale material in porcine meat and bone meal was 0.1%.

Development of PCR primers for the detection of porcine DNA in feed using mtATP6 as the target sequence.

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 0.01% and 0.001% 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 official analytical method for feed in Japan.

Risk of introduction of BSE into Japan by the historical importation of live cattle from the United Kingdom.

All cattle imported from the United Kingdom to Japan since 1980 and slaughtered before 2002 were traced (n=33), and the number of cattle that were possibly infected with BSE and entered the animal feed chain was calculated. Because there was no effective system to avoid recycling of the BSE agent via animal feed until the early 1990s, of the 33 cattle imported from the UK into Japan, most probably 7 or 8 were infected and entered the animal feed chain, 2 of which entered the animal feed chain in each of 1992 and 1993. In terms of infectivity, 400-550 cattle oral ID(50) of the BSE agent entered the feed chain in each of these years. The amount of infectivity that entered the feed chain in 1989, 1991 and 1995 was smaller but still substantial, suggesting that the BSE agent might have entered the Japanese feed chain in any of these years.

High GC contents of primer 5'-end increases reaction efficiency in polymerase chain reaction.

Many studies have suggested that regulation of the polymerase chain reaction (PCR) is influenced by several factors. However, the understanding of reaction efficiency factors is not sufficient. Here we propose that high GC contents of primer 5'-end increases reaction efficiency in PCR. Using 71 primers (45 pairs), we analyzed factors that affect reaction efficiency, and statistically tested the correlation between the amplification signals and several factors. As a result, there were significant correlations between the amplification signals and the GC contents in the first 1 approximately 3 bps of primer 5'-end.

Developing PCR primers using a new computer program for detection of multiple animal-derived materials in feed.

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.