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.