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

Ministry of Agriculture, Forestry andFisheries: 1 2 1 Kasumigaseki, Chiyoda-ku, Tokyo 100 8950, Japan; Corresponding authorIn Japan, PCR identification of species-specific, animal group-specific and plant DNA isemployed as part of the audit program to ensure compliance with the feed ban in place for thecontrol of bovine spongiform encephalopathy (BSE). Since October 2001, animal proteins otherthan dairy proteins, egg proteins and gelatin have been prohibited to be used in feed forruminants. Meat-and-bone meal (MBM) derived from poultry, pig and/or fish is allowed to be usedin feed for poultry, pigs and fish. Porcine MBM is permitted in feed for domestic animals otherthan cattle since April 2005. Given the fact that pigs and cattle are the two major sources of MBMin Japan, the identification of porcine DNA with high specificity and sensitivity has becomeincreasingly important to ensure that MBM products are free from ruminant materials. Two PCRprimer sets (PPA8 and PPA6) were newly designed using mtATP8 and mtATP6 as the targetsequences, with relatively short amplification sizes. PPA8 and PPA6 were able to specificallydetect 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/fragmentedduring rendering procedures. The PCR method using these primer sets is registered as the o$cialanalytical method for feed in Japan.Key words: animal feed; meat-and-bone meal; PCR; porcine DNA; primerIntroductionBovine spongiform encephalopathy (BSE) is a pro-gressive and fatal disease that a#ects the central ner-vous system of cattle. BSE was first confirmed in theUnited Kingdom (UK) in 1986, and subsequentlyreported in several other countries in the 1990s. Meat-and-bone meal (MBM) contaminated with a scrapie-likeagent is considered to be the vehicle of BSE infection