A highly sensitive and specific tetraplex PCR assay for soybean, poultry, horse and pork species identification in sausages: Development and validation

• The first report of development and validation of Tetraplex PCR assays for horse, soybean, pork and poultry species detection in a single reaction.
• We report a highly sensitive and specific Tetraplex PCR assay as reported in literature.
• We report the developed system was successfully applied to commercial sausages.

A tetraplex PCR assay was developed for a rapid and reliable identification of horse, soybean, poultry, and pork species in sausages simultaneously. The method merges the use of horse (Equus caballus), soybean (Glycine max), poultry (Gallus gallus), and pork (Sus scrofa) specific primers that amplify small fragments (horse; 85 bp, soybean; 100 bp, poultry; 183 bp and pork; 212 bp) of the mitochondrial cyt b, lectin, 12S rRNA and ATPase subunit 6 genes respectively. Good quality DNA was isolated from reference sausage to optimize the assay. Tetraplex analysis of the reference sausage samples showed that the detection limit of the assay was 0.01% for each species. Taken together, all data indicated that this tetraplex PCR assay was a simple, rapid, sensitive, specific, and cost-effective detection method for horse, soybean, poultry, and pork species in commercial sausages.