Multiplex real-time PCR for the identification and quantification of DNA from duck, pig and chicken in Chinese blood curds

- We determined TIANamp® Genomic DNA Kit (Tiangen Biotech) to extract DNA from blood curd.
- The limit of detection (LOD) of the assay was 0.15 ng for each target species.
- The sensitivity of the method was 1% for each species analyzed.
- Simultaneous species identification was achieved with greater sensitivity.
- The assay could be applied in practice and contribute to the food authenticity.

Food authentication and quality control requires sensitive species-specific identification and quantification of DNA from unknown sources. An accurate and reliable multiplex TaqMan® real-time quantitative PCR (qPCR) method modified from Köppel et al. (2009) based on the specific sites of nuclear DNA has been developed to identify duck, pig and chicken DNA in blood curds. Total DNA in blood curd samples was extracted using three methods: TIANamp® Blood DNA Kit, TIANamp® Genomic DNA Kit and a modified phenol/chloroform extraction method. The concentration and purity of DNA were compared regarding DNA yields and purity. A modified phenol/chloroform extraction method and the TIANamp® Genomic DNA Kit gave improved extraction efficiencies compared with the TIANamp® Blood DNA Kit. The limit of detection (LOD) of the assay was 0.15 ng for each target species (1:103 dilution). Analysis of experimental mixtures demonstrated that the sensitivity of the assay was 1% for each species analyzed. This system proved its accuracy, precision and applicability for the examination of different types of animal blood in blood curd products of the type presently on the market.