Co-amplification and sequencing of a cytochrome b fragment affecting the identification of cattle in PCR-RFLP food authentication studies

Abstract

Food authentication studies based on PCR-RFLP analysis are frequently targeted to well-conserved mitochondrial sequences, such as certain regions of the cytochrome b (cytb) gene. The use of mitochondrial L14841/H15149 universal cytb-targeted primers in PCR-RFLP assays revealed the existence of a complex restriction pattern in three genetically-unrelated Iberian (Northern Spain) cows, this being due to the simultaneous co-amplification of two 359 bp cytb fragments. Microsatellite analysis of 11 bovine-specific loci confirmed no familiar linkage among the animals investigated. Both co-amplification products were successfully separated by specific cleavage with endonucleases RsaI and MvaI, which allowed the recovery of each amplification product, respectively. The new co-amplified cytb fragment described in this study was successfully sequenced, and exhibited a significantly high homology (95.1–99.3% range) with respect to mitochondrial sequences previously described for a Bos indicus specimen and for another two Asian Bos taurus, this underlining that its presence in cattle may be more extended than initially thought. In contrast, the homology with the cytb sequence widely accepted for B. taurus was only 89.6%. The results presented in this work imply that food authentication studies by PCR-RFLP analysis may be complicated in the case of cattle by the co-amplification of two different cytb fragments