Development and application of a tetra-ARMS PCR assay for detecting indel polymorphisms in the bovine PRNP gene

Insertion/deletion (indel) polymorphisms in the promoter region (23 bp) and intron 1 (12 bp) of the bovine PRNP gene influence gene expression and susceptibility to bovine spongiform encephalopathy (BSE). Conventional detection strategies dependent on DNA sequencing are cumbersome and costly. A tetra-primer amplification refractory mutation system PCR (tetra-ARMS PCR) assay was developed which can enable efficient and cost-effective genotyping of each indel locus. Optimized tetra-ARMS PCR primers and multiplex conditions allowed electrophoretic genotyping of the 23 bp indel via the size and presence of 422 bp, 259 bp, and 193 bp amplicons, whereas the 12 bp indel was genotyped based on the size and presence of 598 bp, 472 bp, and 153 bp fragments. Furthermore, the compatibility of these primer sets was preliminarily investigated, demonstrating the potential for co-amplification in a single-tube multiplex format. Validation against Sanger sequencing using 62 randomly selected cattle-derived retail samples demonstrated complete concordance. This straightforward, specific and cost-effective method requires only conventional PCR instrumentation, thereby establishing a robust and accessible platform for PRNP-assisted breeding and cattle product genotyping.