A rapid visual detection method for indel polymorphisms in the bovine PRNP gene based on a duplex MIRA-LFD assay

Insertion/deletion (indel) polymorphisms in the bovine PRNP gene promoter region (23 bp) and intron 1 (12 bp) influence gene expression and susceptibility to bovine spongiform encephalopathy (BSE). Conventional detection methods based on DNA sequencing are cumbersome and time-consuming. This study developed a visual detection method for these two polymorphic loci using multienzyme isothermal rapid amplification (MIRA) combined with lateral flow dipstick (LFD) technology, enabling efficient, low-cost, and rapid analysis. We designed specific MIRA primers and LFD probes for the bovine PRNP gene and optimized the reaction system. This allows the 12 bp and 23 bp indel polymorphisms to be detected visually based on the presence or absence of colored bands on test strips. By employing specific probes for each locus on a single duplex strip, the assay facilitates the simultaneous visual detection and haplotyping of both polymorphic loci. Tests on 62 randomly selected commercial cattle-derived products showed that the genotyping results for both loci were completely consistent with sequencing results. The established method is simple, convenient, highly specific, and easy to operate. It has minimal requirements for laboratory equipment, providing a practical technical platform for PRNP-assisted breeding in cattle and for the genotyping analysis of cattle products.