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

Area of Science:

• Genetics
• Molecular Biology
• Animal Science

Background:

• Insertion/deletion (indel) polymorphisms in the bovine PRNP gene promoter and intron 1 affect gene expression and susceptibility to Bovine Spongiform Encephalopathy (BSE).
• Conventional DNA sequencing for detecting these polymorphisms is laborious and time-consuming.

Purpose of the Study:

• To develop a rapid, visual, and cost-effective method for detecting 12 bp and 23 bp indel polymorphisms in the bovine PRNP gene.
• To enable simultaneous genotyping and haplotyping of these two polymorphic loci.

Main Methods:

• Development of a visual detection assay combining multienzyme isothermal rapid amplification (MIRA) with lateral flow dipstick (LFD) technology.
• Design of specific MIRA primers and LFD probes for the bovine PRNP gene.
• Optimization of the reaction system for visual detection of colored bands on test strips.

Main Results:

• The established MIRA-LFD method allows for visual detection of 12 bp and 23 bp indel polymorphisms in the bovine PRNP gene.
• A single duplex strip enables simultaneous visual detection and haplotyping of both polymorphic loci.
• Genotyping results from 62 cattle products perfectly matched sequencing data, demonstrating high specificity and accuracy.

Conclusions:

• The developed MIRA-LFD assay is simple, convenient, highly specific, and easy to operate with minimal equipment requirements.
• This method provides a practical platform for PRNP-assisted breeding in cattle.
• The assay is valuable for genotyping analysis of cattle products, potentially aiding in BSE risk assessment.