A Dual Fluorescent Multiprobe Assay for Prion Protein Genotyping in Sheep

Overview

Journal BMC Infect Dis

Publisher Biomed Central

Specialty Infectious Diseases

Date 2005 Mar 17

PMID 15769289

Citations 14

Authors

Mario Van Poucke

Jo Vandesompele

Marc Mattheeuws

Alex Van Zeveren

Luc J Peelman

Affiliations

Soon will be listed here.

Abstract

Background: Scrapie and BSE belong to a group of fatal, transmissible, neurodegenerative diseases called TSE. In order to minimize the risk of natural scrapie and presumed natural BSE in sheep, breeding programmes towards TSE resistance are conducted in many countries based on resistance rendering PRNP polymorphisms at codons 136 (A/V), 154 (R/H) and 171 (R/H/Q). Therefore, a reliable, fast and cost-effective method for routine PRNP genotyping in sheep, applicable in standard equipped molecular genetic laboratories, will be a vital instrument to fulfill the need of genotyping hundreds or thousands of sheep.

Methods: A dual fluorescent multiprobe assay consisting of 2 closed tube PCR reactions containing respectively 4 and 3 dual-labelled fluorescent ASO probes for the detection in real-time of the 7 allelic variants of sheep PRNP mentioned above.

Results: The assay is successfully performed using unpurified DNA as a template for PCR, without any post-PCR manipulations and with semi-automatic determination of the PRNP genotypes. The performance of the assay was confirmed via PCR-RFLP and sequencing in a cross-validation study with 50 sheep.

Conclusions: We report the development and validation of a robust, reliable and reproducible method for PRNP genotyping of a few to many sheep samples in a fast, simple and cost-effective way, applicable in standard equipped molecular genetic laboratories. The described primer/probe design strategy can also be applied for the detection of other polymorphisms or disease causing mutations.

Citing Articles

Identification of a Novel Idiopathic Epilepsy Risk Locus and a Variant in the Gene in the Dutch Partridge Dog.

Beckers E, Bhatti S, Van Poucke M, Polis I, Farnir F, Van Nieuwerburgh F Animals (Basel). 2023; 13(5).

PMID: 36899667 PMC: 10000155. DOI: 10.3390/ani13050810.

A Nonsense Variant in the Gene Causes X-Linked Muscular Dystrophy in the Maine Coon Cat.

Beckers E, Cornelis I, Bhatti S, Smets P, Shelton G, Guo L Animals (Basel). 2022; 12(21).

PMID: 36359052 PMC: 9653713. DOI: 10.3390/ani12212928.

The prevalence of the ABCB1-1Δ variant in a clinical veterinary setting: The risk of not genotyping.

Beckers E, Casselman I, Soudant E, Daminet S, Paepe D, Peelman L PLoS One. 2022; 17(8):e0273706.

PMID: 36037240 PMC: 9423603. DOI: 10.1371/journal.pone.0273706.

qPCR assays with dual-labeled probes for genotyping honey bee variants associated with varroa resistance.

Claeys Bouuaert D, Van Poucke M, Smet L, Verbeke W, de Graaf D, Peelman L BMC Vet Res. 2021; 17(1):179.

PMID: 33931072 PMC: 8086294. DOI: 10.1186/s12917-021-02886-x.

A feline orthologue of the human MYH7 c.5647G>A (p.(Glu1883Lys)) variant causes hypertrophic cardiomyopathy in a Domestic Shorthair cat.

Schipper T, Van Poucke M, Sonck L, Smets P, Ducatelle R, Broeckx B Eur J Hum Genet. 2019; 27(11):1724-1730.

PMID: 31164718 PMC: 6871078. DOI: 10.1038/s41431-019-0431-4.

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