A dual fluorescent multiprobe assay for prion protein genotyping in sheep.
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.
Van Poucke
Mario
M
Department of Animal Genetics and Breeding, Faculty of Veterinary Medicine, Ghent University, Heidestraat 19, B-9820 Merelbeke, Belgium. Mario.VanPoucke@UGent.be
Vandesompele
Jo
J
Mattheeuws
Marc
M
Van Zeveren
Alex
A
Peelman
Luc J
LJ
D000483:Alleles; D000818:Animals; D005456:Fluorescent Dyes / Q000032:analysis*; D014644:Genetic Variation; D005838:Genotype; D017096:Prion Diseases / Q000175:diagnosis / Q000662:veterinary*; D011328:Prions / Q000032:analysis* / Q000235:genetics*; D012757:Sheep Diseases / Q000175:diagnosis*
D005456:Fluorescent Dyes; D011328:Prions