title: Real-time qPCR is a powerful assay to estimate the 171 R/Q alleles at the PrP locus directly in a flock's raw milk: a comparison with the targeted next-generation sequencing.
abstract: The hazard to human health represented by transmissible spongiform encephalopathies in sheep is one of the major reasons for implementing the genetic selection plan to break down prion diseases. The problem is particularly important because of the risk of disease transmission from ewe to lamb via milk or colostrum. In order to establish an active and convenient monitoring of the flocks already undergone genetic selection and thus, indirectly increase consumers' security, the challenge of the work was quantifying the classical scrapie risk in bulk milk. A new quantitative real-time PCR assay for the estimation of the 171 R and Q allelic frequencies in a DNA pool representative of all the lactating ewes present in a flock was optimized and validated "in field". The repeatability range was 3.69-5.27 for R and 4.20-5.75 for Q. The ruggedness of the allele frequencies resulted 4.26 for R and 4.77 for Q. Regarding the validation "in field", none of the considered sources of variability (flock, month, number of genotyped animals and somatic cell count) showed a significant effect on flock and milk at the linear model. The targeted next-generation sequencing was also tested to evaluate its applicability in this context. Results show that the real-time PCR assay could represent a valid tool for the determination of 171 R/Q allele frequencies in bulk milk. The implementation of a service for breeder self-control along the production chain would aim to increase the production of high-security dairy products, while monitoring over time of the effects of genetic selection in the flocks.
authors: Istituto Sperimentale Italiano "Lazzaro Spallanzani", Località La Quercia, 26027 Rivolta d'Adda, Cremona, Italy. Electronic address: maria.feligini@istitutospallanzani.it.
authors: Maria
authors: M
authors: Feligini
authors: Istituto Sperimentale Italiano "Lazzaro Spallanzani", Località La Quercia, 26027 Rivolta d'Adda, Cremona, Italy.
authors: Graziella
authors: G
authors: Bongioni
authors: Istituto Sperimentale Italiano "Lazzaro Spallanzani", Località La Quercia, 26027 Rivolta d'Adda, Cremona, Italy.
authors: Eva
authors: E
authors: Brambati
authors: Istituto Sperimentale Italiano "Lazzaro Spallanzani", Località La Quercia, 26027 Rivolta d'Adda, Cremona, Italy.
authors: Alessandra
authors: A
authors: Amadesi
authors: Istituto Sperimentale Italiano "Lazzaro Spallanzani", Località La Quercia, 26027 Rivolta d'Adda, Cremona, Italy.
authors: Caterina
authors: C
authors: Cambuli
authors: Istituto Sperimentale Italiano "Lazzaro Spallanzani", Località La Quercia, 26027 Rivolta d'Adda, Cremona, Italy.
authors: Simona
authors: S
authors: Panelli
authors: Istituto Sperimentale Italiano "Lazzaro Spallanzani", Località La Quercia, 26027 Rivolta d'Adda, Cremona, Italy.
authors: Cesare
authors: C
authors: Bonacina
authors: Istituto Sperimentale Italiano "Lazzaro Spallanzani", Località La Quercia, 26027 Rivolta d'Adda, Cremona, Italy.
authors: Andrea
authors: A
authors: Galli
mesh_terms: D000483:Alleles; D019943:Amino Acid Substitution; D000818:Animals; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods; D008894:Milk Proteins / Q000235:genetics*; D017096:Prion Diseases / Q000175:diagnosis*; D011328:Prions / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012756:Sheep; D014730:Veterinary Medicine / Q000379:methods*
chemical_list: D008894:Milk Proteins; D011328:Prions
keywords: Milk; Next-generation sequencing; PRNP gene; Real-time PCR; Scrapie; Sheep