A decade of using small-to-medium throughput allele discrimination assay to determine prion protein gene ( Prnp ) genotypes in sheep in Slovenia

Classical scrapie is a form of fatal transmissible spongiform encephalopathy affecting sheep and goats that is characterized by accumulation of the misfolded pathogenic form of the protease-resistant prion protein (PrP^Sc) derived from the host-encoded membrane glycoprotein (PrP^c). ² The main etiologic agent ¹⁹ of scrapie is predicted to be PrP^Sc; however, in sheep, the genetic susceptibility determined by the prion protein (PrP) genotype plays an important role in the onset of classical scrapie.^1,10,11 The gene encoding PrP (Prnp) is polymorphic at different amino acid positions, some of which are linked to susceptibility to classical scrapie. ¹⁰ Polymorphisms at codons 136 alanine (A) or valine (V), 154 arginine (R) or histidine (H), and 171 glutamine (Q) or arginine (R) or histidine (H), play a major role in genetic susceptibility to classical scrapie. Sheep with the genotype ARR/ARR are considered the most resistant to scrapie, in contrast to sheep with at least one VRQ allele, which determines susceptibility to scrapie with the shortest incubation period.^1,11 By contrast, sheep showing resistance to classical scrapie are susceptible to atypical scrapie, the susceptibility to which is affected by a polymorphism at codons 141 and 154. ¹⁸ Sheep naturally infected with bovine spongiform encephalopathy (BSE) have never been diagnosed; however, experimentally infected sheep show similar genetic susceptibility to BSE as to classical scrapie, with genotype ARR/ARR as most resistant and ARQ/ARQ as most susceptible to BSE. ⁹

Classical scrapie outbreaks are controlled through the culling of affected animals, movement restrictions of animals from the affected herd, and selective breeding of sheep resistant to classical scrapie. ⁵ At the European Union (EU) level, regulations introduced the requirement for Prnp genotyping of a random sample of sheep. ⁶ The first survey of the Prnp genotype in sheep breeds in all EU member states was launched in 2003. ⁴ Breeding programs in sheep were developed during the period in which the main concern was to prevent the spread of BSE in sheep flocks and to also eradicate classical scrapie in sheep. From 2005 until the end of June 2007, breeding programs were mandatory for EU member states. Thereafter, it was voluntary and has stopped in many EU member states. ⁷

Various methods are available to determine the Prnp genotype, including restriction-fragment length polymorphisms, ⁸ sequencing, ¹² dot-blot hybridization, ¹³ denaturing gradient gel electrophoresis analysis, ¹ TaqMan assay,^3,8,15–17 and temperature gradient gel electrophoresis. ¹⁴ We describe herein the results of our small-to-medium throughput allele discrimination assay as used in our laboratory beginning in 2006 to determine Prnp genotypes in sheep in Slovenia in the genotyping program.

Samples were collected from sheep (35,138 from 239 farms, 2006–2015; Fig. 1) included in the regular Prnp genotyping program, determined by order of the Ministry of Agriculture, Forestry and Food of the Republic of Slovenia; some samples from breeders were also included. Blood samples were collected in tubes containing anticoagulant (dipotassium or tripotassium EDTA or lithium heparin; Vacuette, Greiner Bio-One, Kremsmuenster, Austria). After delivery to the laboratory, samples were stored at 2–8°C for up to 77 d. Muscle samples were collected from dead sheep randomly sampled at the pathology units and stored at −15°C before DNA extraction. Blood samples were used to determine only 3 codons (136, 154, and 171); the additional codon 141 was determined for muscle samples from 2009 (n = 814).

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Figure 1.

Numbers of sheep with Prnp genotypes determined each year (2006–2015; n = 35,138).

Genomic DNA was extracted from 300 μL of whole blood when performed manually (Wizard genomic DNA purification kit, Promega, Madison, WI) or from 50 μL of whole blood for semiautomatic extraction according to the manufacturer’s instructions (ABI Prism 6100 nucleic acid prep station, Applied Biosystems, Foster City, CA). DNA was resuspended in 100 μL or 200 μL of elution solution at an average concentration of 40 ng/μL or 5 ng/μL, respectively. Only the manual procedure was used for DNA extraction from ~3 mm³ of muscle tissue from dead sheep.

Oligonucleotide primers and probes for the discrimination of Prnp alleles 136, 141, 154, and 171 were designed by a commercial service (Assay by Design Service, Applied Biosystems) for the sheep Prnp gene (GenBank accession AJ223072). Some of the sequences were also described by other authors.^{8,13,15–17,20} (Table 1). The annealing of primer and probe sequences to the sheep Prnp gene is shown in Figure 2. Probes were labeled with the fluorescent dye VIC and alternative probes with FAM. Four 0.5-mL tubes were prepared for each genotype to determine the alleles at positions 136 (1 tube), 154 (1 tube), and 171 (2 tubes), and an additional tube was prepared to determine position 141 of Prnp. For each tube, the following reagents were combined (per sample): 3.75 μL of DNase-free water, 5.0 μL of polymerase (TaqMan genotyping master mix or TaqMan universal master mix, Applied Biosystems, Thermo Fisher Scientific, Waltham, MA), and 0.25 μL of prepared mixture of probes and primers for each position (Applied Biosystems). From this mixture, 9 μL for each Prnp position was transferred to a tube or well in a microtiter plate, and 1 μL of DNA sample was added. A standard PCR program was used on a real-time PCR reaction system (ABI PRISM 7000 sequence detection system, Applied Biosystems), with heating for 2 min at 50°C and 10 min at 95°C, followed by 40 cycles of denaturation at 95°C for 15 s, and 1 min elongation at 60°C. When 2 sets of primers and probes produced an increase in the fluorescence signal and ΔCt was larger than 3 cycles, the allele was not considered heterozygous.

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Table 1.

Sets of primers and probes for the determination of the sheep Prnp alleles.

Position 136 (A/V)
Primers	PrP-136F: 5′-GCCTTGGTGGCTACATGCT-3′15,16 PrP-136R: 5′-CGGTCCTCATAGTCATTGCCAAAAT-3′15,16
Probes	PrP-136-Ala-VIC: 5′-CTCATGGCACTTCC 3′ 16 PrP-136-Val-FAM: 5′-CTCATGACACTTCC 3′8,16,20
Position 141 (F/L)
Primers	PrP-141F: 5′-TGGCTACATGCTGGGAAGTG-3′ PrP-141R: 5′-GGTCCTCATAGTCATTGC-3′
Probes	PrP-141-Phe-FAM: 5′-AGCAGGCCTTTTAT-3′ PrP-141-Leu-VIC: 5′-AGCAGGCCTCTTAT-3′
Position 154 (R/H)
Primers	PrP-154 F: 5′-TGGCAATGACTATGAGGACCG-3′17,20 PrP-154 R: 5′-TGGTCTGTAGTACACTTGGTTGGG-3′17,20
Probes	PrP-154-Arg-FAM:5′-ACTATCGTGAAAACAT-317,20 PrP-154- His-VIC: 5′-TACTATCATGAAAACATG-3′8,20
Position 171 (H/Q)
Primers	PrP-171F: 5′-ACCCCAACCAAGTGTACTACAGA-3′ 16 PrP-171R: 5′-GTCATGCACAAAGTTGTTCTGGT-3′
Probes	PrP-171-Gln-VIC: 5′-CCAGTGGATCAGTATAGT-3′ 16 PrP-171-His-FAM: 5′-CCAGTGGATCATTATAGT-3′ 16
Position 171 (R/Q)
Primers	PrP-171F: 5′-ACCCCAACCAAGTGTACTACAGA-3′ 16 PrP171R: 5′-GTCATGCACAAAGTTGTTCTGGT-3′
Probes	PrP-171-Gln-VIC: 5′-CCAGTGGATCAGTATAGT-3′ 16 PrP-171-Arg-FAM: 5′-CAGTGGATCGGTATAGT-3′
Sequencing of 669-bp fragment of Prnp
Primers	SPrP-1: 5′-CATCATGGTGAAAAGCCACATAGGC-3′ 13 T2: 5′-CTGGTACTGGGTGATGCACATTTGC-3′

A = alanine; F = phenylalanine; H = histidine; L = lysine; Prnp = prion protein gene; Q = glutamine; R = arginine; V = valine. Superscript numbers are references.

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Figure 2.

The annealing of primers and labeled probes for codons 136, 141, 154, and 171 on sequences of the Prnp gene (segment of the Ovis aries breed Texel chromosome 13, NCBI reference sequence: NC_019470.2). Positions of primer sequences are in boxes; positions of probes are written in italics and marked in gray. Bold letters indicate positions of polymorphisms in the region of the Prnp gene. Underlined small bold letters are those that are detected in PCRs.

To evaluate the reliability of the assay, direct sequencing of a 669-bp fragment of the Prnp gene was performed. The sequence of the Prnp gene from nucleotide 110 to nucleotide 779 was amplified by PCR in a 13.5-μL reaction volume containing 1 μL of genomic DNA, 11.25 μL of ready-to-use mixture DNA polymerase, salts, magnesium, and dNTPs for PCR amplification (Platinum PCR SuperMix, Thermo Fisher Scientific), and 0.5 μL (10 pmol/μL) of each primer. The following protocol was used: 94°C for 10 min, followed by 35 cycles of 1 min at 95°C, 1 min at 55°C, and 2 min at 72°C. This PCR product was diluted 1:10, and 1 μL of the dilution was used in combination with 32 pmol primers named SPrP-1 ¹³ and T2, respectively, for direct sequencing (BigDye Terminator v1.1 cycle sequencing kit, Thermo Fisher Scientific) according to the manufacturer’s instructions. The reaction, consisting of 25 cycles of 1-min denaturation at 96°C, 20 s annealing at 50°C, and 4 min polymerization at 60°C, was performed on a thermocycler (Biometra, Göttingen, Germany). Sequencing was performed (ABI Prism 310 genetic analyzer, Applied Biosystems) and then analyzed (Sequencing Software Chromas, Technelysium, Brisbane, Australia).

Relative trueness of the procedure was assessed on samples with a known Prnp genotype from proficiency testing (Animal & Plant Health Agency [APHA] Quality Assurance Unit, Sutton Bonington, Great Britain; 10 samples per year; 10 y; n = 100). Repeatability and reproducibility of the procedure was verified on routine blood samples obtained for Prnp genotyping (n = 10). For these, 2 operators performed allelic discrimination assays in triplicate on 2 different days. In addition, results of proficiency testing and routine samples included in the validation were verified by sequencing. Robustness of the procedure was assessed by the inclusion of different operators, different methods for DNA extraction, different times of sample storage in refrigerators (up to 77 d for blood samples), and different matrices of samples (blood from live animals and muscle taken at autopsy).

Allele and genotype frequencies in the Slovenian sheep population were calculated by counting the determined alleles and genotypes (allelic frequencies = twice the frequency of its homozygote plus one-half the sum of the frequencies of all heterozygotes in which it was determined; genotype frequencies = number of animals with a specific genotype divided by the total number of animals). The obtained frequencies were multiplied by 100 and expressed in percentages. The difference between frequencies in years 2006 and 2015 was analyzed using the chi-square test.

Consistent allelic variants of the Prnp gene at positions 136, 141, 154, and 171 were detected in all samples tested in the validation study either from proficiency testing or routine samples (Supplementary Fig. 1) as determined by proficiency testing results and/or results of sequencing. The use of a different form of anticoagulant did not influence the results of the testing procedure, nor did the use of different DNA extraction methods. Reliable results were obtained when DNA was extracted from whole blood samples that were stored in a refrigerator for up to 77 d. By contrast, the quality of DNA from muscle tissue obtained from dead sheep was often not suitable. When DNA from muscle was used as starting material, 8% of the allelic discrimination assays did not produce results, probably because of advanced tissue autolysis.

In our small-scale laboratory (1 full-time and 2 part-time persons), we perform testing of up to 500 blood samples per week. The processing of samples usually starts with DNA extraction at the end of the working day and is completed the next day, although the entire genotyping procedure can be completed in 1 d. The method is very robust with respect to the quality of samples for DNA extraction or choice of DNA extraction method. The results are easy to read and interpret. The method allows for the determination of nucleotides on separate positions of Prnp. To control classical scrapie in sheep, only 3 codons are routinely determined, and if atypical scrapie is in question, the additional codon 141 is included in testing.

The reliability of the method is demonstrated by the complete agreement of results obtained in 10 y of proficiency testing and by direct sequencing of the samples. When considering the critical points of the procedure that could cause errors in the final result, we determined that data entry errors and the accidental exchange of samples are the most important for the testing procedure.

The trends in the number of tested animals per year from 2006 to 2015 and the obtained frequencies of separate Prnp alleles and genotypes per year are shown in Figure 3A and 3B. In 2007 and 2008, there was an increase in the frequency of the ARQ (wild type ⁹ ) allele (p < 0.01), which changed in 2009, and the frequency has significantly decreased (p < 0.01) until the end of 2015. A contrasting trend was observed for the ARR allele showing resistance to classical scrapie. The frequency significantly increased (p < 0.01) from the year 2009. The frequencies of the alleles affecting susceptibility to atypical scrapie did not change significantly during this period. The frequency of phenylalanine (F) at position 141 of Prnp in tested sheep was 0.5% (8 alleles from 1,628 alleles in 814 animals tested in 6 y; from these, 4 alleles were detected in sheep with atypical scrapie), and the frequency of histidine (H) at position 154 was 8.1% in 2015.

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Figure 3.

A. Obtained frequencies of separate Prnp alleles in sheep per year. B. Percentages of genotypes per year obtained from genotyping blood samples from live sheep and muscle samples obtained from randomly sampled dead sheep.

From 2006 to 2008, when the selection of classical scrapie–resistant sheep was first introduced into breeding programs, >5,000 sheep per year were genotyped. Almost all animals of high genetic merit in Slovenia were genotyped during the first 3 y of selection; therefore, from 2009 to 2015, only newly selected animals were genotyped, and the number of tested animals was halved. In 2016, only potential breeding rams (a few hundred) were included in Prnp genotyping in Slovenia in the context of the breeding program.

The frequency of genotype ARR/ARR, which is the most resistant to classical scrapie, and ARR/ARQ have increased significantly (p < 0.01) in tested animals in the 10-y period from 2006 to 2015 (from 6.7 and 27.1% of tested sheep to 12.1 and 32.4%, respectively). By contrast, the frequencies of genotypes ARQ/ARQ and ARQ/VRQ in tested animals decreased significantly (p < 0.01) in this period (from 36.4 and 3.5% to 31.1 and 1.8%, respectively). Genotype VRQ/VRQ, which determines the highest susceptibility to classical scrapie, is poorly represented in sheep in Slovenia; in all years, it was <0.5% of tested animals and has not changed significantly (p < 0.5). On the basis of the known genotypes, culling of susceptible sheep and breeding of resistant sheep was performed within the infected flocks in the frame of classical scrapie eradication. In Slovenia, the national breeding program for resistance to classical scrapie was first implemented in 2006. In the breeding programs, animals are genotyped, and those carrying favorable Prnp alleles for resistance are used as breeding animals in order to increase the frequency of the resistant allele in the next generations. A selection scheme focusing on the improvement of resistance to classical scrapie is possible because the relationship between specific Prnp alleles and resistance to scrapie is strong.^1,10,11 In Slovenia and in some EU countries (Cyprus, France, The Netherlands, United Kingdom) where breeding programs for resistance were implemented and frequency of the resistant alleles increased, the occurrence of classical scrapie in sheep decreased significantly. ⁷ The described method is successfully used in Slovenia for Prnp genotyping as a tool for classical scrapie eradication and selection of resistant sheep in the framework of the national breeding programs.