A pooled testing system to rapidly identify cattle carrying the elite controller BoLA-DRB3*009:02 haplotype against bovine leukemia virus infection

Abstract

As genetically resistant individuals, the "elite controllers" (ECs) of human immunodeficiency virus infection have been focused on as the keys to developing further functional treatments in medicine. In the livestock production field, identifying the ECs of bovine leukemia virus (BLV) infection in cattle is desired to stop BLV transmission chains on farms. Cattle carrying the bovine leukocyte antigen (BoLA)-DRB3*009:02 allele (DRB3*009:02) have a strong possibility of being BLV ECs. Most of cattle carrying this allele maintain undetectable BLV proviral loads and do not shed virus even when infected. BLV ECs can act as transmission barriers when placed between uninfected and infected cattle in a barn. To identify cattle carrying DRB3*009:02 in large populations more easily, we developed a pooled testing system. It employs a highly sensitive, specific real-time PCR assay and TaqMan MGB probes (DRB3*009:02-TaqMan assay). Using this system, we determined the percentage of DRB3*009:02-carrying cattle on Kyushu Island, Japan. Our pooled testing system detected cattle carrying the DRB3*009:02 allele from a DNA pool containing one DRB3*009:02-positive animal and 29 cattle with other alleles. Its capacity is sufficient for herd-level screening for DRB3*009:02-carrying cattle. The DRB3*009:02-TaqMan assay showed high-discriminative sensitivity and specificity toward DRB3*009:02, making it suitable for identifying DRB3*009:02-carrying cattle in post-screening tests on individuals. We determined that the percentage of DRB3*009:02-carrying cattle in Kyushu Island was 10.56%. With its ease of use and reliable detection, this new method strengthens the laboratory typing for DRB3*009:02-carrying cattle. Thus, our findings support the use of BLV ECs in the field.

Keywords: BoLA-DRB3*009:02; TaqMan assay; allele-specific PCR; bovine leukemia virus; elite controllers; pooled samples.

FIGURE 1

Primer and probe design for discriminating DRB3*009:02 from other alleles. (A) Schematic diagram showing the discrimination strategy used for DRB3*009:02. The efficiency of HiDi Taq DNA polymerase decreased dramatically when the 3′ terminal nucleotides in the primers and sample DNA differed. We therefore selected the 71st G for the target 3′ terminal nucleotide in the forward primer and 245th T for the target 3′ terminal nucleotide in the reverse primer. From 357 alleles, eight alleles including DRB3*009:02 were amplifiable by this primer. By merging the probe's discrimination ability, only DRB3*009:02 and DRB3*163:01 generated positive signals. (B) The primer and probe positions in the DRB3*009:02 sequence are shown. Gray highlight indicates the primer sites and underlining indicates the probe sites. The 3′ terminal nucleotides in the primers are indicated in red. (C) Sequences in the probe's site for eight alleles that were amplified by the primers are shown. Gray highlight indicates the different nucleotides in DRB3*009:02

FIGURE 2

Performance of the designed primer set in cPCR. Electrophoresis results from cPCR using the designed primer set. 1: Genomic DNA from cattle heterozygous for DRB3*009:02 and 015:01. 2: Plasmid DNA encoding DRB3*009:01. 3: Genomic DNA from cattle heterozygous for DRB3*034:01 and 005:03. 4: Genomic DNA from cattle heterozygous for DRB3*001:01 and 14:01:01. 5: Negative control (PCR‐grade water)

FIGURE 3

Discriminating DRB3*009:02 from 009:01 using the DRB3*009:02‐TaqMan assay. Amplification plot for the 50 ng samples of genomic DNA from cattle heterozygous for DRB3*009:02 and 015:01 and for 100 ng of plasmid DNA encoding the DRB3*009:01 sequence. Only genomic DNA from the cattle heterozygous for DRB3*009:02 and 015:01 produced positive signals

FIGURE 4

Performance of the DRB3*009:02‐TaqMan assay versus the SYBR Green assay. Amplification plots for 13 DRB3*009:02‐positive samples and 51 other samples. (A) DRB3*009:02‐TaqMan assay. (B) SYBR Green assay. (C) Post‐PCR melting curve for the samples obtained after PCR of (B). Red and blue lines in (A)–(C) indicate samples with DRB3*009:02 and other alleles, respectively. (D) Box and scatter plot of the Tm values from the melting peaks from the SYBR Green assay for 17 DRB3*009:02‐positive samples versus 133 others. The Y‐axis indicates the Tm and the gray points indicate each sample. Red and blue bars indicate the first and second trials, respectively. The black bars in the boxes indicate the median Tm values

FIGURE 5

Performance of the DRB3*009:02‐TaqMan assay at detecting DRB3*009:02‐carrying cattle DNA from pooled DNA. Amplification plots for the 500, 250, 100, 50, 10, and 1 ng of DRB3*009:02‐containing DNA pool and DRB3*009:02‐NOT‐containing DNA pool are shown. Red and blue lines indicate the DRB3*009:02‐containing DNA pool and DRB3*009:02‐NOT‐containing DNA pool, respectively. The blank area underneath the DNA amounts in the reaction mixtures indicates the amount of DNA used for detecting DRB3*009:02. Ct values above 0.15 ΔRn in the DRB3*009:02‐containing DNA pool were 33.85 (500 ng), 33.66 (250 ng), 34.82 (100 ng), 35.91 (50 ng), and 37.74 (10 ng); 1 ng = not detected