Technical note: Droplet digital PCR as a new molecular method for a simple and reliable diagnosis of freemartinism in cattle

摘要

Freemartinism is the most common type of disorderof sex development in cattle. It leads to sterility in thefemale co-twin in heterosexual twin pregnancy, and isthus a serious problem in cattle production. The incidenceof freemartin syndrome is directly dependenton the prevalence of twinning, which has increased indairy cattle populations in recent years. Thus, earlyand rapid identification of freemartins is needed toreduce economic loss. Of the various methods used todiagnose this condition, identifying the XX and XYcell lines in blood samples using cytogenetic techniquesis the gold standard; however, this technique is timeconsuming. Faster and more reliable techniques arethus being sought. Droplet digital PCR (ddPCR) is athird-generation PCR method and it has not previouslybeen used to detect XX/XY leukocyte chimerism incattle. The aim of the present study was to verify theusefulness of ddPCR to detect and quantify leukocytechimerism in this species. The X and Y copy numberswere estimated by identifying the copy numbers of 2genes located on the sex chromosomes: amelogenin Xlinked(AMELX) on the X chromosome and amelogeninY-linked (AMELY) on the Y chromosome. In the firststep, we performed ddPCR on samples prepared fromfemale DNA mixed with male DNA in serially dilutedproportions. We determined that the sensitivity of thismethod was sufficient to detect a low-frequency (<5%)cell line. In the next step, ddPCR was used to analyze22 Holstein Friesian freemartins. Cytogenetic evaluationof these cases revealed leukocyte chimerism; theproportion of XX and XY metaphase spreads variedover a wide range, from XX (98%)/XY (2%) to XX(4%)/XY (96%). The use of ddPCR facilitated the preciseestimation of the ratio of the copy number of X toY sex chromosomes. In all cases, the XX/XY chimerismdetected by cytogenetic analysis was confirmed usingddPCR. The method turned out to be very simple, accurate,and sensitive. In conclusion, we recommend theddPCR method for fast and reliable detection of XX/XY leukocyte chimerism in cattle.