我们通过PCR评估了从母体血液样品中提取cffDNA的可行性,以评估孕牛胎儿性别鉴定所需的阈值浓度。在四次审判中,我们1)比较了七种方法的提取效率,这些方法使用携带雄性fetii(妊娠150-240d)牛牙釉素(bAML)和Y特异性基因序列的牛的新鲜血浆/血液,2)确定了用于胎儿性别鉴定的PCR所需的掺入cffDNA的最小量,3)确定了三种商业试剂盒中的最佳方案,用于从纯血浆和加标血浆样品(妊娠181-240d)中提取cffDNA,以进行Y特异性序列的PCR检测;4)在不同阶段的怀孕母牛上测试了Y特异性序列PCR妊娠(60-150对151-240d怀孕)。在这些实验中,来自未饲养奶牛母牛的血液样本(加拿大荷斯坦,n=10),怀孕的奶牛(加拿大荷斯坦,妊娠60-240d,n=25与男性的fetii),流产肉牛(安格斯十字架,n=5,怀孕100-150天)用于DNA提取,尖峰,和PCR。在所有试验中,从未繁殖的小母牛(n=5)和小牛(n=5)的血液样本中提取的DNA作为对照。在第一次审判中,DNeasy血液和组织,QiagenDSP病毒,在从新鲜收获的孕妇血浆/血液中分离cffDNA的七种方法中,NucleoMagcfDNA分离试剂盒相对成功。在试验2中,使用连续稀释的来自牛血浆样品中掺入的雄性ftii的cffDNA,当血浆中掺入的cffDNA浓度>31.3pg/ml和>2ng/ml时,观察到通过PCR靶向Y特异性序列和bAML基因的阳性和明确检测,分别。在第三次审判中,DNeasy血液和组织试剂盒最成功地提取了母体血浆中最低浓度的cffDNA,随后进行了Y特异性序列的PCR。在我们的第四次审判中,在妊娠后半期(9/10)显示Y特异性PCR阳性结果的奶牛多于前半期(3/9,Fischer精确检验;P<0.05,90%;CI:55.5-99.75vs33%;CI:7.5-70.1)。总之,我们在产前性别鉴定的PCR中观察到不同DNA提取方法和妊娠阶段结果之间的差异。因此,目前的PCR方法对于检测怀孕母牛的cffDNA是不可靠的。此外,对于Y特异性PCR和bAMLPCR,牛母体血浆中需要≥10(≥31.3pg/mlcffDNA)和≥648(≥2ng/mlcffDNA)个完整胎儿基因组拷贝,分别。
We evaluated the feasibility of
cffDNA extraction from the maternal blood samples regarding the threshold concentrations required for fetal sexing in pregnant cattle by PCR. In four trials, we 1) compared the extraction efficiency of seven methods using freshly harvested plasma/blood of cows carrying male fetii (150-240 d gestation) bovine amelogenin (bAML) and Y-specific gene sequences, 2) identified the minimum amounts of spiked
cffDNA needed for a PCR for fetal sexing, 3) determined the most optimal protocol among three commercial kits for cffDNA extraction from neat and spiked plasma samples (181-240 d gestation) for PCR detection of Y-specific sequence and 4) tested Y-specific sequence PCR on pregnant cows at different stages of gestation (60-150 versus 151-240 d pregnant). In these experiments, blood samples from unbred dairy heifers (Canadian Holstein, n = 10), pregnant dairy cows (Canadian Holstein, 60-240 d gestation, n = 25 with male fetii), and aborting beef cows (Angus cross, n = 5, 100-150 d pregnant) were used for DNA extraction, spiking, and PCR. Extracted DNA from the blood samples of unbred heifers (n = 5) and bull calves (n = 5) served as controls in all trials. In the first trial, DNeasy Blood and Tissue, Qiagen DSP Virus, and NucleoMag cfDNA isolation kits were relatively successful among seven methods to isolate cffDNA from freshly harvested maternal plasma/blood of pregnant cows. In trial 2, using serial dilutions of
cffDNA from male fetii spiked in cow plasma samples, a positive and unambiguous detection by PCR targeting Y-specific sequence and bAML gene was observed when spiked cffDNA concentration in plasma was >31.3 pg/ml and >2 ng/ml, respectively. In the third trial, the DNeasy Blood and Tissue kit was most successful in extracting cffDNA spiked at the minimum concentrations in maternal plasma and subsequent PCR for Y-specific sequence. In our fourth trial, more cows in the second half (9/10) of gestation showed a positive Y-specific PCR outcome than those in the first half (3/9, Fischer\'s exact test; P < 0.05, 90%; CI: 55.5-99.75 vs 33%; CI: 7.5-70.1). In conclusion, we observed variability between different DNA extraction methodologies and stages of gestation results in the PCR for prenatal sexing. Thus, the current PCR methodologies are unreliable for detecting
cffDNA in pregnant cows. Additionally, ≥10 (≥31.3 pg/ml of
cffDNA) and ≥648 (≥2 ng/ml of
cffDNA) copies of the whole fetal genome in bovine maternal plasma are needed for Y-specific PCR and bAML PCR, respectively.