Study On Sex Identification Of Fetus By Using Free Fetal DNA In Cow Peripheral Blood

Sex controlling plays an important role in accelerating the breeding speed of excellent female animals and improving the production efficiency of limited trait animal husbandry.Sex identification are generally divided into early embryonic sex identification and early-mid term fetal sex identification.Amniocentesis or chorionic membrane sampling were performed for fetal sex identification during early-mid term,which may cause a certain degree of damage to the mother and fetus.Studies have shown the presence of fetal free DNA in the peripheral blood serum of pregnant cows,so it provides a theoretical basis for the establishment of non-traumatic prenatal fetal sex identification technology by detecting bull-specific genes in fetal free DNA.In the present study,DNA was extracted from plasma or serum of cows at different stages of pregnancy by three extraction methods,and the bull-specific genes（TSPY gene or SRY gene）were detected by PCR,fluorescence quantitative PCR or loop mediated isothermal amplification（LAMP）assay.The obtained results are as following:1.Comparison of different methods for free DNA extracted from cow plasma and serumIn the present study,three methods were used to extract DNA,including chloroform-phenol method,heating method,and commercial plasma/serum free DNA extraction kit method.A total of 70 DNA samples were extracted from pregnant bovine plasma and serum.The results showed that concentrations of free DNA extracted from the serum and plasma by the kit method were 7.67±4.22 ng/μL and 5.91±2.89 ng/μL,respectively,in which the DNA from the serum was significantly higher than that in plasma（P<0.01）;serum and plasma DNA extracted by phenol-chloroform method were 81.80±112.11 ng/μL and 80.00±205.62 ng/μL,respectively,with no significant difference between these two types of samples（P>0.05）;the serum and plasma DNA concentrations obtained by heat extraction were 160.20±52.01 ng/μL and 151.00±78.10 ng/μL,respectively,also with no significant difference in the two samples（P>0.05）.In addition,the purity of free DNA in serum and plasma extracted by the three types of methods were low,the ratio of OD₂₆₀/OD₂₈₀ was only around 1.0,although some of them were between 1.8～2.0.Comparing the above three extraction methods,the chloroform-phenol method takes about 24 h per 10 samples,the heating method takes about 40 min,and the kit method takes about 1 h.2.Detection of fetal-specific TSPY genes and SRY genes in serumThe fetal-specific multi-copy TSPY gene was detected in 60 samples of pregnant bovine serum（the labor result was 30 male and female calves）by ordinary PCR and quantitative PCR methods,respectively.（1）Ordinary PCR assayWhen the DNA samples extracted by heating method were used as templates,the detect results showed that only 5 samples were positive for TSPY genes with a detection rate of 16.7%（5/30）,and the false negative rate is 83.3%（25/30）,while the accuracy rate is 58.3%（35/60）.When the DNAs extracted by phenol-chloroform method were used as templates,9 samples were positive for TSPY gene,the detection rate is 30%（9/30）,the false negative rate is 70%（21/60）,and the accuracy rate is 65%（39/60）.When the DNA extracted by the commercial kit method is used as a template,there are11 samples with positive TSPY genes detected by PCR,with a detection rate of 36.7%（11/30）,and 19 false-negative samples,with a false-negative rate of 63.3%（19/30）and an accuracy rate of 68.3%（41/60）.The above results show that the DNA template extracted from the commercial kit is more suitable for the following identification.It takes about 3 h to finish the sex identification when using the ordinary PCR method,from the beginning of PCR amplification to agarose gel electrophoresis.（2）Real-time quantitative PCR assayFirstly,recombinant plasmid containing the bull TSPY gene was constructed,a standard curve was generated（R²=0.998,E=98.2%）.The dynamic amplification curve shows a good linear relationship when the concentration range is between1.18×10⁸ and 1.18×10³ copies/μL,and the sensitivity is 11.8 copies/μL.Using the DNAs extracted from the commercial kit as templates,28 of the 30 serum samples of pregnant cattle（male calves）were TSPY gene positive,with a detection rate of 93.3%（28/30）,a false-negative rate of 6.7%（2/30）,and an accuracy rate of 96.7%（58/60）.The number of copies of the TSPY gene ranged from 34.55 to 154.53 copies/μL,with an average of 102.91±36.03 copies/μL.The time for fluorescence quantitative PCR detection is about 2 h.（3）LAMP assayLoop-mediated isothermal amplification（LAMP）was used to detect the bull-specific single copy SRY gene in the samples,and the amplification temperature was first optimized.The results showed that the amplification curve was observed at 13 min on the machine when using same amount of template and the reaction temperature set at 63°C.The sensitivity between the LAMP assay and ordinary PCR assay was compared by using the bull genome as the template.The sensitivity of LAMP method was 5×10^-7 ng,while the sensitivity of PCR detection was 5×10^-3 ng.In addition,the SRY gene in 60 samples was further detected by the optimized LAMP method,and the results showed that 37 samples were positive for SRY gene,of which 7 were false positive（37/30）with an accuracy rate of 88.3%（53/60）.Lamp assay requires about 1h.3.Comparison of sensitivity,specificity and accuracy of the three detection methodsThis study further compared the detection results of bull-specific TSPY geneby PCR,real-time q PCR and LAMP in cattle serum collected at different gestational stages.The results showed that TSPY gene was not detected in all 7 seral samples of pregnant cattle（2 samples from cattle with male calves and 5 samples from cattle with female calves）by both PCR and real time q PCR methods during the first 1～2 months of pregnancy,which was detectable using the LAMP method（2/2）with a 100%of accuracy（7/7）.During the 3 to 4 months of pregnancy,TSPY gene was not detected in20 serums（10 samples from cattle with male and female calves,respectively）by the PCR assay,which could be detectable by the real-time q PCR with 100%of accuracy or LAMP assay with a accuracy rate of 85%（17/20,3 samples were false positive）.During 5 to 6 months of pregnancy,the bull-specific gene was detected by using the three methods,the sensitivity of is only 61%（11/18,7 samples are false negative）for PCR method,with an accuracy rate of 78%（26/33）,and the accuracy for real time q PCR assay is 100%（33/33）;for the LAMP assay,the accurate rate was 87%（29/33）with 4 samples false positive.The above results show that peripheral bloods were collected from pregnant cattle by non-traumatic method,and DNA can be extracted from serum and plasma samples by chloroform-phenol method,heating method and kit extraction method.Although the DNA content obtained by the commercial kit is low,but the detection accuracy is higher.The sensitivity of LAMP assay is more sensitive for samples from the first trimester（during the first 1-2 months）and is less time-consuming;while the detecting accuracy of the real time q PCR method is higher for samples from cattle during the 3 to 4 months of pregnancy.This study provides reference data for the establishment of a non-invasive,accurate and rapid method for early fetal sex identification in cattle.