Detection Of Fetal Free-DNA From Maternal Peripheral Blood Of Bovine And The Application Of Fetal DNA In Sex Identification

Objective: Fetal free-DNA can be detected from the maternal peripheral blood In pregnant women, which can be used for noninvasive prenatal diagnose. There were no studies were been done in other mammal. In our study, we detected the fetal free-DNA in Peripheral blood of maternal cow at sometime, and determining when it is arisen, if exist. Moreover, try to make use of it for noninvasive prenatal sex identification.Methods: Fifty blood samples from pregnant bovine during gestational weeks 9-38 were investigated, including thirteen early pregnant bovine , sixteen medium-term pregnant bovine and twenty one late pregnant bovine. Three pairs of nested PCR primers(six pairs of primers) was designed with Primer premier 5.0 in the 3'end of SRY gene, on the HMG-box sequence and the sequence which include HMG-box core sequence. Peripheral blood of bovine in difference pregnancy period were collected, gain plasma and serum after the blood were treated with. Cell-free fetal DNA was extracted from maternal plasma or serum with four different methods (blood DNA mini kits , normal phenol/chloroform, heated up , template directly with plasma/serum), The nested PCR that detects fetal free DNA from peripheral blood Possesses the advantages of sensitivity, specificity and veracity. The effect was achieved under the ultraviolet transmission instrument.Results: The fetus in early pregnant maternal are still not born. The detect result in plasma accord with the actual sex in middle pregnancy , late pregnancy is 87.5% (14/16) , 95.24%(20/21,respectively; but in serum, the result is75.0%( 12/16),90.48%( 19/21 ),The total identify ratio in plasma and in serum is 91.89% (34/37) and 83.87% (31/37); The template DNA was extracted with different methods, the test ratio is different, the highest exact ratio is 91.89%(34/37) , the template DNA is used in this methods which was extracted with blood mini DNA extracted kits, the second is 83.87% (31/37) ,heating-up methods was applied in this test, the lowest ratio is 75.68% (28/37) ,which applied normal phenol/chloroform means. There was nothing achieved when directly amplified with plasma/serum. The outcome is homology with three different pairs of primers, but the primer on the HMG-box sequence is conservative, which can also be used to amplify the SRY gene of mouse.Conclusion: 1. Fetal free DNA can be detected in pregnant bovine's plasma and Serum, which can be used for noninvasive prenatal sex identification; 2. in this test, the first appearance of fetal free-DNA in maternal is about three months after pregnant; 3. The sex identify ratio which use...