塔里木马鹿胎儿性别鉴定
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摘要
为建立一种简单、准确的塔里木马鹿早期胎儿性别鉴定新方法,本研究利用妊娠马鹿血浆中胎儿游离的DNA为模板,通过常规PCR扩增牙釉蛋白特异序列,对89头妊娠12-14周龄塔里木马鹿进行胎儿性别鉴定,鉴定结果与实际产犊性别进行比较。结果表明:成功从X染色体上扩增出282 bp的片段,从Y染色体上扩增出252 bp的特异性片段;89头妊娠母鹿胎儿检测结果显示,50头为公,39头为母,与实际产仔性别比对后,鉴定准确率为91.01%。由此可知,利用胎儿游离DNA和牙釉蛋白基因对塔里木马鹿进行早期胎儿性别鉴定,方法简单且准确率高。
To establish a simple and accurate method for the sex identification of the Cervus elapghus yarkandensis early fetal.The free DNA of fetal fetus as a template was used to amplify the enamel protein specific sequence by routine PCR, and then to identify the sex of 89 pregnant Cervus elaphus yarkandensis fetuses with 12-14 weeks of age, finally the identification results and actual calving sex were compared in this study. The results showed that the 282 bp fragment was successfully amplified from X chromosome and 252 bp fragment was successfully amplified from Y chromosome; The fetal sex identification results of 89 pregnant Cervus elapghus showed that 50 Cervus elapghus were male and 39 were female. The identification accuracy was 91.01%, compared with the actual calving sex. Therefore, the plasma free DNA as the template, and the amelogenin gene can be used to identify the fetal sex, and the method is simple and high accuracy.
To establish a simple and accurate method for the sex identification of the Cervus elapghus yarkandensis early fetal.The free DNA of fetal fetus as a template was used to amplify the enamel protein specific sequence by routine PCR, and then to identify the sex of 89 pregnant Cervus elaphus yarkandensis fetuses with 12-14 weeks of age, finally the identification results and actual calving sex were compared in this study. The results showed that the 282 bp fragment was successfully amplified from X chromosome and 252 bp fragment was successfully amplified from Y chromosome; The fetal sex identification results of 89 pregnant Cervus elapghus showed that 50 Cervus elapghus were male and 39 were female. The identification accuracy was 91.01%, compared with the actual calving sex. Therefore, the plasma free DNA as the template, and the amelogenin gene can be used to identify the fetal sex, and the method is simple and high accuracy.
引文
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[18]蒋立斌.绵羊外周血中游离DNA应用于胎儿性别鉴定以及基因分型的初步研究[D].石河子:石河子大学,2014:5-10.
[19]Chan K C,Zhang A B,Hui N,et al.Size distributions of maternal and fetal DNA in maternal plasma[J].Clin Chem,2004,50(1):88-92.
[2]Chrenek P,Boulanger L,Heyman Y,et al.Sexing and multiple genotype analysis from a single cell of bovine embryo[J].Theriogenology,2001,55(5):1071-1081.
[3]Gibson C W,Golub E E,Abrams W R,et al.Bovine amelogenin message heterogeneity:alternative splicing and Y-chromosomal gene transcription[J].Biochemistry,1992,31(35):8384-8388.
[4]Sullivan K M,Mannucci A,Kimpton C P,et al.A rapid and quantitative DNA sex test:fluorescence-based PCR analysis of X-Y homologous gene amelogenin[J].Biotechniques,1993,15(4):636-638,640-641.
[5]Buel E,Wang G,Schwartz M.PCR amplification of animal DNA with human X-Y amelogenin primers used in gender determination[J].J Forensic Sci,1995,40(4):641-644.
[6]Chen C,Hu C L,Wang C H,et al.Gender determination in single bovine blastomeres by polymerase chain reaction amplification of sex-specific polymorphic fragments in the amelogenin gene[J].Mol Reprod Dev,1999,54(3):209-214.
[7]Zeleny R,Bernreuther A,Schimmel H,et al.Evaluation of PCR-based beef sexing methods[J].J Agric Food Chem,2002,50(15):4169-4175.
[8]Sembon S,Suzuki S,Fuchimoto D,et al.Sex identification of pigs using polymerase chain reaction amplification of the amelogenin gene[J].Zygote,2008,16(4):327-332.
[9]Hasegaw T,Sato F,Ishida N,et al.Sex determination by simultaneous amplification of equine SRY and amelogenin genes[J].J Vet Med Sci,2000,62(10):1109-10.
[10]Pfeiffer I,Brenig B.X-and Y-chromosome specific variants of the amelogenin gene allow sex determination in sheep(Ovis aries)and European red deer(Cervus elaphus)[J].BMC Genet,2005,6(1):16.
[11]Zhu B,Sun Q W,Lu Y C,et al.Prenatal fetal sex diagnosis by detecting amelogenin gene in maternal plasma[J].Prenat Diagn,2005,25(7):577-81.
[12]Gurgul A,Radko A,Slota E.Characteristics of X-and Ychromosome specific regions of the amelogenin gene and a PCR-based method for sex identification in red deer(Cervus elaphus)[J].Mol Biol Rep,2010,37(6):2915-2918.
[13]杨麒巍,于杉,张桂珍.孕妇外周血中游离胎儿DNA富集与分离方法及在无创性产前诊断中的应用[J].中国实验诊断学,2014,18(2):345-348.
[14]幸宇云,陈从英,黄路生,等.应用牙釉蛋白(AML)基因鉴别牛早期胎儿性别[J].农业生物技术学报,2005,13(6):759-762.Xing Y Y,Chen C Y,Huang L S,et al.Gender determination in bovine preimplantation embryosby amplification of amelogenin(AML)gene[J].Journal of Agricultural Biotechnology,2005,13(6):759-762.
[15]于韶荣,史美祺,刘宝瑞,等.全血分离时间对肠癌患者血浆游离DNA浓度的影响[J].成都医学院学报,2014,9(5):554-557.Yu S R,Shi M Q,Liu B R,et al.The influence of whole blood isolation time on plasma free DNA concentration in patients with intestinal cancer[J].Journal of Chengdu Medical College,2014,9(5):554-557.
[16]席继锋,王香祖,贾斌,等.PCR扩增妊娠奶牛牙釉蛋白基因进行胎儿性别鉴定的研究[J].中国奶牛,2011(14):1-4.Xi J F,Wang X Z,Jia B,et al.Study of embryo sex identification by PCR amplification for AML gene in pregnant dairy cows[J].China Dairy Cattle,2011(14):1-4.
[17]李勤,张毅,丁宇烽,等.孕妇血样采集后体外存放对血浆中胎儿游离DNA水平的影响[J].第二军医大学学报,2008,29(9):1042-1045.Li Q,Zhang Y,Ding Y F,et al.Influence of delay in bloodprocessing on the level of cell-free fetal DNA in maternal plasma sample[J].Academic Journal of Second Military Medical University,2008,29(9):1042-1045.
[18]蒋立斌.绵羊外周血中游离DNA应用于胎儿性别鉴定以及基因分型的初步研究[D].石河子:石河子大学,2014:5-10.
[19]Chan K C,Zhang A B,Hui N,et al.Size distributions of maternal and fetal DNA in maternal plasma[J].Clin Chem,2004,50(1):88-92.