猪性控精子纯度实时荧光定量PCR检测方法的建立

Establish on Real-time Quantitative PCR Method for Purity Detectionof Porcine Sex-controlled Sperm

研究旨在利用实时荧光定量PCR法检测杜洛克猪性控精子,以期建立一种快速、准确且经济高效的性控精子纯度检测方法。选取位于猪Y染色体上的Y染色体性别决定区(sex-determining region Y,SRY)基因和位于X染色体上的A-激酶锚定蛋白4(A-kinase anchoring protein 4,AKAP4)基因,以猪耳组织样提取的基因组DNA为模板进行PCR扩增验证引物的特异性,利用胶回收试剂盒进行回收并质粒小提,将获得的两种质粒经检测后稀释至相同浓度(20 ng/μL),混合构建含有不同比例SRY和AKAP4基因的质粒模板,用于绘制检测精子纯度所用标准曲线的反应模板。分别使用SRY和AKAP4基因特异引物检测3个混合的X精子(P.x_gro1、P.x_gro2、P.x_gro3)和3个混合的Y精子(P.y_gro1、P.y_gro2、P.y_gro3)的纯度。结果显示,用SRY基因特异性引物检测P.x_gro1、P.x_gro2、P.x_gro3精子的纯度分别为91.44%、91.93%、88.99%,P.y_gro1、P.y_gro2、P.y_gro3精子的纯度分别为89.91%、87.31%、88.71%;用AKAP4基因特异性引物检测P.x_gro1、P.x_gro2、P.x_gro3精子的纯度分别为91.44%、91.93%、88.99%,P.y_gro1、P.y_gro2、P.y_gro3精子的纯度分别为89.91%、87.31%、88.71%;卡方适合性检验结果显示,两次检测结果之间差异不显著,表明使用这种方法进行精子纯度检测所得结果准确。本试验通过实时荧光定量PCR法准确检测了经流式细胞仪分选后的精子的纯度,建立了一种利用实时荧光定量PCR法检测猪精液中X精子和Y精子比例的新方法。

The aim of this study was to use Real-time quantitative PCR technique to detect sexually controlled sperm of Duroc pigs,in order to establish a rapid,accurate and cost-effective method for sperm purity detection.The sex-determining region Y(SRY)gene located on the Y chromosome and A-kinase anchoring protein 4(AKAP4)gene located on the X chromosome of pig were selected,using genomic DNA extracted from pig ear tissue samples as a template for PCR amplification for specific primer verification.The gel recovery kit was used to recover the gel,the plasmid were extracted.After testing,the obtained two plasmids were diluted to the same concentration(20 ng/μL),and mixed to construct plasmid templates containing different proportions of SRY and AKAP4 genes,which were used to construct the reaction template for the standard curve of sperm purity detection.The purity of 3 mixed X spermatozoa(P.x_gro1,P.x_gro2 and P.x_gro3)and 3 mixed Y spermatozoa(P.y_gro1,P.y_gro2 and P.y_gro3)were detected by SRY and AKAP4 genes specific primers,respectively.The results showed that the purity obtained using SRY primers of P.x_gro1,P.x_gro2 and P.x_gro3 sperm groups were 91.44%,91.93%and 88.99%,and the purity of P.y_gro1,P.y_gro2 and P.y_gro3 sperm groups were 89.91%,87.31%and 88.71%,respectively.The purity obtained by using AKAP4 primers of P.x_gro1,P.x_gro2,P.x_gro3 sperm groups were 91.44%,91.93%and 88.99%,and the purity of P.y_gro1,P.y_gro2,P.y_gro3 sperm groups were 89.91%,87.31%and 88.71%,respectively.Independent sample t-test analysis showed that no significant difference between the two test results,indicating that the results were accurate for sperm purity detection.In this experiment,Real-time quantitative PCR was used to accurately detect the purity of sperm sorted by flow cytometry,and established a new method for detecting the ratio of X sperm and Y sperm in porcine sperm samples by Real-time quantitative PCR.