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实时荧光定量PCR检测猪源Mx1方法的建立及应用

DOI: 10.7685/j.issn.1000-2030.2013.01.016, PP. 92-96

Keywords: 猪源Mx1,实时荧光定量PCR,标准曲线

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Abstract:

根据GenBank中的猪源抗病毒蛋白Mx1基因(poMx1)序列设计1对特异性引物,从Mx1基因全长片段中扩增和克隆了特异性的115bp核苷酸片段。测序鉴定正确后将重组质粒进行定量并10倍倍比稀释作为扩增模板,通过SYBRGreenⅠ实时荧光定量PCR方法,建立Mx1的标准曲线及回归方程。结果显示该标准曲线的回归方程为Y=-2.9863lgX+38.2393(R2=0.9985),灵敏度为1×102μL-1;应用该方法对猪瘟兔化弱毒苗接种猪肾细胞(PK-15)后产生的poMx1mRNA进行定量分析,发现病毒感染细胞4h后,poMx1的表达量最高(P<0.01),然后随着时间的增加而缓慢减少。结论建立的SYBRGreenⅠ实时荧光定量PCR方法可以检测poMx1mRNA的表达水平。

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