• 中文核心期刊
  • CSCD来源期刊
  • 中国科技核心期刊
  • CA、CABI、ZR收录期刊

鸭骨形态发生蛋白SYBR Green Ⅰ荧光定量RT-PCR检测方法的建立

A SYBR Green I-based qRT-PCR Assay for Detecting Duck BMPs

  • 摘要:
      目的  旨在建立一种检测鸭骨形态发生蛋白(BMPs)mRNA转录水平的SYBR Green Ⅰ实时荧光定量RT-PCR检测方法。
      方法  根据GenBank中BMP1、BMP2、BMP3、BMP4、BMP5、BMP6、BMP7、BMP8aBMP10和BMP15的核苷酸序列设计并合成特异性引物进行PCR扩增,产物回收后克隆至pMD-18-T载体,构建阳性重组质粒作为标准品,优化SYBR Green Ⅰ荧光定量PCR反应条件,建立其标准曲线,对建立的方法进行特异性、敏感性和重复性试验。
      结果  建立的BMP1、BMP2、BMP3、BMP4、BMP5、BMP6、BMP7、BMP8aBMP10和BMP15基因实时荧光定量检测方法特异性强,无引物二聚体及非特异性产物,熔解曲线为单峰,相关系数R2均大于0.998,组间和组内变异系数均小于1%。应用该方法检测BMPs基因在半番鸭组织中的表达水平,结果显示,BMP1、BMP2、BMP5和BMP7基因在心脏中表达量较高,分别为5.5×104、1.1×105、5.1×105和7.7×105拷贝·μL−1BMP6和BMP10基因在肝脏中表达量较高,分别为7.5×104和7.6×103拷贝·μL−1;而BMP3、BMP4、BMP8aBMP15分别在法氏囊、胸腺、喙和脾脏中表达量最高,分别为1.5×105、2.1×104、1.8×103和3.3×103拷贝·μL−1
      结论  本研究建立的实时荧光定量PCR方法特异性强、重复性好,为检测鸭BMPs表达水平提供了技术手段。

     

    Abstract:
      Objective   An assay for detecting mRNA transcription of bone morphogenetic proteins (BMPs) in ducks using the SYBR Green Ⅰ-based qRT-PCR was developed.
      Methods   Specific primers were designed and synthesized according to the nucleotide sequences of duck BMPs in GenBank. The PCR amplified products were cloned into pMD-18-T vector, and the recombinant plasmids DNA used to establish standard curves. Specificity, sensitivity, and repeatability of the methodology were examined.
      Result   The newly developed qRT-PCR assay showed singe specific melting peaks of BMP1, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8a, BMP10, and BMP15 separately with correlation coefficients (R2) higher than 0.998. The coefficients of variation within and between groups were less than 1%. The mRNA expressions of the BMPs were detected in different tissues of hybrid Muscovy ducks. The expressions of BMP1, BMP2, BMP5, and BMP7 in the heart were significantly higher than those in other tissues at 5.5×104, 1.1×105, 5.1×105, and 7.7×105 copies·μL−1, respectively. BMP6 and BMP10 were highly expressed in the liver at 7.5×104 and 7.6×103 copies·μL−1, respectively, while BMP3 in Bursa of Fabricius, BMP4 in the thymus, BMP8a in the beak, and BMP15 in the spleen at 1.5×105, 2.1×104, 1.8×103, and 3.3×103 copies·μL−1, respectively.
      Conclusion  The newly developed qRT-PCR assay for the determination of mRNA expression of BMPs in ducks was specific, sensitive, repeatable, and applicable.

     

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