A SYBR Green I-based qRT-PCR Assay for Detecting Duck BMPs
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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、BMP8a、BMP10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、BMP8a、BMP10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−1；BMP6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、BMP8a和BMP15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。 -
关键词:
- 鸭 /
- 骨形态发生蛋白（BMPs） /
- 荧光定量PCR /
- SYBR Green I
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. -
Key words:
- Duck /
- bone morphogenetic proteins /
- qRT-PCR /
- SYBR Green I
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图 1 BMPs基因PCR扩增结果
M：DNA DL 500 相对分子质量标准；1～10分别为BMP1、BMP2、BMP3、BMP4、BMP5、BMP6、BMP7、BMP8a、BMP10和BMP15。
Figure 1. PCR amplification of BMPs
M: DL500 DNA marker; 1-10: BMP1, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8a, BMP10, and BMP15, respectively.
图 2 BMP基因的SYBR Green I荧光定量PCR扩增曲线
A：1～6分别为6.03×108～6.03×103 copies·μL−1，7为DEPC水；B：1～6分别为6.41×108～6.41×103 copies·μL−1，7为DEPC水；C：1～6分别为1.73×108～1.73×103 copies·μL−1，7为DEPC水；D：1～6分别为6.22×107～6.22×102 copies·μL−1，7为DEPC水；E：1～6分别为1.84×108～1.84×103 copies·μL−1，7为DEPC水；F：1～6分别为1.85×107～1.85×102 copies·μL−1，7为DEPC水；G：1～6分别为1.49×107～1.49×102 copies·μL−1，7为DEPC水；H：1～6分别为7.01×107～7.01×102 copies·μL−1，7为DEPC水；I：1～6分别为6.03×108～6.03×103 copies·μL−1，7为DEPC水；J：1～6分别为2.86×107～2.86×102 copies·μL−1，7为DEPC水。
Figure 2. Amplification curves of BMPs by SYBR Green I qRT-PCR
A: 1–6 represent 6.03×108–6.03×103 copies·μL−1, respectively, 7 represents DEPC water; B: 1–6 represent 6.41×108–6.41×103 copies·μL−1, respectively, 7 represents DEPC water; C: 1–6 represent 1.73×108–1.73×103 copies·μL−1, respectively, 7 represents DEPC water; D: 1–6 represent 6.22×107–6.22×102 copies·μL−1, respectively, 7 represents DEPC water; E: 1–6 represent 1.84×108–1.84×103 copies·μL−1, respectively, 7 represents DEPC water; F: 1–6 represent 1.85×107–1.85×102 copies·μL−1, respectively, 7 represents DEPC water; G: 1–6 represent 1.49×107–1.49×102 copies·μL−1, respectively, 7 represents DEPC water; H: 1–6 represent 7.01×107–7.01×102 copies·μL−1, respectively, 7 represents DEPC water; I: 1–6 represent 6.03×108–6.03×103 copies·μL−1, respectively, 7 represents DEPC water; J: 1–6 represent 2.86×107–2.86×102 copies·μL−1, respectively, 7 represents DEPC water.
图 3 BMP基因SYBR Green I荧光定量PCR的标准曲线
Figure 3. Standard curves of BMPs by SYBR Green I qRT-PCR
图 4 BMP基因SYBR Green I荧光定量PCR的熔解曲线
Figure 4. Melting curves of BMPs by SYBR Green I qRT-PCR
图 5 荧光定量PCR检测不同组织中BMPs表达量
Figure 5. Expressions of BMPs in different tissues detected by qRT-PCR
表 1 BMP基因的扩增引物
Table 1. BMP amplification primers
引物名称
Primer name引物序列（5′-3′）
Primer sequence扩增长度
Amplification length/bp参考序列编号
GenBank No.BMP1-F CTTGAAGATGGAGCCGGAG 182 XM038166990 BMP1-R TGTCTCCTTTGCTCAGCCTC BMP2-F CCACGAAGAAGTTTTGGAAG
CTGTTGTTCTCAAAGGCTCC156 XM027453308 BMP2-R BMP3-F ATGAACTTCTGATCGGTGTC 170 XM038178529 BMP3-R GACCCTTGTCAGAGGATTAC BMP4-F CCACCATGAAGAGCACCTGG
TTTATCCGGTGGAAGCCCCT180 XM038179087 BMP4-R BMP5-F CCCAACAGGGATGCAGACTT
ACTTCGACCGTCCCCAGTTT165 XM038177391 BMP5-R BMP6-F CAAACAGATCCTCTGACCTG
TTTACACTGAAACCGTCCTG169 XM027451601 BMP6-R BMP7-F AAAGCATAGACGGGCAAAGC
TTTGGATCGATTCTGGCTCC167 XM005010474 BMP7-R BMP8a-F GCACCCTGCACATCAGCATC
AGCCCATCATCCGTCTCCAC202 XM038167176 BMP8a-R BMP10-F TCCTGGACTTGGAGAACCTG 181 XM038167140 BMP10-R GAATCCCAGCCAATCTCCTT BMP15-F ACGCCGTGGTGCAGAACTTGGT 157 XM027465364 BMP15-R CAGGACTCTGCGATCATGTTCT 
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表 2 实时荧光定量PCR敏感性检测
Table 2. Sensitivity of qRT-PCR assay
基因
Gene质粒浓度
Concentration of
plasmid standard /
(拷贝·μL−1)Ct值($ \overline { x}\pm { \rm {SD}}$)
Ct value ($ \overline { x}\pm { \rm {SD}}$)变异系数
Coefficient of
variation/%BMP1 6.03×103 26.48±0.05 0.18 6.03×102 29.94±0.10 0.33 6.03×101 33.63±0.56 1.67 BMP2 6.41×103 28.93±0.03 0.11 6.41×102 32.36±0.23 0.71 6.41×101 33.97±0.62 1.83 BMP3 1.73×103 26.11±0.03 0.12 1.73×102 29.40±0.21 0.72 1.73×101 33.10±0.72 2.20 BMP4 6.22×103 29.07±0.10 0.34 6.22×102 32.09±0.10 0.34 6.22×101 33.94±0.55 1.64 BMP5 1.84×103 28.38±0.16 0.58 1.84×102 30.10±0.24 0.82 1.84×101 31.17±0.42 1.36 BMP6 1.85×103 28.45±0.02 0.08 1.85×102 31.92±0.09 0.27 1.85×101 36.35±0.46 1.28 BMP7 1.49×103 29.39±0.07 0.25 1.49×102 32.72±0.10 0.31 1.49×101 36.30±0.58 1.59 BMP8a 7.01×103 26.44±0.04 0.15 7.01×102 30.10±0.07 0.24 7.01×101 33.98±0.48 1.41 BMP10 1.34×104 24.53±0.07 0.31 1.34×102 27.43±0.18 0.68 1.34×101 30.44±0.53 1.74 BMP15 2.86×103 28.11±0.07 0.23 2.86×102 32.14±0.19 0.58 2.86×101 35.93±0.80 2.23
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表 3 荧光定量PCR重复性试验结果
Table 3. Reproducibility of qRT-PCR assay
基因
Gene组内重复性试验Ct值
The Ct values of intra-assay组间重复性试验Ct值
The Ct values of inter-assay平均值±标准差
Means ± SD变异系数
CV/%平均值±标准差
Means ± SD变异系数
CV/%BMP1 20.19±0.02 0.12 20.04±0.08 0.42 BMP2 26.71±0.03 0.11 26.90±0.19 0.69 BMP3 28.88±0.08 0.28 28.79±0.12 0.41 BMP4 29.20±0.08 0.28 29.32±0.16 0.56 BMP5 31.06±0.06 0.20 31.37±0.23 0.72 BMP6 26.87±0.04 0.13 26.81±0.20 0.74 BMP7 23.40±0.03 0.12 23.52±0.11 0.47 BMP8a 32.14±0.07 0.22 32.11±0.19 0.60 BMP10 27.81±0.07 0.24 27.69±0.18 0.65 BMP15 25.13±0.01 0.06 25.17±0.08 0.32
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