Accurate quantitation of circulating cell-free mitochondrial DNA in plasma by droplet digital PCR
Accurate quantitation of circulating cell-free mitochondrial DNA in plasma by droplet digital PCR
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通过液滴数字 PCR 准确定量血浆中循环游离线粒体 DNA
DOI：
10.1007/s00216-017-0217-x
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发表时间：
2017-02
期刊：
影响因子：
--
通讯作者：
Jianxin Lyu
中科院分区：
文献类型：
--
作者：
Chu Liu;Chaowei Wen;Wei Li;Jianxin Lyu
为了建立一种准确定量血浆中循环游离线粒体DNA （ccf-mtDNA）的方法，我们设计了一种通过扩增线粒体nd1 （MT-ND1）来测定ccf-mtDNA拷贝数的ddPCR方法。为了评估该方法的敏感性和特异性，分别使用含有mt - nd1序列的重组pMD18-T质粒和mtdna缺失（ρ0）的HeLa细胞。随后，制备不同的血浆样品进行ddPCR，以评估检测血浆ccf-mtDNA的可行性。结果表明，ddPCR方法具有较高的敏感性和特异性。当ddPCR前从血浆中提取DNA时，ccf-mtDNA拷贝数高于未提取时的拷贝数。这种差异不是由于PCR抑制剂，如血浆中的抗凝血剂EDTA-Na2，因为标准EDTA-Na2浓度（5 mM）并没有显著抑制ddPCR反应。这一差异可能与血浆外泌体mtDNA有关，其含量为4.21±0.38拷贝/μL，占血浆ccf-mtDNA的约19%。因此，ddPCR可以快速可靠地检测血浆中ccf-mtDNA，并预先进行DNA提取，从而更准确地检测ccf-mtDNA。在ddPCR中，直接使用血浆作为模板适用于检测血浆内的外源性无细胞核酸，但不适用于具有囊泡相关形式的核酸，如外泌体mtDNA。图形摘要：本作品的设计。*：模块1，#：模块2，&：模块3
To establish a method for accurate quantitation of circulating cell-free mitochondrial DNA (ccf-mtDNA) in plasma by droplet digital PCR (ddPCR), we designed a ddPCR method to determine the copy number of ccf-mtDNA by amplifying mitochondrialND1(MT-ND1). To evaluate the sensitivity and specificity of the method, a recombinant pMD18-T plasmid containingMT-ND1sequences and mtDNA-deleted (ρ0) HeLa cells were used, respectively. Subsequently, different plasma samples were prepared for ddPCR to evaluate the feasibility of detecting plasma ccf-mtDNA. In the results, the ddPCR method showed high sensitivity and specificity. When the DNA was extracted from plasma prior to ddPCR, the ccf-mtDNA copy number was higher than that measured without extraction. This difference was not due to a PCR inhibitor, such as EDTA-Na2, an anti-coagulant in plasma, because standard EDTA-Na2concentration (5 mM) did not significantly inhibit ddPCR reactions. The difference might be attributable to plasma exosomal mtDNA, which was 4.21 ± 0.38 copies/μL of plasma, accounting for ∼19% of plasma ccf-mtDNA. Therefore, ddPCR can quickly and reliably detect ccf-mtDNA from plasma with a prior DNA extraction step, providing for a more accurate detection of ccf-mtDNA. The direct use of plasma as a template in ddPCR is suitable for the detection of exogenous cell-free nucleic acids within plasma, but not of nucleic acids that have a vesicle-associated form, such as exosomal mtDNA.Graphical AbstractDesigns of the present work. *: Module 1, #: Module 2, &: Module 3
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影响因子：
64.8
作者：
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影响因子：
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作者：
Beck, Julia;Bierau, Sarah;Schuetz, Ekkehard
通讯作者：
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作者：
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影响因子：
5.4
作者：
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通讯作者：
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DOI：
10.1073/pnas.1409328111
发表时间：
2014-10-28
影响因子：
11.1
作者：
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通讯作者：
Makova, Kateryna D.