Evaluation of droplet digital PCR for characterizing plasmid reference material used for quantifying ammonia oxidizers and denitrifiers.
Evaluation of droplet digital PCR for characterizing plasmid reference material used for quantifying ammonia oxidizers and denitrifiers.
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用于表征用于定量氨氧化剂和反硝化剂的质粒参考材料的液滴数字 PCR 评估
DOI：
10.1007/s00216-013-7546-1
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发表时间：
2014-02
影响因子：
4.3
通讯作者：
Liu, Yingying
中科院分区：
文献类型：
--
作者：
Dong, Lianhua;Meng, Ying;Wang, Jing;Liu, Yingying
关键词：
具有标准值的DNA标准物质在DNA测量的许多分析过程中起着至关重要的作用。氨氧化细菌(AOB)和古细菌(AOA)中的amoA基因以及反硝化细菌中的NirSandnosZ基因的定量对于确定它们在自然环境中的分布和丰度是非常重要的。我们开发了含有nirS、nosZ、amoA-AOB和amoA-AOA的质粒标准品，以提供拷贝数浓度的DNA标准，以确保这些基因定量的可比性和可靠性。评价滴状数字聚合酶链式反应(DdPCR)对质粒标准物质的鉴定。结果表明，质粒的限制性内切酶消化可以提高扩增效率，减小ddPCR的测量偏差。与质粒的构象相比，含有靶序列的DNA片段的大小和限制性内切酶切点相对于靶序列的位置不是影响ddPCR定量的显著因素。采用高效液相色谱-同位素稀释质谱(LC-IDMS)为质粒标准物质的定量提供独立数据。用ddPCR和LC-IDMS测定的质粒拷贝数浓度一致，提高了质粒标准物质的准确性和可靠性。通过对两次独立测量结果的平均，确定了该质粒标准物质的参考值及其扩展不确定度(k= 2)为(5.19 ± 0.41) × 10 9拷贝数μL−1。考虑到本研究中揭示的因素，可以提高ddPCR的可靠性和准确性，因此，该方法具有准确定量DNA标准物质的潜力。
DNA reference materials of certified value have a critical function in many analytical processes of DNA measurement. Quantification ofamoAgenes in ammonia oxidizing bacteria (AOB) and archaea (AOA), and ofnirSandnosZgenes in the denitrifiers is very important for determining their distribution and abundance in the natural environment. A plasmid reference material containingnirS,nosZ,amoA-AOB, andamoA-AOA is developed to provide a DNA standard with copy number concentration for ensuring comparability and reliability of quantification of these genes. Droplet digital PCR (ddPCR) was evaluated for characterization of the plasmid reference material. The result revealed that restriction endonuclease digestion of plasmids can improve amplification efficiency and minimize the measurement bias of ddPCR. Compared with the conformation of the plasmid, the size of the DNA fragment containing the target sequence and the location of the restriction site relative to the target sequence are not significant factors affecting plasmid quantification by ddPCR. Liquid chromatography–isotope dilution mass spectrometry (LC–IDMS) was used to provide independent data for quantifying the plasmid reference material. The copy number concentration of the digested plasmid determined by ddPCR agreed well with that determined by LC–IDMS, improving both the accuracy and reliability of the plasmid reference material. The reference value, with its expanded uncertainty (k= 2), of the plasmid reference material was determined to be (5.19 ± 0.41) × 109copies μL−1by averaging the results of two independent measurements. Consideration of the factors revealed in this study can improve the reliability and accuracy of ddPCR; thus, this method has the potential to accurately quantify DNA reference materials.
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影响因子：
11.4
作者：
Gilbert, Yan;Le Bihan, Yann;Lessard, Paul
通讯作者：
Lessard, Paul
影响因子：
4.4
作者：
Liu, XD;Tiquia, SM;Zhou, JZ
通讯作者：
Zhou, JZ
影响因子：
64.8
作者：
LASHOF, DA;AHUJA, DR
通讯作者：
AHUJA, DR
DOI：
10.1073/pnas.0506625102
发表时间：
2005-10-11
影响因子：
11.1
作者：
Francis, CA;Roberts, KJ;Oakley, BB
通讯作者：
Oakley, BB
DOI：
10.3358/shokueishi.42.24
发表时间：
2001-02-01
期刊：
JOURNAL OF THE FOOD HYGIENIC SOCIETY OF JAPAN
影响因子：
--
作者：
Matsuoka, T;Kuribara, H;Hino, A
通讯作者：
Hino, A