Quantitation of adenovirus DNA and virus particles with the PicoGreen fluorescent dye
Quantitation of adenovirus DNA and virus particles with the PicoGreen fluorescent dye
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DOI：
10.1006/abio.1999.4282
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发表时间：
1999-10-15
影响因子：
2.9
通讯作者：
McCaman, MT
中科院分区：
文献类型：
--
作者：
Murakami, P;McCaman, MT
一种用于腺病毒DNA快速定量的微孔板测定法已经使用选择性结合双链DNA的荧光染料PicoGreen开发。该方法首先应用于提取的腺病毒DNA，然后扩展到完整的，纯化的腺病毒样品后，裂解的病毒衣壳与离子去污剂SDS。利用腺病毒DNA和完整颗粒之间的化学计量关系，然后推导出样品的完整病毒的物理颗粒计数。这种基于PicoGreen的检测方法具有出色的重现性、线性和灵敏度。在其现有形式中，该检测试剂盒的定量限为10.3 ng/ml病毒DNA，预计相当于2.6 x 10（8）个病毒颗粒/ml。将该方法与广泛使用的光谱法进行比较，其中用SDS裂解样品并在260 nm处读取吸光度，发现其灵敏度高10至20倍。染料结合试验还使用相当少的样品体积（
A microplate assay for the rapid quantitation of adenovirus DNA has been developed using the fluorescent dye PicoGreen, which selectively binds double-stranded DNA. The method was first applied to extracted adenoviral DNA and then extended to samples of intact, purified adenovirus after lysis of the viral capsid with the ionic detergent SDS. Utilizing the stoichiometric relationship between adenovirus DNA and intact particles, a physical particle count of intact virus is then derived for the sample. This PicoGreen-based assay has excellent reproducibility, linearity, and sensitivity. In its present form, this assay has a limit of quantitation of 10.3 ng/ml viral DNA, predicted to correspond to 2.6 x 10(8) virus particles/ml. This procedure was compared to a widely utilized spectroscopic method, in which samples are lysed with SDS and absorbance is read at 260 nm, and found to be 10- to 20-fold more sensitive. The dye binding assay also uses considerably less sample volume (