Comparison of reverse-transcriptase qPCR and droplet digital PCR for the quantification of dengue virus nucleic acid
Comparison of reverse-transcriptase qPCR and droplet digital PCR for the quantification of dengue virus nucleic acid
复制标题
DOI：
10.1016/j.biologicals.2018.01.001
复制
发表时间：
2018-03-01
期刊：
影响因子：
1.7
通讯作者：
Nougarede, Nolwenn
中科院分区：
文献类型：
--
作者：
Abachin, Eric;Convers, Samantha;Nougarede, Nolwenn
聚合酶链反应（PCR）是一种重要的体外扩增核酸的分子生物学技术。已经开发了逆转录酶定量PCR（RT-qPCR）和最近的逆转录酶数字液滴PCR（RT-ddPCR）用于核酸的定量。我们开发了一种定量减毒登革病毒血清2型核酸的RT-ddPCR方法，并将其与常规RTqPCR方法进行了比较。虽然常规RT-qPCR试验靶向NS 5基因，但选择E基因用于优化RT-ddPCR试验条件。单独使用减毒登革病毒血清型2和在存在其他三种登革血清型的情况下证明了测定的特异性。发现来自减毒登革病毒血清型2的25个样品的两种测定的结果相当，来自线性回归分析的11.2> 0.98。RT-ddPCR测定的主要优点是它允许核酸的定量，而不需要标准曲线。RT-ddPCR可用于疫苗生产过程中登革热疫苗病毒核酸的绝对定量。
Polymerase chain reaction (PCR) is an important molecular biology technique for in vitro amplification of nucleic acids. Reverse transcriptase quantitative PCR (RT-qPCR) and more recently reverse transcriptase digital droplet PCR (RT-ddPCR) have been developed for the quantification of nucleic acids. We developed an RT-ddPCR assay for the quantification of attenuated dengue virus serotype 2 nucleic acid and compared it with a routine RTqPCR assay. While the routine RT-qPCR assay targets the NS5 gene, the E gene was selected for the optimization of the RT-ddPCR assay conditions. The specificity of the assay was demonstrated using the attenuated dengue virus serotype 2 alone and in the presence of the other three dengue serotypes. The results from both assays for 25 samples of the attenuated dengue virus serotype 2 were found to be comparable, with an 11.2 from the linear regression analysis of > 0.98. A major advantage of the RT-ddPCR assay is that it allows quantification of nucleic acid, without the need of a standard curve. RT-ddPCR can be implemented for the absolute quantification of dengue vaccine virus nucleic acid during the vaccine manufacturing process.