Comparison of reverse-transcriptase qPCR and droplet digital PCR for the quantification of dengue virus nucleic acid

被引：31

作者：

Abachin, Eric ^{[1
]}

Convers, Samantha ^{[1
]}

Falque, Stephanie ^{[1
]}

Esson, Raphael ^{[1
]}

Mallet, Laurent ^{[1
]}

Nougarede, Nolwenn ^{[1
]}

机构：

[1] Sanofi Pasteur, Analyt R&D Dept, Microbiol Characterizat R&D Unit, 1541 Ave Marcel Merieux, F-69280 Marcy Letoile, France

来源：

BIOLOGICALS | 2018年 / 52卷

关键词：

Droplet digital PCR; Dengue virus; RT-qPCR; RT-ddPCR; Viral quantification; POLYMERASE-CHAIN-REACTION; DNA COPY NUMBER; SYSTEM; RNA;

D O I：

10.1016/j.biologicals.2018.01.001

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

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.

引用

页码：49 / 54

页数：6