Article
New approaches for the standardization and validation of a real-time qPCR assay using TaqMan probes for quantification of yellow fever virus on clinical samples with high quality parameters
Fecha
2015Registro en:
MONTEIRO, Alice G. Fernandes; et al. New approaches for the standardization and validation of a real-time qPCR assay using TaqMan probes for quantification of yellow fever virus on clinical samples with high quality parameters. Human Vaccines & Immunotherapeutics, v.11, n.7, p.1865--1871, July 2015.
2164-5515
10.4161/21645515.2014.990854
2164-554X
Autor
Monteiro, Alice G. Fernandes
Trindade, Gisela F.
Yamamura, Anna M. Y.
Moreira, Otacilio C.
Paula, Vanessa S. de
Duarte, Ana Cláudia M.
Britto, Constança
Lima, Sheila Maria B.
Institución
Resumen
The development and production of viral vaccines, in general, involve several steps that need the monitoring of viral
load throughout the entire process. Applying a 2-step quantitative reverse transcription real time PCR assay (RT-qPCR),
viral load can be measured and monitored in a few hours. In this context, the development, standardization and
validation of a RT-qPCR test to quickly and efficiently quantify yellow fever virus (YFV) in all stages of vaccine
production are extremely important. To serve this purpose we used a plasmid construction containing the NS5 region
from 17DD YFV to generate the standard curve and to evaluate parameters such as linearity, precision and specificity
against other flavivirus. Furthermore, we defined the limits of detection as 25 copies/reaction, and quantification as 100
copies/reaction for the test. To ensure the quality of the method, reference controls were established in order to avoid
false negative results. The qRT-PCR technique based on the use of TaqMan probes herein standardized proved to be
effective for determining yellow fever viral load both in vivo and in vitro, thus becoming a very important tool to assure
the quality control for vaccine production and evaluation of viremia after vaccination or YF disease.