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

Abstract

Abbreviation: PCR, polymerase chain reaction; RT-qPCR, reverse transcriptase quantitative polymerase chain reaction; YFV, yellow fever virus; NS5, protein of the viral polyprotein, it is the largest and the most highly conserved among the flaviviral proteins; 17DD, strain used to yellow fever vaccine; YF, yellow fever; RNA, ribonucleic acid; C, capsid protein; prM/M, membrane protein; E, envelope protein; NS, nonstructural protein; ELISA, enzyme-linked immunosorbent assay; DNA, deoxyribonucleic acid; DL, detection limit; QL, quantification limit; ANVISA, Brazilian Health Surveillance Agency; CV, coefficient of variation; RNAse P, human constitutive gene; Ct, cycle threshold; EXO IPC, exogenous internal positive control; PFU, plaque former unit; FDA, food and drug administration agency; JEV, japanese encephalitis virus; DENV, dengue virus; MuV, mumps virus; MV, measles virus; MOI, multiplicity of infection; WNV, West Nile VirusThe 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.

Montelukast, an Anti-asthmatic Drug, Inhibits Zika Virus Infection by Disrupting Viral Integrity

“…The EV71, an unenveloped RNA virus, was included as negative control. The primers used to detect the RNA sequences of ZIKV, DENV-2, YFV, and EV71 were described previously (Lok et al, 2012;Fernandes-Monteiro et al, 2015;Fischer et al, 2017;Yu et al, 2017;Wu et al, 2018;Yuan et al, 2018) and listed in Table 1.…”

Salmonella entericaserovar Typhi and gallbladder cancer: a case–control study and meta‐analysis

“…PCR‐grade water was used as the negative control in all tests. Human RNAse P gene was used as the internal quality control to ensure proper DNA extraction and the absence of PCR inhibitors .…”

A sensitive one-step TaqMan amplification approach for detection of rubella virus clade I and II genotypes in clinical samples

“…While the ratio of infectious virions to genome copies is hardly one to one, due to the generation of defective interfering viral particles, a correlation between these two parameters was reported for yellow fever virus [10]. In this study, the number of PFU or FFU per ml, as a calculation of infectious virus particles, was contrasted over time of culture to genome copies per ml for different RV strains with or without cytopathogenicity on Vero cells.…”