title: 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: 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.
authors.affiliation: a Laboratório de Tecnologia Virológica (LATEV); Instituto de Tecnologia em Imunobiológicos; Biomanguinhos/Fiocruz ; Rio de Janeiro , Brazil.
authors.forename: Alice G
authors.initials: AG
authors.lastname: Fernandes-Monteiro
authors.forename: Gisela F
authors.initials: GF
authors.lastname: Trindade
authors.forename: Anna M Y
authors.initials: AM
authors.lastname: Yamamura
authors.forename: Otacilio C
authors.initials: OC
authors.lastname: Moreira
authors.forename: Vanessa S
authors.initials: VS
authors.lastname: de Paula
authors.forename: Ana Cláudia M
authors.initials: AC
authors.lastname: Duarte
authors.forename: Constança
authors.initials: C
authors.lastname: Britto
authors.forename: Sheila Maria B
authors.initials: SM
authors.lastname: Lima
mesh_terms: D000818:Animals; D000918:Antibody Specificity; D002522:Chlorocebus aethiops; D006801:Humans; D010957:Plasmids / Q000235:genetics; D011786:Quality Control; D012367:RNA, Viral / Q000276:immunology / Q000302:isolation & purification; D060888:Real-Time Polymerase Chain Reaction / Q000592:standards*; D012015:Reference Standards; D015203:Reproducibility of Results; D014709:Vero Cells; D019562:Viral Load; D014766:Viremia / Q000821:virology; D015004:Yellow Fever / Q000276:immunology* / Q000517:prevention & control* / Q000821:virology; D022341:Yellow Fever Vaccine / Q000235:genetics* / Q000276:immunology / Q000592:standards*; D015005:Yellow fever virus / Q000235:genetics* / Q000276:immunology
chemical_list: D012367:RNA, Viral; D022341:Yellow Fever Vaccine
keywords: 17DD, strain used to yellow fever vaccine; ANVISA, Brazilian Health Surveillance Agency; C, capsid protein; CV, coefficient of variation; Ct, cycle threshold; DENV, dengue virus; DL, detection limit; DNA, deoxyribonucleic acid; E, envelope protein; ELISA, enzyme-linked immunosorbent assay; EXO IPC, exogenous internal positive control; FDA, food and drug administration agency; JEV, japanese encephalitis virus; MOI, multiplicity of infection; MV, measles virus; MuV, mumps virus; NS, nonstructural protein; NS5, protein of the viral polyprotein, it is the largest and the most highly conserved among the flaviviral proteins; PCR, polymerase chain reaction; PFU, plaque former unit; QL, quantification limit; RNA, ribonucleic acid; RNAse P, human constitutive gene; RT-qPCR; RT-qPCR, reverse transcriptase quantitative polymerase chain reaction; WNV, West Nile Virus; YF, yellow fever; YFV, yellow fever virus; molecular diagnosis; prM/M, membrane protein; viral load; viral vaccines; yellow fever virus