title: Development of one-step real-time PCR assay for titrating trivalent live attenuated influenza vaccines.
abstract: Traditionally, infectivity of a trivalent live attenuated influenza vaccines (LAIVs) is titrated by determining the 50% egg infectious dose assay (EID50) or plaque forming units (PFU), which requires specific monoclonal antibodies to neutralize 2 strains while estimating the titer of the non-neutralized strain. Compared to this time-consuming, laborious, subjective and variable process, reverse transcription-quantitative real-time PCR (RT-qPCR) technology has advantages of rapidity, sensitivity, reproducibility and reduced contamination, thus has been applied widely for detecting pathogens and measuring viral titers. In this study, the critical harvest time was determined to be 18 h post-infection (hpi) for type A influenza and 12 hpi for type B influenza, but no significant difference between titers at 12 hpi and 18 hpi for the type B strain was observed. In conclusion, trivalent LAIVs can be titrated simultaneously within 24 h by this one-step RT-qPCR assay, which yielded titers comparable to those obtained by the traditional EID50 assay. Therefore, the RT-qPCR assay may be used as a highly specific, sensitive, precise and rapid alternative to the EID50 assay for titering LAIVs.
authors.affiliation: a National Engineering Laboratory for AIDS Vaccine; School of Life Sciences ; Jilin University ; Changchun , China.
authors.forename: Yang
authors.initials: Y
authors.lastname: Zang
authors.forename: Dongchuan
authors.initials: D
authors.lastname: Du
authors.forename: Peng
authors.initials: P
authors.lastname: Ge
authors.forename: Yongqing
authors.initials: Y
authors.lastname: Xu
authors.forename: Xintao
authors.initials: X
authors.lastname: Liu
authors.forename: Yan
authors.initials: Y
authors.lastname: Zhang
authors.forename: Weiheng
authors.initials: W
authors.lastname: Su
authors.forename: Irina
authors.initials: I
authors.lastname: Kiseleva
authors.forename: Larisa
authors.initials: L
authors.lastname: Rudenko
authors.forename: Fei
authors.initials: F
authors.lastname: Xu
authors.forename: Wei
authors.initials: W
authors.lastname: Kong
authors.forename: Chunlai
authors.initials: C
authors.lastname: Jiang
mesh_terms: D006801:Humans; D007252:Influenza Vaccines / Q000032:analysis / Q000592:standards*; D009975:Orthomyxoviridae / Q000302:isolation & purification*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014613:Vaccines, Attenuated / Q000592:standards
chemical_list: D007252:Influenza Vaccines; D014613:Vaccines, Attenuated
keywords: 50% egg infectious dose assay; CV%, coefficient of variation; Ct, Cycle threshold; E, efficiency; EID50, 50% egg infectious dose assay; HA, hemagglutinin; LAIVs, live attenuated influenza vaccines; NA, neuraminidase; R2, Correlation coefficient values; RT-qPCR, reverse transcription-quantitative real-time PCR; SD, standard deviation; hpi, hour post-infection; infectivity; live attenuated influenza vaccine; quantitative real-time PCR; titration