Detection of yellow fever virus: a comparison of quantitative real-time PCR and plaque assay.
Yellow fever virus quantitation is performed routinely by cultivation of virus containing samples using susceptible cells. Counting of the resulting plaques provides a marker for the number of infectious particles present in the sample. This assay usually takes up to 5 days before results are obtained and must be carried out under L2 or L3 laboratory conditions, depending on the yellow fever virus strain used. For clinical diagnosis of yellow fever virus infections the cell culture-based approach takes too long and is of limited practical relevance. Recently, due to its considerable sensitivity, PCR has become a promising method for virus detection. However, whilst PCR can detect virus-specific nucleic acids, it does not allow conclusions to be drawn regarding the infectious potential of the virus detected. Nonetheless, for diagnostic purposes, a rapid, specific and sensitive virus PCR is preferable. Therefore, two independent yellow fever virus-specific real-time PCR assays were established and compared the viral RNA loads to the results of a traditional plaque assay. The estimated ratio of yellow fever virus genomes to infectious particles was between 1000:1 and 5000:1; both approaches displayed a comparable precision of <45%. A significant correlation between genome number as determined by real-time PCR and the corresponding number of plaques in paired samples was found with a Pearson coefficient of correlation of r=0.88 (P<0.0001).
Bae
Hi-Gung
HG
Robert Koch-Institut, Nordufer 20, D-13353, Berlin, Germany.
Nitsche
Andreas
A
Teichmann
Anette
A
Biel
Stefan S
SS
Niedrig
Matthias
M
D000818:Animals; D002460:Cell Line; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000032:analysis* / Q000097:blood*; D015203:Reproducibility of Results; D019914:Taq Polymerase / Q000378:metabolism; D010948:Viral Plaque Assay; D015004:Yellow Fever / Q000821:virology*; D022341:Yellow Fever Vaccine; D015005:Yellow fever virus / Q000235:genetics / Q000302:isolation & purification*
D012367:RNA, Viral; D022341:Yellow Fever Vaccine; D019914:Taq Polymerase