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Real‐time polymerase chain reaction as a rapid and efficient alternative to estimation of picornavirus titers by tissue culture infectious dose 50% or plaque forming units
Author(s) -
Jonsson Nina,
Gullberg Maria,
Lindberg A. Michael
Publication year - 2009
Publication title -
microbiology and immunology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.664
H-Index - 70
eISSN - 1348-0421
pISSN - 0385-5600
DOI - 10.1111/j.1348-0421.2008.00107.x
Subject(s) - titer , virology , biology , real time polymerase chain reaction , plaque forming unit , picornavirus , infectious dose , nucleic acid , enterovirus , virus quantification , virus , polymerase chain reaction , serial dilution , rna , medicine , gene , biochemistry , alternative medicine , pathology
Quantification of viral infectious units is traditionally measured by methods based on forming plaques in semisolid media (PFU) or endpoint dilution of a virus‐containing solution (TCID 50 ), methods that are laborious, time‐consuming and take on average 3–7 days to carry out. Quantitative real‐time PCR is an established method to quantify nucleic acids at high accuracy and reproducibility, routinely used for virus detection and identification. In the present study, a procedure was developed using a two‐step real‐time PCR and the SYBR Green detection method to study whether there are correlations between TCID 50 /ml, PFU/ml and Ct values generated by real‐time PCR enabling rapid and efficient calculation of titer equivalents when working with viruses in the research laboratory. In addition, an external standard with known concentrations was included using in vitro transcribed viral RNA, thus allowing the calculation of the amount of RNA copies needed for various applications (i.e. per plaque or TCID 50 ).The results show that there is a correlation between the three quantification methods covering a wide range of concentration of viruses. Furthermore, a general regression line between TCID 50 and Ct values was obtained for all viruses included in the study, which enabled recording titer equivalents using real‐time PCR. Finally, by including an external standard, the amount of RNA genomes generating one TCID 50 or PFU for each enterovirus serotype included was determined.