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.
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 (TCID50), 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 TCID50/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 TCID50).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 TCID50 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 TCID50 or PFU for each enterovirus serotype included was determined.
Jonsson
Nina
N
School of Pure and Applied Natural Sciences, University of Kalmar, SE-391 82 Kalmar, Sweden.
Gullberg
Maria
M
Lindberg
A Michael
AM
D000818:Animals; D002460:Cell Line; D002522:Chlorocebus aethiops; D010849:Picornaviridae / Q000737:chemistry / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012367:RNA, Viral / Q000032:analysis*; D012015:Reference Standards; D010948:Viral Plaque Assay; D014773:Virology / Q000379:methods*; D014776:Virus Cultivation
D012367:RNA, Viral