Quantitative real-time PCR for rhinovirus, and its use in determining the relationship between TCID50 and the number of viral particles.
The development of a quantitative real-time PCR (qPCR) assay for human rhinovirus serotype 16 (HRV16) is described using the plasmid pR16.11, which contains the full-length genome of HRV16. A standard curve was generated by plotting the critical threshold (C(t)) against numbers of plasmid. The limit of sensitivity was less than10 cDNA copies, and the curve showed a high degree of linearity over a range of 10(1) to 10(6) cDNA copies with r(2)≥0.9989. Amplification efficiency of the qPCR was greater than 97.6 percent. The standard curve was highly reproducible with low intra- and inter-assay coefficients of variation. Standard curves were also generated from cDNA derived from two viral suspensions of known TCID(50), and were exactly parallel to those generated from the plasmid. Comparison of the curves generated from the plasmid or viral cDNA showed that for the two suspensions, TCID(50) corresponded to either 142 or 2088 viral particles. This new qPCR will permit quantitative assessments of interactions between virus and epithelium such as determinations of the affinity and number of viral binding sites or of the number of virus produced per infected cell.
Department of Physiology and Membrane Biology, University of California-Davis, Davis, CA 95616-8643, USA. lasachs@ucdavis.edu
Lorne A
LA
Sachs
David
D
Schnurr
Shigeo
S
Yagi
Marrah E
ME
Lachowicz-Scroggins
Jonathan H
JH
Widdicombe
D002478:Cells, Cultured; D004847:Epithelial Cells / Q000821:virology; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D012229:Rhinovirus / Q000235:genetics / Q000302:isolation & purification* / Q000472:pathogenicity*; D019562:Viral Load / Q000379:methods*; D014774:Virulence