Abstract
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 (Ct) 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 101 to 106 cDNA copies with r2≥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 TCID50, 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, TCID50 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.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 212-218 |
| Number of pages | 7 |
| Journal | Journal of Virological Methods |
| Volume | 171 |
| Issue number | 1 |
| DOIs | |
| State | Published - Jan 2011 |
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Keywords
- Airway epithelium
- Quantitative PCR
- Rhinovirus
- TCID
ASJC Scopus subject areas
- Virology
Cite this
Quantitative real-time PCR for rhinovirus, and its use in determining the relationship between TCID50 and the number of viral particles. / Sachs, Lorne A.; Schnurr, David; Yagi, Shigeo; Lachowicz-Scroggins, Marrah E.; Widdicombe, Jonathan.
In: Journal of Virological Methods, Vol. 171, No. 1, 01.2011, p. 212-218.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Quantitative real-time PCR for rhinovirus, and its use in determining the relationship between TCID50 and the number of viral particles
AU - Sachs, Lorne A.
AU - Schnurr, David
AU - Yagi, Shigeo
AU - Lachowicz-Scroggins, Marrah E.
AU - Widdicombe, Jonathan
PY - 2011/1
Y1 - 2011/1
N2 - 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 (Ct) 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 101 to 106 cDNA copies with r2≥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 TCID50, 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, TCID50 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.
AB - 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 (Ct) 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 101 to 106 cDNA copies with r2≥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 TCID50, 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, TCID50 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.
KW - Airway epithelium
KW - Quantitative PCR
KW - Rhinovirus
KW - TCID
UR - http://www.scopus.com/inward/record.url?scp=78650534344&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=78650534344&partnerID=8YFLogxK
U2 - 10.1016/j.jviromet.2010.10.027
DO - 10.1016/j.jviromet.2010.10.027
M3 - Article
C2 - 21070809
AN - SCOPUS:78650534344
VL - 171
SP - 212
EP - 218
JO - Journal of Virological Methods
JF - Journal of Virological Methods
SN - 0166-0934
IS - 1
ER -