TY - JOUR
T1 - Real-time multiplex PCR assay to quantify hepatitis C virus RNA in peripheral blood mononuclear cells
AU - Pugnale, Paolo
AU - Latorre, Patrizia
AU - Rossi, Christine
AU - Crovatto, Katia
AU - Pazienza, Valerio
AU - De Gottardi, Andrea
AU - Negro, Francesco
PY - 2006/5
Y1 - 2006/5
N2 - Ultrasensitive methods to measure very low levels of hepatitis C virus (HCV) RNA in biological samples may have diagnostic and prognostic significance and be useful to evaluate the response to antiviral treatment. A sensitive assay to quantify HCV RNA in peripheral blood mononuclear cells (PBMCs) was developed and validated using the iCycler iQ™ Detection System (Bio-Rad) coupled with TaqMan® chemistry. HCV was co-amplified with the endogenous control glyceraldehyde-3-phosphate dehydrogenase in a multiplex reaction. Calculated PCR amplification efficiencies for both target and control genes were used in a mathematical model for relative quantitation of HCV RNA. A linear relationship between input RNA and CT values over 6 log dilutions was observed for both HCV- and GAPDH-specific products (R2 ≥ 0.99). As few as 1.5 IU/reaction could be detected, with high accuracy (CV ≤ 3.94%) and reproducibility (CV ≤ 2.20%). Quantitation of HCV RNA levels ranging from 103 to 107 IU/ml as measured in 47 plasma samples was highly correlated with values obtained by the COBAS Amplicor™ HCV Monitor test, v2.0 (Roche) (R2 = 0.977). In conclusion, this assay provides an excellent tool to determine accurately HCV kinetics in PBMCs during antiviral therapy and to assess the long-term significance of different patterns of response to treatment.
AB - Ultrasensitive methods to measure very low levels of hepatitis C virus (HCV) RNA in biological samples may have diagnostic and prognostic significance and be useful to evaluate the response to antiviral treatment. A sensitive assay to quantify HCV RNA in peripheral blood mononuclear cells (PBMCs) was developed and validated using the iCycler iQ™ Detection System (Bio-Rad) coupled with TaqMan® chemistry. HCV was co-amplified with the endogenous control glyceraldehyde-3-phosphate dehydrogenase in a multiplex reaction. Calculated PCR amplification efficiencies for both target and control genes were used in a mathematical model for relative quantitation of HCV RNA. A linear relationship between input RNA and CT values over 6 log dilutions was observed for both HCV- and GAPDH-specific products (R2 ≥ 0.99). As few as 1.5 IU/reaction could be detected, with high accuracy (CV ≤ 3.94%) and reproducibility (CV ≤ 2.20%). Quantitation of HCV RNA levels ranging from 103 to 107 IU/ml as measured in 47 plasma samples was highly correlated with values obtained by the COBAS Amplicor™ HCV Monitor test, v2.0 (Roche) (R2 = 0.977). In conclusion, this assay provides an excellent tool to determine accurately HCV kinetics in PBMCs during antiviral therapy and to assess the long-term significance of different patterns of response to treatment.
KW - Hepatitis C virus
KW - Multiplex real-time PCR
KW - Peripheral blood mononuclear cell
KW - qPCR
UR - http://www.scopus.com/inward/record.url?scp=33645313471&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=33645313471&partnerID=8YFLogxK
U2 - 10.1016/j.jviromet.2005.11.007
DO - 10.1016/j.jviromet.2005.11.007
M3 - Article
C2 - 16384611
AN - SCOPUS:33645313471
VL - 133
SP - 195
EP - 204
JO - Journal of Virological Methods
JF - Journal of Virological Methods
SN - 0166-0934
IS - 2
ER -