Quantitative TaqMan assay for the detection and monitoring of cytomegalovirus infection in organ transplant patients.
Quantitative polymerase chain reaction assays have become the most common methods in the determination of viral load during cytomegalovirus (CMV) infection of transplant patients. In recent years, the development of automated nucleic acid extraction devices together with the introduction of real-time technology have been important elements for improvements of these assays. This chapter describes a method for the quantitation of CMV DNA viral load in the plasma samples of organ transplant patients. The method is based on MagNA Pure LC nucleic acid extraction system (Roche) and the TaqMan real-time technology. MagNA Pure LC is highly automated procedure with which 32 plasma samples could be processed within 1.5 h. TaqMan chemistry and Sequence Detector System 7900HT device (Applied Biosystems) are used for the quantitative amplification of the CMV genome. The chapter also describes preparation of the plasmid, which is needed to achieve a quantitative standard curve for quantitation.
Piiparinen
Heli
H
Department of Virology, Helsinki University Central Hospital and University of Helsinki, Helsinki, Finland.
Lautenschlager
Irmeli
I
D003587:Cytomegalovirus; D003586:Cytomegalovirus Infections / Q000175:diagnosis*; D004279:DNA, Viral / Q000032:analysis / Q000302:isolation & purification; D006801:Humans; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D019914:Taq Polymerase; D014180:Transplantation; D019562:Viral Load / Q000379:methods*
D004279:DNA, Viral; D019914:Taq Polymerase