A non-radioisotopic quantitative competitive polymerase chain reaction method: application in measurement of human herpesvirus 7 load

Quantitative-competitive polymerase chain reaction (QCPCR) is a well-optimi sed and objective methodology for the determination of viral load in clinic al specimens. A major advantage of QCPCR is the ability to control for the differential modulation of the PCR process in the presence of potentially i nhibitory material. QCPCR protocols were developed previously for CMV, HHV- 6, HHV-7 and HHV-8 and relied upon radioactively labelled primers, followed by autoradiography of the separated and digested PCR products to quantify viral load. Whilst this approach offers high accuracy and dynamic range, no n-radioactive approaches would be attractive. Here, an alternative detectio n system is reported, based on simple ethidium bromide staining and compute r analysis of the separated reaction products, which enables its adoption i n the analysis of a large number of samples. In calibration experiments usi ng cloned HHV-7 DNA, the ethidium bromide detection method showed an improv ed correlation with known copy number over that obtained with the isotopic method. In addition, 67 HHV-7 PCR positive blood samples, derived from immu nocompromised patients, were quantified using both detection techniques. Th e results showed a highly significant correlation with no significant diffe rence between the two methods. The applicability of the computerised densit ometry method in the routine laboratory is discussed. (C) 2000 Published by Elsevier Science B.V. All rights reserved.