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Panminerva Medica 2006 June;48(2):119-27


language: English

Development of a quantitative-competitive PCR for quantification of human cytomegalovirus load and comparison with antigenaemia, viraemia and pp67 RNA detection by nucleic acid sequence-based amplification

Bergallo M. 1, Costa C. 1, Tarallo S. 1, Daniele R. 1, Merlino C. 1, Segoloni G. P. 2, Negro Ponzi A. 1, Cavallo R. 1

1 Virology Unit Department of Public Health and Microbiology University of Turin, Turin, Italy 2 Renal Transplant Unit Department of Internal Medicine University of Turin, Turin, Italy


Aim. The human cytomegalovirus (HCMV) is an important pathogen in immunocompromised patients, such as transplant recipients. The use of sensitive and rapid diagnostic assays can have a great impact on antiviral prophylaxis and therapy monitoring and diagnosing active disease. Quantification of HCMV DNA may additionally have prognostic value and guide routine management. The aim of this study was to develop a reliable internally-controlled quantitative-competitive PCR (QC-PCR) for the detection and quantification of HCMV DNA viral load in peripheral blood and compare it with other methods: the HCMV pp65 antigenaemia assay in leukocyte fraction, the HCMV viraemia, both routinely employed in our laboratory, and the nucleic acid sequence-based amplification (NASBA) for detection of HCMV pp67-mRNA.
Methods. Quantitative-competitive PCR is a procedure for nucleic acid quantification based on co-amplification of competitive templates, the target DNA and a competitor functioning as internal standard. In particular, a standard curve is generated by amplifying 102 to 105 copies of target pCMV-435 plasmid with 104 copies of competitor pCMV-C plasmid. Clinical samples derived from 40 kidney transplant patients were tested by spiking 104 copies of pCMV-C into the PCR mix as internal control, and comparing results with the standard curve.
Results. Of the 40 patients studied, 39 (97.5%) were positive for HCMV DNA by QC-PCR. While the correlation between the number of pp65-positive cells and the number of HCMV DNA genome copies/mL and the former and the pp67mRNA-positivity were statistically significant, there was no significant correlation between HCMV DNA viral load assayed by QC-PCR and HCMV viraemia.
Conclusion. The QC-PCR assay could detect from 102 to over 107 copies of HCMV DNA with a range of linearity between 102 e 105 genomes.

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