Improved real-time PCR assay for detection and quantification of all 54 known types of human adenoviruses in clinical samples
Iwona Dorota Bil-Lula, Nicola De Franceschi, Krzysztof Pawlik, Mieczysław Woźniak
Med Sci Monit 2012; 18(6): BR221-228
Available online: 2012-06-01
Background: Detection and quantification of adenoviruses (AdVs) causing life-threatening complications are important abilities in recognition of infection and management of immunocompromised patients. Due to the rapid increase in the number of known AdV types, most commercial tests for detection and identification of AdVs are outdated.
Material/Methods: We designed an improved, easier and faster real-time quantitative polymerase chain reaction (RQ-PCR) method for detection and quantification of 54 types of human AdVs. A wide validation effort was undertaken to ensure confidence in highly sensitive and specific detection of AdVs in compromised patients. The validation process included evaluation of the method’s suitability and reliability for use in routine diagnostics.
Results: Due to high sensitivity (9.2×102 copies/ml) and broad dynamic range (7 log) we are able to detect specific viral DNA in large amounts of cell-free body fluids. The new assay is characterized by high precision and low variation within and between individual virus tests (CV=0.036%, CV=1.29%), low bias error (4%) and no cross-reactivity with other pathogens.
Conclusions: The implementation of this new assay in clinical and laboratory practice provides a rapid, reliable and less laborious method for detection and monitoring of AdV replication in immunocompromised patients. Moreover, it offers the ability to distinguish between active and latent infection and assess treatment efficiency.
Keywords: Reference Values, Real-Time Polymerase Chain Reaction - methods, Genotype, DNA, Viral - genetics, DNA Primers - metabolism, Child, Preschool, Child, Biological Assay - methods, Adult, Adolescent, Adenoviruses, Human - isolation & purification, Reproducibility of Results, Sensitivity and Specificity, young adult