Comparison of Three Adenovirus Quantitative PCR Assays with ATCC Reference Strains and Clinical Samples

Adenoviruses (AdV) have been associated with a variety of human diseases and are recognized as causing significant morbidity and mortality in immunocompromised or transplant patients. Quantification of AdV DNA in plasma is notoriously difficult due to the genetic diversity of the 71 different serotypes identified to date. There is no World Health Organization standard available to harmonize quantitative data, so results between labs vary widely. In this study, we compared a laboratory-developed multiplex PCR assay with primers and probes specific for each group (A to G) and subgroup E4 (Octaplex) to one with a single primer and probe set (modified from N. Jothikumar et al., Appl Environ Microbiol 71:3131-3136, 2005) and one utilizing bisulfite pretreatment of DNA to reduce variation prior to amplification (Genetic Signatures). Our Octaplex assay detected all low-copy-number clinical samples, while the other two assays had subsets of samples that did not amplify. The modified Jothikumar assay failed to efficiently amplify three of the high-copy-number cultured strains, while the Genetic Signatures 3base assay had a positive bias, resulting in higher copies/ml (>0.5 log₁₀) for all culture fluids tested. All three assays resulted in endpoint detection of the available 51 AdV types. Using two different materials to generate a standard curve revealed that the Octaplex TaqMan assay and the modified Jothikumar assay both consistently gave adenovirus levels lower than the commercial platform for AdV culture fluids but not patient samples. This study highlights the differences in detection of AdV between laboratories that can be attributed to both the PCR method, as well as the reference material used for quantitation.