Development of multiplexed real-time quantitative polymerase chain reaction assay for detecting human adenoviruses

Abstract

Adenoviruses (AdVs) have been associated with a wide variety of human disease and are increasingly recognized as viral pathogens that can cause significant morbidity and mortality in immunocompromised patients. Early detection of AdV DNA in plasma and sterile fluids has been shown to be useful for identifying patients at risk for invasive AdV disease. Because of the large number of existing Adv types, few real-time quantitative AdV polymerase chain reaction (PCR) assays published effectively cover all AdV types. We designed a series of AdV PCR primers and probes and empirically multiplexed them into 2 separate real-time PCR assays to quantitatively detect all 49 serotypes of human AdV (types 1–49) available from American Type Culture Collection. We then subsequently multiplexed all the primers and probes into 1 reaction. The sensitivity of these assays was determined to be less than 10 copies per reaction (500 copies/mL plasma). In a retrospective evaluation, we detected all 84 clinical AdV isolates isolated in cell culture from patients undergoing hematopoietic stem cell transplantation between 1981 and 1987. Prospective analysis of 46 consecutive clinical samples submitted for AdV testing showed greater sensitivity and equal specificity of the AdV PCR than viral culture. This real-time PCR assay allows rapid, sensitive, and specific quantification of all currently defined AdVs into either 2 or 1 multiplex assay for clinical samples.

Introduction

Adenoviruses (AdVs) are nonenveloped double-stranded DNA viruses. Fifty-one human AdVs have been identified on the basis of their resistance to neutralization antibodies. The different serotypes are classified into 6 species, AF, based on their ability to agglutinate red blood cells and their DNA homology. The DNA sequence homology within AdV species ranges from 50% to almost 100%. The sequence homology between species can be as low as 4% (Horwitz, 2001, Schilham et al., 2002, Shenk, 2001).

The diseases caused by AdVs include respiratory illness, gastroenteritis, hemorrhagic cystitis, and meningoencephalitis. In the immunosuppressed patients, disseminated AdV infections are well described and frequently result in death (Erard et al., 2007, Hierholzer, 1992). Recent surveys suggest that AdV infections are increasing, especially among patients undergoing T-cell–depleted and unrelated cord blood stem cell transplantation. Most recognized AdV infections are due to viruses with A, B, and C species (Horwitz, 2001). However, the other serotype viruses have also been shown to cause diseases, and the association of specific serotypes and patient groups or clinical diseases is not clear-cut (Hierholzer, 1992, Horwitz, 2001).

The current standard laboratory methods for diagnosis of AdV infection have limitations. Viral culture is the most common approach, cytopathic effect and identification takes several days, and several AdVs are fastidious and do not replicate in commonly used cell cultures (Horwitz, 2001). Antigen detection techniques such as direct immunofluorescence, detection of viral antigen by agglutination of antibody-coated latex beads, and dot blot hybridization tend to have low levels of sensitivity and are not amenable to high throughput.

Several polymerase chain reaction (PCR)-based methods have been developed to measurement of the AdV load in respiratory specimens, tissues, and body fluids (Cooper et al., 1999, Echavarria et al., 1998, Leruez-Ville et al., 2004, Lion et al., 2003, Schilham et al., 2002, Vabret et al., 2004). Real-time AdV PCR to detect and quantify AdV in peripheral blood has been shown to be useful in the characterization of the clinical risk status of immunosuppressed patients, the determination of appropriate treatment regimens, and the assessment of the therapeutic responsiveness (Leruez-Ville et al., 2004, Lion et al., 2003, Schilham et al., 2002, Vabret et al., 2004). The number of real-time quantification AdV PCR methods have been published using degenerate/consensus primers and probes (Heim et al., 2003, Lion et al., 2003, Miura-Ochiai et al., 2007) or multiplexing multiple primers and probes into 1 or 2 different reactions (Chmielewicz et al., 2005, Ebner et al., 2006, Ebner et al., 2005, Gu et al., 2003). The degenerate or consensus primers may amplify different viruses with different efficiency, and multiplexing PCR tends to be less efficient and prone to inhibition. In this study, we designed group-specific primers and probes first, and optimized and multiplexed these primer/probe sets into 2 reactions to detect and quantify all pathogenic human AdVs. Lastly, we also evaluated the possibility to multiplex all 5 sets of primers and probes into 1 reaction (penta-plex) using commercially available PCR master mixture designed for multiplexing PCR.