Quantitative real-time PCR assays for detection and type-specific identification of the endemic species C human adenoviruses

Abstract

Human adenoviruses (HAdVs) are medically important respiratory pathogens. Among the 7 recognized species (A-G), species C HAdVs (serotypes 1, 2, 5 and 6) are globally endemic and infect most people early in life. Species C HAdV infections are most often subclinical or mild and can lead to persistent shedding from the gastrointestinal and upper respiratory tracts. They can also cause severe disseminated disease in newborn and immunocompromised persons, where rapid and quantitative detection and identification of the virus would help guide therapeutic intervention. To this end, we developed quantitative type-specific real-time PCR (qPCR) assays for HAdV-1, -2, -5 and -6 targeting the HAdV hexon gene. All type-specific qPCR assays reproducibly detected as few as 5 copies/reaction of quantified hexon recombinant plasmids with a linear dynamic range of 8 log units (5-5×10 copies). No non-specific amplifications were observed with concentrated nucleic acid from other HAdV types or other common respiratory pathogens. Of 199 previously typed HAdV field isolates and positive clinical specimens, all were detected and correctly identified to type by the qPCR assays; 10 samples had 2 HAdV types and 1 sample had 3 types identified which were confirmed by amplicon sequencing. The species C HAdV qPCR assays permit rapid, sensitive, specific and quantitative detection and identification of four recognized endemic HAdVs. Together with our previously developed qPCR assays for the epidemic respiratory HAdVs, these assays provide a convenient alternative to classical typing methods.

Comparative evaluation of a laboratory-developed real-time PCR assay and RealStar® Adenovirus PCR Kit for quantitative detection of human adenovirus

“…Our in-house assay achieved a high sensitivity (98.1%) when compared to the commercial kit, and there correlation of the two assays is very good (R 2 = 0.984). Our in-house assay has a linear range up to 9 log 10 copies/mL, which is comparable to other studies [ 21 , 24 , 25 ]. There were very low intra- and inter-assay variations as reflected in the %CV values.…”

Adenovirus: Epidemiology, Global Spread of Novel Types, and Approach to Treatment

“…PCR-based techniques targeting the fiber genes 254 or hypervariable regions of the hexon 340 and/or sequencing of hexon genes allow definitive identification of the type/species. 64,341,342 Molecular typing by PCR amplification and sequencing of both hexon and fiber genes has proved to be extremely valuable for the identification of intertypic recombinants. 253…”

Perspective on Adenoviruses: Epidemiology, Pathogenicity, and Gene Therapy

“…In addition to these techniques, molecular detection of HAdV infection is often performed using the polymerase chain reaction (PCR) assay [7,86,87,88], which amplifies small segments of viral DNA, enabling laboratory technicians and doctors to detect the presence of HAdV in blood, stool, and/ or mucous samples [89,90]. In 2017, Bennett and Gunson developed a single multiplex assay to detect viral gastroenteritis from patients’ stool samples [87].…”