Development of a Rapid Isothermal Assay for Detection of Adenovirus Types Important in Respiratory Infections

Abstract

Background: Nucleic acid amplification tests (NAATs) for human adenoviruses (HAdVs) causing respiratory infections usually target the hexon gene. However, new HAdV types with substantial variations in the hexon gene may not be detected. Thus, we focus on NAATs based on a conserved region in the penton gene to detect all HAdV types causing respiratory infections.

Methods: A highly conserved region at the 3' end of the penton gene was chosen as a target for NAAT. Primers and probes for quantitative polymerase chain reaction (qPCR) and isothermal recombinase polymerase amplification (RPA) were designed for the detection of all HAdV types causing respiratory infections.

Results: Two highly sensitive qPCR assays were established, one for the detection of HAdV-E4 and HAdV-B types and another for the detection of HAdV-C types (LOD < 10 standard DNA copies for both assays). Furthermore, a one-tube RPA with a universal RPA probe was developed for rapid detection of all HAdV types causing respiratory infections (LOD ≤ 244 standard DNA copies). All three assays were used for testing clinical nasopharyngeal swabs obtained from SARS-CoV-2-negative children with respiratory disease symptoms. Eight out of 243 samples tested were found to be HAdV positive by qPCR and by one-tube RPA, except for one sample with a very low viral load of 30 genome equivalents.

Conclusions: Penton gene-based NAAT systems were developed and successfully used for the detection of HAdV in clinical samples. The newly developed one-tube RPA assay offers the possibility for rapid and simple detection of respiratory HAdV infections at the point of need.

Keywords: HAdV‐B; HAdV‐C; HAdV‐E; human adenovirus (HAdV); penton gene; recombinase polymerase amplification (RPA); respiratory infections.

FIGURE 1

Primers and probes for the detection of human adenovirus (HAdV) types causing respiratory infections. A conserved region in the penton gene of HAdV types relevant in respiratory infections (Table S1) of Species B and E and Species C was identified by multiple alignment using Clustal Omega. Shown are representative sequences for each type. Sequences of HAdV‐E4 (2), HAdV‐B7, HAdV‐B14, HAdV‐B16, HAdV‐B68, and HAdV‐C2 correspond to sequences in GenBank accessions KY996450, JX423383, MK568772, JN860680, AY601636, and JX173084, which were also used for preparation of DNA standards. Sequence mismatches of primers and probes found in all penton gene sequences available in the nucleotide database of NCBI GenBank for each depicted HAdV type are shown in black squares. ^: identical nucleotides across types of Species B and Type E4, *: identical nucleotides across types of Species C, +: identical nucleotides across types of species B, Type E4 and types of Species C. Primers and probes (qPCR, depicted in blue; recombinase polymerase amplification [RPA], depicted in green, Table S2) were chosen with the help of Primer 3 and PrimedRPA software. X: abasic site for Exonuclease III cleavage in RPA probe.

FIGURE 2

Analytical sensitivity of the qPCR assays. (A) Calibration curves calculated by linear regression for qPCR results using the penton gene standard DNA as template. HAdV‐qPCR results exhibit a linear dependency of C _q values with respect to Log₁₀ (DNA copies) over the tested DNA standard range (10⁷–10⁰ standard DNA copies, n = 8 for each amount of standard DNA). (B) Probit analyses for the calculation of limits of detection (LODs). The LODs with 95% detection probability (0.95) are marked with dashed lines. Penton gene DNA standards tested: HAdV‐B7 (one mismatch in PCR_BE_rev primer), HAdV‐B14 (two mismatches in PCR_BE_rev primer), HAdV‐E4 (2) (one mismatch in PCR_BE_probe), and HAdV‐C2 (no mismatches in PCR primers and probe). Dark blue: detection by HAdV‐B + E qPCR. Light blue: detection by HAdV‐C PCR.

FIGURE 3

Limits of detection (LODs) of the one‐tube RPA assay. Penton gene DNA standards tested: HAdV‐B7 (no mismatches in RPA BE primers and RPA universal probe), HAdV‐B14 (one mismatch in RPA_BE_for_2 primer, two mismatches in RPA_BE_rev_2.3 primer), HAdV‐B16/86 (one mismatch in RPA_universal_probe_1), HAdV‐E4 (2) (one mismatch in RPA_BE_for_2 primer), and HAdV‐C2 (no mismatches in RPA C primers and RPA universal probe). Tested standard DNA range: 10⁴–10⁰ standard DNA copies, n = 7 for each amount of standard DNA. The LODs with 95% detection probability (0.95) calculated by probit analysis are marked with dashed lines.