Use of oligonucleotide microarrays for rapid detection and serotyping of acute respiratory disease-associated adenoviruses

Abstract

The cessation of the adenovirus vaccination program for military trainees has resulted in several recent acute respiratory disease (ARD) outbreaks. In the absence of vaccination, rapid detection methods are necessary for the timely implementation of measures to prevent adenovirus transmission within military training facilities. To this end, we have combined a fluorogenic real-time multiplex PCR assay with four sets of degenerate PCR primers that target the E1A, fiber, and hexon genes with a long oligonucleotide microarray capable of identifying the most common adenovirus serotypes associated with adult respiratory tract infections (serotypes 3, 4, 7, 16, and 21) and a representative member of adenovirus subgroup C (serotype 6) that is a common cause of childhood ARD and that often persists into adulthood. Analyses with prototype strains demonstrated unique hybridization patterns for representative members of adenovirus subgroups B(1), B(2), C, and E, thus allowing serotype determination. Microarray-based sensitivity assessments revealed lower detection limits (between 1 and 100 genomic copies) for adenovirus serotype 4 (Ad4) and Ad7 cell culture lysates, clinical nasal washes, and throat swabs and purified DNA from clinical samples. When adenovirus was detected from coded clinical samples, the results obtained by this approach demonstrated an excellent concordance with those obtained by the more established method of adenovirus identification as well as by cell culture with fluorescent-antibody staining. Finally, the utility of this method was further supported by its ability to detect adenoviral coinfections, contamination, and, potentially, recombination events. Taken together, the results demonstrate the usefulness of the simple and rapid diagnostic method developed for the unequivocal identification of ARD-associated adenoviral serotypes from laboratory or clinical samples that can be completed in 1.5 to 4.0 h.

FIG. 1.

Electrophoretic profiles of the amplicons obtained by multiplex PCR with representative Ad prototype strains from each subgroup. A 1-kb DNA ladder is presented in the unmarked lane on the far left, and the amplicon designations are given on the right. The Ad serotypes (below the brackets) and Ad subgroups (above the brackets) are also indicated. Fiber 1, amplicons generated by primers AdB1 and AdB2 (primers specific for B-subgroup fiber genes); Fiber 2, amplicons generated by primers AdfibF3 and AdfibR3 (primers designed to amplify Ad4 and Ad6 fiber genes).

FIG. 2.

Combination fluorogenic multiplex PCR and microarray hybridization-based detection scheme for human adenoviruses. (A) Clinical sample collection (throat swab or nasal wash); time elapsed, 0 min. Step 1, generic multiplex PCR amplification reaction setup including Cy5-dCTP and SYBR Green I dye for real-time detection (sample preparation is not necessary for appropriately diluted clinical samples); time elapsed, 10 min. (B and C) Nucleic acid amplification with the R.A.P.I.D. LightCycler instrument. SYBR Green I dye intercalation permits real-time monitoring of the amplification reactions. When real-time monitoring resulted in a positive detection reaction, the amplified material was subsequently hybridized to the microarray for serotype determination; time elapsed, 35 min (laboratory sample) or 58 min (clinical sample). Step 2, 40-cycle R.A.P.I.D. amplification completed; time elapsed, 68 min. (D) Positive reaction sample hybridization and serotype determination. The hybridization results confirmed and provided the serotype determinations for true-positive amplification reactions and/or elucidated false-positive amplicons from the real-time monitoring reactions; time elapsed, 90 min.

FIG. 3.

Microarray hybridization patterns of representative serotypes from adenovirus subgroups B₁, B₂, C, and E. The numbers in the lower left corners of each array indicate the adenovirus serotype. White circles outline weakly positive signals. The white rectangle on the right side of each array outlines a set of unrelated spotted Cy3-labeled probes (+) used for array orientation. All probes specific for a particular serotype are oriented in rows (the serotype designation can be found on the right or left of each array), whereas the unique probe designation (E1 and E2, serotype-specific E1A probes; H1 and H2, serotype-specific hexon probes; and F1 and F2, serotype-specific fiber probes) can be found above each array. All probes targeting the same gene are oriented in columns.

FIG. 4.

Microarray-based detection of Ad3 contamination of an ATCC Ad4 stock (VR-4) and cell culture verification. (A) Multiplex PCR and microarray analysis suggested the presence of two adenoviral serotypes (Ad3 and Ad4) in ATCC VR-4. A-549 cells were inoculated with ATCC VR-4 and immunostained at 72 h postinfection with Ad4-specific rabbit antiserum (B), Ad3-specific rabbit antiserum (C), or Ad7a-specific rabbit antiserum (D) and a fluorescein isothiocyanate-conjugated secondary anti-rabbit antibody. The white arrowheads in panels B and C indicate characteristic adenoviral nuclear inclusions (not seen in panel D), confirming the presence of both Ad3 and Ad4 in ATCC VR-4.