Evaluation and Validation of a Real-Time PCR Assay for Detection and Quantitation of Human Adenovirus 14 from Clinical Samples

In 2007, the Centers for Disease Control and Prevention (CDC) reported that Human adenovirus type 14 (HAdV-14) infected 106 military personnel and was responsible for the death of one U.S. soldier at Lackland Air Force Base in Texas. Identification of the responsible adenovirus, which had not previously been seen in North America and for which rapid diagnostic tools were unavailable, required retrospective analysis at reference laboratories. Initial quarantine measures were also reliant on relatively slow traditional PCR analysis at other locations. To address this problem, we developed a real-time PCR assay that detects a 225 base pair sequence in the HAdV-14a hexon gene. Fifty-one oropharyngeal swab specimens from the Naval Health Research Center, San Diego, CA and Advanced Diagnostic Laboratory, Lackland AFB, TX were used to validate the new assay. The described assay detected eight of eight and 19 of 19 confirmed HAdV-14a clinical isolates in two separate cohorts from respiratory disease outbreaks. The real-time PCR assay had a wide dynamic range, detecting from 102 to 107 copies of genomic DNA per reaction. The assay did not cross-react with other adenoviruses, influenza, respiratory syncytial virus, or common respiratory tract bacteria. The described assay is easy to use, sensitive and specific for HAdV-14a in clinical throat swab specimens, and very rapid since turnaround time is less than four hours to obtain an answer

Range of viral load in NHRC throat swabs.

<p>Range of viral load in NHRC throat swabs.</p

Summary of Human adenovirus 14 detection by conventional and real-time PCR in clinical specimens from the ADL.

<p>Summary of Human adenovirus 14 detection by conventional and real-time PCR in clinical specimens from the ADL.</p

Alignment of species HAdV-B Hexon sequences which span the HAdV-14 primers and TaqMan probe.

<p>Note that the HAdV-14a and HAdV-14p sequences are identical. The forward primer sequence is highlighted in green, the FAM/TAMRA probe is highlighted in aqua, and the reverse primer is highlighted in red. Asterisks underneath sequences highlight identity in the species.</p

Summary of Human adenovirus 14 detection by conventional and real-time PCR in clinical specimens from the NHRC.

<p>Summary of Human adenovirus 14 detection by conventional and real-time PCR in clinical specimens from the NHRC.</p

Sensitivity of Human adenovirus 14 detection by real-time J.B.A.I.D.S. PCR in a series of 10-fold dilutions of genomic HAdV-14 DNA (10² to 10⁷) per PCR reaction.

<p>(A) Amplification profile of real-time J.B.A.I.D.S. PCR. (B) Linear regression of the standard curve.</p