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

Abstract

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.

A comparison of viral fitness and virulence between emergent adenovirus 14p1 and prototype adenovirus 14p strains

“…The IScript One-Step RT-PCR Kit for probes was used (Bio-Rad: Hercules, CA) with Ad Hex forward (5'-GGGTTGAAACTACTGAAGAACG-3'), reverse (5'CATCTGTATCAGTCCACG ATT-3') primers, and probes that target hypervariable regions 1–6 of the hexon gene previously described. 18 The following cycling conditions were followed: initial denaturing cycle at 95.0°C for 15 minutes followed by 45 cycles of denaturation at 95.0°C for 1 s, annealing at 60.0°C for 15 s, and extension at 72.0°C for 10 s. Three different independent assays were run in triplicate.…”

Emerging Respiratory Viruses Other than Influenza

“…Ad14-specific NAATs have been described, but are generally used only for epidemiologic studies. 139 RVs are best identified by RT-PCR targeting the 5 0 UTR performed on respiratory secretions from infected individuals. Although relatively highly conserved in comparison with RV coding regions, sequence variation in the 5 0 UTR between different RV types creates difficulties in designing primer pairs that can satisfactorily detect all RV types and species.…”

The Role of Adenovirus in Respiratory Tract Infections

“…The rapidity of real-time PCR is extremely helpful in the clinical setting, especially in outbreaks and epidemics. Recently, Metzgar et al [34] described the increased utility of real-time PCR, especially in detection of pathogenic serotype 14. Their study showed that PCR had a higher sensitivity and specificity for serotype 14, with no known cross-reactivity with other respiratory viral pathogens [34].…”