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 10(2) to 10(7) 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.
Metzgar
David
D
Department of Respiratory Disease Research, Naval Health Research Center, San Diego, CA, USA.
Skochko
Greg
G
Gibbins
Carl
C
Hudson
Nolan
N
Lott
Lisa
L
Jones
Morris S
MS
D000258:Adenovirus Infections, Human / Q000175:diagnosis* / Q000821:virology*; D000260:Adenoviruses, Human / Q000235:genetics*; D001483:Base Sequence; D015331:Cohort Studies; D004196:Disease Outbreaks; D006801:Humans; D008969:Molecular Sequence Data; D012367:RNA, Viral / Q000235:genetics; D015203:Reproducibility of Results; D012140:Respiratory Tract Diseases / Q000175:diagnosis / Q000821:virology; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D012689:Sequence Homology, Nucleic Acid; D013997:Time Factors
D012367:RNA, Viral