Rapid detection and molecular serotyping of adenovirus by use of PCR followed by electrospray ionization mass spectrometry

Abstract

We have developed a PCR/electrospray ionization mass spectrometry (PCR/ESI-MS) assay for the rapid detection, identification, and serotyping of human adenoviruses. The assay employs a high-performance mass spectrometer to "weigh" the amplicons obtained from PCR using primers designed to amplify known human adenoviruses. Masses are converted to base compositions and, by comparison against a database of the genetic sequences, the serotype present in a sample is determined. The performance of the assay was demonstrated with quantified viral standards and environmental and human clinical samples collected from a military training facility. Over 500 samples per day can be analyzed with sensitivities greater than 100 genomes per reaction. This approach can be applied to many other families of infectious agents for rapid and sensitive analysis.

FIG. 1.

Sensitivity of detection of adenovirus serotype 4 and serotype 7 genomic material by PCR using primer pairs PP769 and PP943 as evaluated by gel-based analysis. Serial dilutions of previously quantified adenovirus serotype 4 and serotype 7 genomic material were tested in PCR assays against PP769 and PP943. The resulting amplicons were analyzed by gel electrophoresis. The concentration of material in each reaction is represented in PFU.

FIG. 2.

Sensitivity of detection of adenovirus serotype 4 genomic material by PCR using primer pairs PP769 and PP943 as evaluated by ESI-MS-based analysis. Adenovirus serotype 4 genomic material was present at 10, 100, or 1,000 PFU per PCR. (A) ESI-MS detection following amplification by PP943. (B) ESI-MS detection following amplification by PP769. The two peaks in each spectrum correspond to sense and antisense strands of the PCR amplicons, which separate under the conditions of ESI.

FIG. 3.

Detection of adenovirus serotype 4a in the presence of an IPC. Approximately 100 genomic copies of adenovirus serotype 4a genome were mixed with 300 copies of IPC in a standard RT-PCR using PP943 (A) or PP769 (B). Peaks in each spectrum correspond to sense and antisense strands of IPC and adenovirus. The masses of the individual DNA strands and the amplicon base compositions are indicated.

FIG. 4.

Detection of adenovirus serotype 4p, using PP769, in the presence of an IPC and a relevant background. (A) Spectrum obtained following PCR/ESI-MS of approximately 100 PFU of adenovirus serotype 4p in the presence of an air background. (B) Spectrum obtained following PCR/ESI-MS of approximately 100 PFU of adenovirus serotype 4p in the presence of a throat background from healthy volunteers.