Development of a Multiplexed Luminex xMap Assay for Serotype Specific Detection of Five Human Adenoviruses that cause Respiratory Illness.

Session III – Washington, Cicely – Young Investigator Applicant

Title of Contribution: Development of a Multiplexed Luminex xMap Assay for Serotype Specific Detection of Five Human Adenoviruses that cause Respiratory Illness.
Author(s): Washington C1; Hazbón MH1; Houng HS1; Metzgar D2; Binn L1; Seriwatana J1; Lyons A1; Lynch J1 and Kuschner R1

Affiliation(s): 1Walter Reed Army Institute of Research, Silver Spring, Maryland; 2Naval Health Research Center, San Diego, California.

Abstract:  Several serotypes of human Adenoviruses often cause acute respiratory illness and severe outbreaks. Timely serotype identification provides valuable epidemiological information.  Widespread outbreaks have been reported worldwide and the prevalence of specific serotypes varies geographically.  Ad3, Ad4, Ad7, Ad14 and Ad21 are the serotypes most commonly found in the adult population.  Unfortunately, conventional serotype identification is a tedious process that takes several weeks. For this reason, new methods for serotype identification are needed and molecular techniques can be developed for this purpose.  The commercially available assays for the detection of Adenoviruses are universal but do not distinguish the different serotypes.  In this work we describe the development of a sensitive and specific assay capable of identifying serotypes Ad3, Ad4, Ad7, Ad14 and Ad21 in a single multiplexed assay.  Two sets of primers were used for the non serotype specific PCR and then serotype specific primers coupled to Luminex TAGs were used for the target specific extension (TSPE).  Previously published sequences of Ad3, Ad4, Ad7, Ad14, Ad21 hexon genes were used to design the primers.  The TSPE products of Ad3, Ad4, Ad7, Ad14, Ad21 were correctly identified using the Luminex xMap fluid microsphere-based array system.  No cross reactivity with other respiratory pathogens and Adeno serotypes was identified.  This multiplexed assay can be expanded to include more serotypes and should assist in a rapid method of indentifying numerous Adenovirus serotype isolates.