Detecting Respiratory Syncytial Virus Using Nanoparticle-Amplified Immuno-PCR

  • Jonas W. Perez
  • Nicholas M. Adams
  • Grant R. Zimmerman
  • Frederick R. Haselton
  • David W. Wright
Protocol
First Online:
Part of the Methods in Molecular Biology book series (MIMB, volume 1026)

Abstract

Early-stage detection is essential for effective treatment of pediatric virus infections. In traditional ­immuno-PCR, a single antibody recognition event is associated with one to three DNA tags, which are subsequently amplified by PCR. In this protocol, we describe a nanoparticle-amplified immuno-PCR assay that combines antibody recognition of traditional ELISA with a 50-fold nanoparticle valence amplification step followed by amplification by traditional PCR. The assay detects a respiratory syncytial virus (RSV) surface fusion protein using a Synagis antibody bound to a 15 nm gold nanoparticle co-functionalized with thiolated DNA complementary to a hybridized 76-base Tag DNA. The Tag DNA to Synagis ratio is 50 to 1. The presence of virus particles triggers the formation of a “sandwich” complex comprised of the gold nanoparticle construct, virus, and a 1 μm antibody-functionalized magnetic particle used for extraction. Virus-containing complexes are isolated using a magnet, DNA tags released by heating to 95 °C, and detected via real-time PCR. The limit of detection of the nanoparticle-amplified immuno-PCR assay was compared to traditional ELISA and traditional RT-PCR using RSV-infected HEp-2 cell extracts. Nanoparticle-amplified immuno-PCR showed a ∼4,000-fold improvement in the limit of detection compared to ELISA and a fourfold improvement in the limit of detection compared to traditional RT-PCR. Nanoparticle-amplified immuno-PCR offers a viable platform for the development of an early-stage diagnostics requiring an exceptionally low limit of detection.

Key words

Immuno-PCR PCR Viral detection Respiratory syncytial virus Gold nanoparticle Magnetic extraction 
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Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Jonas W. Perez
    • 1
  • Nicholas M. Adams
    • 1
  • Grant R. Zimmerman
    • 1
  • Frederick R. Haselton
    • 1
  • David W. Wright
    • 1
  1. 1.Department of ChemistryVanderbilt UniversityNashvilleUSA

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