Detection of respiratory syncytial virus using nanoparticle amplified immuno-polymerase chain reaction.

Perez JW, Vargis EA, Russ PK, Haselton FR, Wright DW
Anal Biochem. 2011 410 (1): 141-8

PMID: 21111702 · PMCID: PMC4208676 · DOI:10.1016/j.ab.2010.11.033

In traditional immuno-polymerase chain reaction (immuno-PCR), a single antibody recognition event is associated with one to three DNA tags, which are subsequently amplified by PCR. Here we describe a nanoparticle-amplified immuno-PCR (NPA-IPCR) assay that combines antibody recognition of enzyme-linked immunosorbent assay (ELISA) with a 50-fold nanoparticle valence amplification step prior to tag amplification by PCR. The assay detects a respiratory syncytial virus (RSV) surface protein using an antibody bound to a 15-nm gold nanoparticle cofunctionalized with thiolated DNA complementary to a hybridized 76-base tag DNA with a tag DNA/antibody ratio of 50:1. The presence of virus particles triggers the formation of a "sandwich" complex composed of the gold nanoparticle construct, virus, and an antibody-functionalized magnetic particle used for extraction. After extraction, DNA tags are released by heating to 95°C and detected via real-time PCR. The limit of detection of the assay was compared with ELISA and reversion transcription (RT) PCR using RSV-infected HEp-2 cell extracts. NPA-IPCR showed an approximately 4000-fold improvement in the limit of detection compared with ELISA and a 4-fold improvement compared with viral RNA extraction followed by traditional RT-PCR. NPA-IPCR offers a viable platform for the development of early-stage diagnostics requiring an exceptionally low limit of detection.

2010 Elsevier Inc. All rights reserved.

MeSH Terms (16)

Animals Antibodies Antigens Base Sequence Biosensing Techniques Cell Extracts Cell Line DNA, Viral Gold Immunoassay Limit of Detection Metal Nanoparticles Polymerase Chain Reaction Quartz Crystal Microbalance Techniques Reproducibility of Results Respiratory Syncytial Viruses

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