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

作者：Perez Jonas W; Vargis Elizabeth A; Russ Patricia K; Haselton Frederick R; Wright David W^*

来源：Analytical Biochemistry, 2011, 410(1): 141-148.

DOI：10.1016/j.ab.2010.11.033

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

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 (RI) 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.

• 出版日期2011-3-1