PMID- 21111702 OWN - NLM STAT- MEDLINE DCOM- 20110419 LR - 20231110 IS - 1096-0309 (Electronic) IS - 0003-2697 (Print) IS - 0003-2697 (Linking) VI - 410 IP - 1 DP - 2011 Mar 1 TI - Detection of respiratory syncytial virus using nanoparticle amplified immuno-polymerase chain reaction. PG - 141-8 LID - 10.1016/j.ab.2010.11.033 [doi] AB - 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 degrees 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. CI - 2010 Elsevier Inc. All rights reserved. FAU - Perez, Jonas W AU - Perez JW AD - Department of Chemistry, Vanderbilt University, Nashville, TN 37235, USA. FAU - Vargis, Elizabeth A AU - Vargis EA FAU - Russ, Patricia K AU - Russ PK FAU - Haselton, Frederick R AU - Haselton FR FAU - Wright, David W AU - Wright DW LA - eng GR - EB009235/EB/NIBIB NIH HHS/United States GR - R01 EB004537/EB/NIBIB NIH HHS/United States GR - T32 HL007751/HL/NHLBI NIH HHS/United States GR - T32 GM065086/GM/NIGMS NIH HHS/United States GR - R21 EB009235/EB/NIBIB NIH HHS/United States PT - Journal Article PT - Research Support, N.I.H., Extramural PT - Research Support, Non-U.S. Gov't DEP - 20101125 PL - United States TA - Anal Biochem JT - Analytical biochemistry JID - 0370535 RN - 0 (Antibodies) RN - 0 (Antigens) RN - 0 (Cell Extracts) RN - 0 (DNA, Viral) RN - 7440-57-5 (Gold) SB - IM MH - Animals MH - Antibodies/immunology MH - Antigens/chemistry/immunology MH - Base Sequence MH - Biosensing Techniques/*methods MH - Cell Extracts MH - Cell Line MH - DNA, Viral/analysis/genetics/immunology MH - Gold/*chemistry MH - Immunoassay/*methods MH - Limit of Detection MH - Metal Nanoparticles/*chemistry MH - Polymerase Chain Reaction/*methods MH - Quartz Crystal Microbalance Techniques MH - Reproducibility of Results MH - Respiratory Syncytial Viruses/*genetics/immunology/*isolation & purification PMC - PMC4208676 MID - NIHMS261878 EDAT- 2010/11/30 06:00 MHDA- 2011/04/20 06:00 PMCR- 2014/10/24 CRDT- 2010/11/30 06:00 PHST- 2010/08/31 00:00 [received] PHST- 2010/11/10 00:00 [revised] PHST- 2010/11/21 00:00 [accepted] PHST- 2010/11/30 06:00 [entrez] PHST- 2010/11/30 06:00 [pubmed] PHST- 2011/04/20 06:00 [medline] PHST- 2014/10/24 00:00 [pmc-release] AID - S0003-2697(10)00746-3 [pii] AID - 10.1016/j.ab.2010.11.033 [doi] PST - ppublish SO - Anal Biochem. 2011 Mar 1;410(1):141-8. doi: 10.1016/j.ab.2010.11.033. Epub 2010 Nov 25.