Detection of respiratory syncytial virus using nanoparticle amplified immuno-polymerase chain reaction.
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.
Department of Chemistry, Vanderbilt University, Nashville, TN 37235, USA.
Jonas W
JW
Perez
Elizabeth A
EA
Vargis
Patricia K
PK
Russ
Frederick R
FR
Haselton
David W
DW
Wright
D000818:Animals; D000906:Antibodies / Q000276:immunology; D000941:Antigens / Q000737:chemistry / Q000276:immunology; D001483:Base Sequence; D015374:Biosensing Techniques / Q000379:methods*; D002457:Cell Extracts; D002460:Cell Line; D004279:DNA, Viral / Q000032:analysis / Q000235:genetics / Q000276:immunology; D006046:Gold / Q000737:chemistry*; D007118:Immunoassay / Q000379:methods*; D057230:Limit of Detection; D053768:Metal Nanoparticles / Q000737:chemistry*; D016133:Polymerase Chain Reaction / Q000379:methods*; D058525:Quartz Crystal Microbalance Techniques; D015203:Reproducibility of Results; D012136:Respiratory Syncytial Viruses / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification*
D000906:Antibodies; D000941:Antigens; D002457:Cell Extracts; D004279:DNA, Viral; D006046:Gold