title: Detection of genetic markers related to high pathogenicity in influenza by SERS.
abstract: We have developed a method for the detection of genetic markers associated with high pathogenicity in influenza. The assay consists of an array of 5'-thiolated ssDNA oligonucleotides immobilized on the surface of a Ag nanorod substrate that serve as capture probes for the detection of synthetic RNA sequences coding for a genetic mutation in the influenza PB1-F2 protein. Hybridization of the DNA probes to their complementary RNA sequences was detected using surface-enhanced Raman spectroscopy (SERS). Multivariate statistical analysis was used to differentiate the spectra of the complementary DNA probe-RNA target hybrids from those of the non-complementary DNA probes containing a single base pair polymorphism. Hierarchical cluster analysis (HCA) was able to distinguish with 100% accuracy the spectra of the complementary DNA probe-RNA target from the spectra of the immobilized DNA probes alone, or the DNA probes incubated with non-complementary RNA sequences. Linearity of response and limits of sensitivity of the SERS-based assays were determined using a partial least squares (PLS) regression model; detection limits computed by PLS was determined to be ~10 nM. The binding affinity of the DNA probes to their complementary RNA sequences was confirmed using enzyme-linked immunosorbent assay (ELISA); however, the detection limits observed using ELISA were approximately 10× higher (~100 nM) than those determined by PLS analysis of the SERS spectra.
authors.affiliation: Department of Chemistry, University of Georgia, Athens, GA 30602, USA.
authors.forename: Pierre
authors.initials: P
authors.lastname: Negri
authors.forename: Richard A
authors.initials: RA
authors.lastname: Dluhy
mesh_terms: D016000:Cluster Analysis; D015342:DNA Probes / Q000737:chemistry / Q000235:genetics; D005819:Genetic Markers / Q000235:genetics; D009980:Influenza A virus / Q000235:genetics* / Q000472:pathogenicity*; D057230:Limit of Detection; D009154:Mutation; D043942:Nanotubes / Q000737:chemistry; D058873:Pandemics; D012367:RNA, Viral / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D012834:Silver / Q000737:chemistry; D013059:Spectrum Analysis, Raman; D013499:Surface Properties; D014764:Viral Proteins / Q000235:genetics*
chemical_list: D015342:DNA Probes; D005819:Genetic Markers; C442816:PB1-F2 protein, Influenza A virus; D012367:RNA, Viral; D014764:Viral Proteins; D012834:Silver