RNA aptamer-based sensitive detection of SARS coronavirus nucleocapsid protein

Abstract

Severe acute respiratory syndrome coronavirus (SARS-CoV) is the etiological agent of a newly emerged disease SARS. The SARS-CoV nucleocapsid (N) protein is one of the most abundant structural proteins and serves as a diagnostic marker for accurate and sensitive detection of the virus. Using a SELEX (systematic evolution of ligand by exponential enrichment) procedure and recombinant N protein, we selected a high-affinity RNA aptamer capable of binding to N protein with a dissociation constant of 1.65 nM. Electrophoretic mobility shift assays and RNA competition experiments showed that the selected aptamer recognized selectively the C-terminal region of N protein with high specificity. Using a chemiluminescence immunosorbent assay and a nanoarray aptamer chip with the selected aptamer as an antigen-capturing agent, we could sensitively detect N protein at a concentration as low as 2 pg/ml. These aptamer-antibody hybrid immunoassays may be useful for rapid, sensitive detection of SARS-CoV N protein.

Aptamer in Bioanalytical Applications

“…In a report by Ahn et.al. , 88 an aptamer nanoarray chip-based chemiluminesce immunosorbent assay was developed for sensitive detection of severe acute respiratory syndome (SARS) coronavirus nucleocapsid protein. Such a sensitive detection approach could be highly valuable for the virus detection, particularly in the light of SARS disease outbreaks a few years ago.…”

Use of Aptamers as Diagnostics Tools and Antiviral Agents for Human Viruses

“…Nucleocapsid (N) protein is the target choice as it is one of the most abundant structural proteins. Both DNA and RNA aptamers against N protein have been developed with Kd of 4.93 and 1.65 nM, respectively [190,191]. DNA aptamer efficiently detects N protein by Western blot suggesting that it could be an alternative to the antibody.…”

“…DNA aptamer efficiently detects N protein by Western blot suggesting that it could be an alternative to the antibody. Ahn et al have developed a nanoarray aptamer chip with the RNA aptamer selected as an antigen-capturing agent, which is able to detect N protein at a concentration as low as 2 pg/mL [190]. …”

Aptamers against pathogenic microorganisms

“…A possibility for the development of a new aptamer-based diagnostic systems was demonstrated by aptamers targeting SARS coronavirus (Ahn et al, 2009), Rift Valley fever virus (Ellenbecker et al, 2012), and foot-and-mouth disease virus (Bruno et al, 2008a). …”

“…LOD is 60 PFU/μL in PBS.Labib et al (2012c) N protein of SARS coronavirusRNANanoarray aptamer chip. LOD is 42 fM in protein solution.Ahn et al (2009)  Salmonella      Pilin structural protein of    S. typhi RNAInhibition of the invasion of pil+ S. Typhi into human monocytes.Pan et al (2005)  Potentiometric aptasensor based on single-walled carbon nanotubes. LOD is 0.2 CFU/ml in bacterial suspension.Zelada-Guillén et al (2009)  Mixture of OMPs of   S. typhimurium DNAAptamers, immobilized on magnetic beads, were applied for the capturing of the pathogen in biological samples with subsequent PCR detection.…”