Development of DNA aptamers to a foot-and-mouth disease peptide for competitive FRET-based detection.
We sought to develop a novel competitive fluorescence resonance energy transfer (FRET)-aptamer-based strategy for detection of foot-and-mouth (FMD) disease within minutes. A 14-amino-acid peptide from the VP1 structural protein, which is conserved among 16 strains of O-serotype FMD virus, was synthesized and labeled with Black Hole Quencher-2 (BHQ-2) dye. Polyclonal FMD DNA aptamers were labeled with Alexa Fluor 546-14-dUTP by polymerase chain reaction and allowed to bind the BHQ-2-peptide conjugate. Following purification of the FRET-aptamer-peptide complex, a "lights off" response was observed within 10 minutes and was sensitive to a level of 25-250 ng/mL of FMD peptide. Ten candidate aptamers were sequenced from the polyclonal family. The aptamer candidates were screened in an enzyme-based plate assay. A high- and low-affinity aptamer candidate were each labeled with Alexa Fluor 546-14-dUTP by asymmetric polymerase chain reaction and used in the competitive FRET assay, but neither matched the sensitivity of the polyclonal FRET response, indicating the need for further screening of the aptamer library.
Bruno
John G
JG
Operational Technologies Corporation, 4100 NW Loop 410, Suite 230, San Antonio, TX 78229, USA. john.bruno@otcorp.com
Carrillo
Maria P
MP
Phillips
Taylor
T
D000818:Animals; D052157:Aptamers, Nucleotide / Q000235:genetics*; D001483:Base Sequence; D001709:Biotechnology; D036022:Capsid Proteins / Q000235:genetics*; D003001:Cloning, Molecular; D004279:DNA, Viral / Q000235:genetics; D004196:Disease Outbreaks / Q000662:veterinary; D004926:Escherichia coli / Q000235:genetics; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005536:Foot-and-Mouth Disease / Q000175:diagnosis* / Q000453:epidemiology / Q000821:virology; D029544:Foot-and-Mouth Disease Virus / Q000235:genetics* / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D052156:SELEX Aptamer Technique; D013050:Spectrometry, Fluorescence
D052157:Aptamers, Nucleotide; D036022:Capsid Proteins; D004279:DNA, Viral; C442501:VP1 protein, Foot-and-mouth disease virus