title: Using surface-enhanced Raman spectroscopy and electrochemically driven melting to discriminate Yersinia pestis from Y. pseudotuberculosis based on single nucleotide polymorphisms within unpurified polymerase chain reaction amplicons.
abstract: The development of sensors for the detection of pathogen-specific DNA, including relevant species/strain level discrimination, is critical in molecular diagnostics with major impacts in areas such as bioterrorism and food safety. Herein, we use electrochemically driven denaturation assays monitored by surface-enhanced Raman spectroscopy (SERS) to target single nucleotide polymorphisms (SNPs) that distinguish DNA amplicons generated from Yersinia pestis, the causative agent of plague, from the closely related species Y. pseudotuberculosis. Two assays targeting SNPs within the groEL and metH genes of these two species have been successfully designed. Polymerase chain reaction (PCR) was used to produce Texas Red labeled single-stranded DNA (ssDNA) amplicons of 262 and 251 bases for the groEL and metH targets, respectively. These amplicons were used in an unpurified form to hybridize to immobilized probes then subjected to electrochemically driven melting. In all cases electrochemically driven melting was able to discriminate between fully homologous DNA and that containing SNPs. The metH assay was particularly challenging due to the presence of only a single base mismatch in the middle of the 251 base long PCR amplicon. However, manipulation of assay conditions (conducting the electrochemical experiments at 10 °C) resulted in greater discrimination between the complementary and mismatched DNA. Replicate data were collected and analyzed for each duplex on different days, using different batches of PCR product and different sphere segment void (SSV) substrates. Despite the variability introduced by these differences, the assays are shown to be reliable and robust providing a new platform for strain discrimination using unpurified PCR samples.
authors.affiliation: Chemistry, University of Southampton , Highfield, Southampton SO17 1BJ, United Kingdom.
authors.forename: Evanthia
authors.initials: E
authors.lastname: Papadopoulou
authors.forename: Sarah A
authors.initials: SA
authors.lastname: Goodchild
authors.forename: David W
authors.initials: DW
authors.lastname: Cleary
authors.forename: Simon A
authors.initials: SA
authors.lastname: Weller
authors.forename: Nittaya
authors.initials: N
authors.lastname: Gale
authors.forename: Michael R
authors.initials: MR
authors.lastname: Stubberfield
authors.forename: Tom
authors.initials: T
authors.lastname: Brown
authors.forename: Philip N
authors.initials: PN
authors.lastname: Bartlett
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D018834:Chaperonin 60 / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004277:DNA, Single-Stranded / Q000235:genetics; D004563:Electrochemistry; D016680:Genome, Bacterial / Q000235:genetics; D008969:Molecular Sequence Data; D010930:Plague / Q000175:diagnosis / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D013045:Species Specificity; D013059:Spectrum Analysis, Raman / Q000379:methods*; D015010:Yersinia pestis / Q000235:genetics*; D015011:Yersinia pseudotuberculosis / Q000235:genetics*; D015012:Yersinia pseudotuberculosis Infections / Q000175:diagnosis / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D018834:Chaperonin 60; D004269:DNA, Bacterial; D004277:DNA, Single-Stranded