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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

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
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
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.
0003-2700
1605-1612
Papadopoulou, Evanthia
235d2d8f-e2af-4fc0-a402-5fc36184f621
Goodchild, Sarah A.
08211454-37d2-43e6-a41f-86efe8497adc
Cleary, David W.
f4079c6d-d54b-4108-b346-b0069035bec0
Weller, Simon A.
64300ded-b09b-475a-bb7b-0d0de6f202b4
Gale, Nittaya
eead6253-2431-407b-ab6b-92e35d41c3ef
Stubberfield, Michael R.
f9bbc099-dfb6-41ac-8e4d-d6b46685a2e0
Brown, Tom
1cd7df32-b945-4ca1-8b59-a51a30191472
Bartlett, Philip N.
d99446db-a59d-4f89-96eb-f64b5d8bb075
Papadopoulou, Evanthia
235d2d8f-e2af-4fc0-a402-5fc36184f621
Goodchild, Sarah A.
08211454-37d2-43e6-a41f-86efe8497adc
Cleary, David W.
f4079c6d-d54b-4108-b346-b0069035bec0
Weller, Simon A.
64300ded-b09b-475a-bb7b-0d0de6f202b4
Gale, Nittaya
eead6253-2431-407b-ab6b-92e35d41c3ef
Stubberfield, Michael R.
f9bbc099-dfb6-41ac-8e4d-d6b46685a2e0
Brown, Tom
1cd7df32-b945-4ca1-8b59-a51a30191472
Bartlett, Philip N.
d99446db-a59d-4f89-96eb-f64b5d8bb075

Papadopoulou, Evanthia, Goodchild, Sarah A., Cleary, David W., Weller, Simon A., Gale, Nittaya, Stubberfield, Michael R., Brown, Tom and Bartlett, Philip N. (2015) 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. Analytical Chemistry, 87 (3), 1605-1612. (doi:10.1021/ac503063c).

Record type: Article

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.

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Accepted/In Press date: 31 December 2014
e-pub ahead of print date: 15 January 2015
Published date: 3 February 2015
Organisations: Electrochemistry

Identifiers

Local EPrints ID: 376462
URI: http://eprints.soton.ac.uk/id/eprint/376462
ISSN: 0003-2700
PURE UUID: 86db7240-5196-4d50-a0f6-73f6b8d8715e
ORCID for David W. Cleary: ORCID iD orcid.org/0000-0003-4533-0700
ORCID for Philip N. Bartlett: ORCID iD orcid.org/0000-0002-7300-6900

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Date deposited: 28 Apr 2015 15:25
Last modified: 18 Feb 2021 17:24

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Contributors

Author: Evanthia Papadopoulou
Author: Sarah A. Goodchild
Author: David W. Cleary ORCID iD
Author: Simon A. Weller
Author: Nittaya Gale
Author: Michael R. Stubberfield
Author: Tom Brown

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