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Detection of non-PCR amplified S. enteritidis genomic DNA from food matrices using a gold-nanoparticle DNA biosensor: a proof-of-concept study.

Vetrone SA, Huarng MC, Alocilja EC - Sensors (Basel) (2012)

Bottom Line: Bacterial pathogens pose an increasing food safety and bioterrorism concern.Non-PCR amplified DNA was hybridized into sandwich-like structures (magnetic nanoparticles/DNA/AuNPs) and analyzed through detection of gold voltammetric peaks using differential pulse voltammetry.Future efforts will focus on further optimization of the DNA extraction method and AuNP-biosensors, to increase sensitivity at lower DNA target concentrations from food matrices comparable to PCR amplified DNA detection strategies.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Whittier College, 13406 E. Philadelphia St., Whittier, CA 90608, USA. svetrone@whittier.edu

ABSTRACT
Bacterial pathogens pose an increasing food safety and bioterrorism concern. Current DNA detection methods utilizing sensitive nanotechnology and biosensors have shown excellent detection, but require expensive and time-consuming polymerase chain reaction (PCR) to amplify DNA targets; thus, a faster, more economical method is still essential. In this proof-of-concept study, we investigated the ability of a gold nanoparticle-DNA (AuNP-DNA) biosensor to detect non-PCR amplified genomic Salmonella enterica serovar Enteritidis (S. enteritidis) DNA, from pure or mixed bacterial culture and spiked liquid matrices. Non-PCR amplified DNA was hybridized into sandwich-like structures (magnetic nanoparticles/DNA/AuNPs) and analyzed through detection of gold voltammetric peaks using differential pulse voltammetry. Our preliminary data indicate that non-PCR amplified genomic DNA can be detected at a concentration as low as 100 ng/mL from bacterial cultures and spiked liquid matrices, similar to reported PCR amplified detection levels. These findings also suggest that AuNP-DNA biosensors are a first step towards a viable detection method of bacterial pathogens, in particular, for resource-limited settings, such as field-based or economically limited conditions. Future efforts will focus on further optimization of the DNA extraction method and AuNP-biosensors, to increase sensitivity at lower DNA target concentrations from food matrices comparable to PCR amplified DNA detection strategies.

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Non-PCR amplified S. enteritidis genomic DNAt can be detected from a mixed DNA culture using the AuNP-DNA biosensor. Graphs represent the average differential current peak values vs. DNAt concentration. (A) TRIzol® extracted genomic DNAt from PBC and (B) phenol/ethanol extracted genomic DNAt from PBC. H2O, blank control; NS-DNA, non-specific PCR amplified B. anthracis DNAt (0.1 ng/μL) negative control; HCl, 1M hydrogen chloride. Graphs represent the average value of duplicate samples for each condition. Error bars represent the standard deviation of the mean.
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f3-sensors-12-10487: Non-PCR amplified S. enteritidis genomic DNAt can be detected from a mixed DNA culture using the AuNP-DNA biosensor. Graphs represent the average differential current peak values vs. DNAt concentration. (A) TRIzol® extracted genomic DNAt from PBC and (B) phenol/ethanol extracted genomic DNAt from PBC. H2O, blank control; NS-DNA, non-specific PCR amplified B. anthracis DNAt (0.1 ng/μL) negative control; HCl, 1M hydrogen chloride. Graphs represent the average value of duplicate samples for each condition. Error bars represent the standard deviation of the mean.

Mentions: As the AuNP-DNA biosensor demonstrated the ability to detect freshly extracted non-PCR amplified genomic DNAt from a single purified bacterial culture (PBC), we next sought to determine if the biosensor could detect non-PCR amplified genomic DNAt from a mixed bacterial culture (MBC) sample. Fresh, PBCs of S. enteritidis, E. coli, and S. boydii were grown individually overnight at 37 °C, and mixed the following day into a MBC. The genomic DNAt was then extracted from the MBC using both the TRIzol® and phenol/ethanol extraction methods to generate the MBC DNAt, that was then hybridized with AuNPs and MNPs, and detected on the AuNP-DNA biosensor using DPV (Figure 3). Analogous to our previous findings using PBC, DNAt was distinguishable from the MBC extraction using both DNA extraction methods. Interestingly, we also observed a hook effect at the higher genomic DNAt concentration of 3 ng/μL, with a detection peak of 4.5 × 10−5 A, when using the TRIzol® method.


Detection of non-PCR amplified S. enteritidis genomic DNA from food matrices using a gold-nanoparticle DNA biosensor: a proof-of-concept study.

Vetrone SA, Huarng MC, Alocilja EC - Sensors (Basel) (2012)

Non-PCR amplified S. enteritidis genomic DNAt can be detected from a mixed DNA culture using the AuNP-DNA biosensor. Graphs represent the average differential current peak values vs. DNAt concentration. (A) TRIzol® extracted genomic DNAt from PBC and (B) phenol/ethanol extracted genomic DNAt from PBC. H2O, blank control; NS-DNA, non-specific PCR amplified B. anthracis DNAt (0.1 ng/μL) negative control; HCl, 1M hydrogen chloride. Graphs represent the average value of duplicate samples for each condition. Error bars represent the standard deviation of the mean.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3472839&req=5

f3-sensors-12-10487: Non-PCR amplified S. enteritidis genomic DNAt can be detected from a mixed DNA culture using the AuNP-DNA biosensor. Graphs represent the average differential current peak values vs. DNAt concentration. (A) TRIzol® extracted genomic DNAt from PBC and (B) phenol/ethanol extracted genomic DNAt from PBC. H2O, blank control; NS-DNA, non-specific PCR amplified B. anthracis DNAt (0.1 ng/μL) negative control; HCl, 1M hydrogen chloride. Graphs represent the average value of duplicate samples for each condition. Error bars represent the standard deviation of the mean.
Mentions: As the AuNP-DNA biosensor demonstrated the ability to detect freshly extracted non-PCR amplified genomic DNAt from a single purified bacterial culture (PBC), we next sought to determine if the biosensor could detect non-PCR amplified genomic DNAt from a mixed bacterial culture (MBC) sample. Fresh, PBCs of S. enteritidis, E. coli, and S. boydii were grown individually overnight at 37 °C, and mixed the following day into a MBC. The genomic DNAt was then extracted from the MBC using both the TRIzol® and phenol/ethanol extraction methods to generate the MBC DNAt, that was then hybridized with AuNPs and MNPs, and detected on the AuNP-DNA biosensor using DPV (Figure 3). Analogous to our previous findings using PBC, DNAt was distinguishable from the MBC extraction using both DNA extraction methods. Interestingly, we also observed a hook effect at the higher genomic DNAt concentration of 3 ng/μL, with a detection peak of 4.5 × 10−5 A, when using the TRIzol® method.

Bottom Line: Bacterial pathogens pose an increasing food safety and bioterrorism concern.Non-PCR amplified DNA was hybridized into sandwich-like structures (magnetic nanoparticles/DNA/AuNPs) and analyzed through detection of gold voltammetric peaks using differential pulse voltammetry.Future efforts will focus on further optimization of the DNA extraction method and AuNP-biosensors, to increase sensitivity at lower DNA target concentrations from food matrices comparable to PCR amplified DNA detection strategies.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Whittier College, 13406 E. Philadelphia St., Whittier, CA 90608, USA. svetrone@whittier.edu

ABSTRACT
Bacterial pathogens pose an increasing food safety and bioterrorism concern. Current DNA detection methods utilizing sensitive nanotechnology and biosensors have shown excellent detection, but require expensive and time-consuming polymerase chain reaction (PCR) to amplify DNA targets; thus, a faster, more economical method is still essential. In this proof-of-concept study, we investigated the ability of a gold nanoparticle-DNA (AuNP-DNA) biosensor to detect non-PCR amplified genomic Salmonella enterica serovar Enteritidis (S. enteritidis) DNA, from pure or mixed bacterial culture and spiked liquid matrices. Non-PCR amplified DNA was hybridized into sandwich-like structures (magnetic nanoparticles/DNA/AuNPs) and analyzed through detection of gold voltammetric peaks using differential pulse voltammetry. Our preliminary data indicate that non-PCR amplified genomic DNA can be detected at a concentration as low as 100 ng/mL from bacterial cultures and spiked liquid matrices, similar to reported PCR amplified detection levels. These findings also suggest that AuNP-DNA biosensors are a first step towards a viable detection method of bacterial pathogens, in particular, for resource-limited settings, such as field-based or economically limited conditions. Future efforts will focus on further optimization of the DNA extraction method and AuNP-biosensors, to increase sensitivity at lower DNA target concentrations from food matrices comparable to PCR amplified DNA detection strategies.

Show MeSH
Related in: MedlinePlus