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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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AuNP-DNA biosensor detection of non-PCR amplified S. enteritidis genomic DNAt. (A) Differential pulse voltammogram of PCR-amplified DNAt from S. enteritidis at various concentrations. (B) DNAt concentration vs. average differential current peak values from the voltammogram in A. (C) Differential pulse voltammogram of TRIzol® extracted S. enteritidis genomic DNAt at various concentrations. (D) DNAt concentration vs. average differential current peak values from the voltammogram in C. H2O, blank control; NS-DNA, non-specific PCR amplified B. anthracis DNAt (0.1 ng/μL) negative control; HCl, 1 M 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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f2-sensors-12-10487: AuNP-DNA biosensor detection of non-PCR amplified S. enteritidis genomic DNAt. (A) Differential pulse voltammogram of PCR-amplified DNAt from S. enteritidis at various concentrations. (B) DNAt concentration vs. average differential current peak values from the voltammogram in A. (C) Differential pulse voltammogram of TRIzol® extracted S. enteritidis genomic DNAt at various concentrations. (D) DNAt concentration vs. average differential current peak values from the voltammogram in C. H2O, blank control; NS-DNA, non-specific PCR amplified B. anthracis DNAt (0.1 ng/μL) negative control; HCl, 1 M hydrogen chloride. Graphs represent the average value of duplicate samples for each condition. Error bars represent the standard deviation of the mean.

Mentions: With the successful extraction of genomic S. enteritidis DNAt using both extraction methods, we next explored the ability of the AuNP-DNA biosensor system to detect the non-PCR amplified genomic DNAt products. For these experiments, extracted genomic DNAt samples were diluted to three concentrations (3 ng/μL, 1 ng/μL, and 0.1 ng/μL), denatured, and then mixed with functionalized magnetic MNPs and AuNPs (both contained immobilized/conjugated ssDNA probes specific for the Iel insertion element of S. enteritidis on their surface), and allowed to hybridize to create a sandwich structure due to their specificity (Figure 1). Following hybridization, sandwich structures were isolated and washed using magnetic separation, and then added to individual electrode SPCE chips for DPV readout. Current peaks were observed between 0.30 and 0.35 V, the reduction peak of gold ions. We found that similar to the detection of PCR amplified DNAt (Figure 2A), the AuNP-DNA biosensor was also able to detect non-PCR amplified PBC extracted DNAt (Figure 2B). Although there was some variability between duplicate samples, the results demonstrated a trend in average differential peak values of 5.0 × 10−7 A, 6.0 × 10−6 A, and 1.1 × 10−5 A, for the various S. enteritidis PBC genomic DNAt concentrations (3 ng/μL, 1 ng/μL, and 0.1 ng/μL, respectively). In addition, the specificity of our AuNP-DNA biosensor was also revealed as the detection peak of our negative control, PCR amplified B. anthracis non-specific DNA (NS-DNA: 3.0 × 10−6, 0.1 ng/μL) was found to be comparable to that of the H2O blank (2.5 × 10−6 A) (Figure 2A). Surprisingly, a hook effect was observed at the highest DNAt concentration tested, 3 ng/μL, in both the PCR amplified and non-PCR amplified DNAt detection, with detection peaks occurring at lower levels than those produced from the lower DNAt concentrations tested.


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)

AuNP-DNA biosensor detection of non-PCR amplified S. enteritidis genomic DNAt. (A) Differential pulse voltammogram of PCR-amplified DNAt from S. enteritidis at various concentrations. (B) DNAt concentration vs. average differential current peak values from the voltammogram in A. (C) Differential pulse voltammogram of TRIzol® extracted S. enteritidis genomic DNAt at various concentrations. (D) DNAt concentration vs. average differential current peak values from the voltammogram in C. H2O, blank control; NS-DNA, non-specific PCR amplified B. anthracis DNAt (0.1 ng/μL) negative control; HCl, 1 M 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

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getmorefigures.php?uid=PMC3472839&req=5

f2-sensors-12-10487: AuNP-DNA biosensor detection of non-PCR amplified S. enteritidis genomic DNAt. (A) Differential pulse voltammogram of PCR-amplified DNAt from S. enteritidis at various concentrations. (B) DNAt concentration vs. average differential current peak values from the voltammogram in A. (C) Differential pulse voltammogram of TRIzol® extracted S. enteritidis genomic DNAt at various concentrations. (D) DNAt concentration vs. average differential current peak values from the voltammogram in C. H2O, blank control; NS-DNA, non-specific PCR amplified B. anthracis DNAt (0.1 ng/μL) negative control; HCl, 1 M hydrogen chloride. Graphs represent the average value of duplicate samples for each condition. Error bars represent the standard deviation of the mean.
Mentions: With the successful extraction of genomic S. enteritidis DNAt using both extraction methods, we next explored the ability of the AuNP-DNA biosensor system to detect the non-PCR amplified genomic DNAt products. For these experiments, extracted genomic DNAt samples were diluted to three concentrations (3 ng/μL, 1 ng/μL, and 0.1 ng/μL), denatured, and then mixed with functionalized magnetic MNPs and AuNPs (both contained immobilized/conjugated ssDNA probes specific for the Iel insertion element of S. enteritidis on their surface), and allowed to hybridize to create a sandwich structure due to their specificity (Figure 1). Following hybridization, sandwich structures were isolated and washed using magnetic separation, and then added to individual electrode SPCE chips for DPV readout. Current peaks were observed between 0.30 and 0.35 V, the reduction peak of gold ions. We found that similar to the detection of PCR amplified DNAt (Figure 2A), the AuNP-DNA biosensor was also able to detect non-PCR amplified PBC extracted DNAt (Figure 2B). Although there was some variability between duplicate samples, the results demonstrated a trend in average differential peak values of 5.0 × 10−7 A, 6.0 × 10−6 A, and 1.1 × 10−5 A, for the various S. enteritidis PBC genomic DNAt concentrations (3 ng/μL, 1 ng/μL, and 0.1 ng/μL, respectively). In addition, the specificity of our AuNP-DNA biosensor was also revealed as the detection peak of our negative control, PCR amplified B. anthracis non-specific DNA (NS-DNA: 3.0 × 10−6, 0.1 ng/μL) was found to be comparable to that of the H2O blank (2.5 × 10−6 A) (Figure 2A). Surprisingly, a hook effect was observed at the highest DNAt concentration tested, 3 ng/μL, in both the PCR amplified and non-PCR amplified DNAt detection, with detection peaks occurring at lower levels than those produced from the lower DNAt concentrations tested.

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