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A direct detection of Escherichia coli genomic DNA using gold nanoprobes.

Padmavathy B, Vinoth Kumar R, Jaffar Ali BM - J Nanobiotechnology (2012)

Bottom Line: Though conventional DNA amplification using PCR can provide fast results, it is not widely practised in diagnostic laboratories partially because it requires skilled personnel and expensive equipment.It was found to be 100% sensitive and proves to be highly specific without any cross reaction with non-Escherichia coli strains.The salient feature of this approach includes low-cost, robust reagents and simple colorimetric detection of pathogen.

View Article: PubMed Central - HTML - PubMed

Affiliation: AU-KBC Research Centre, M.I.T.Campus of Anna University, Tamil Nadu, India.

ABSTRACT

Background: In situation like diagnosis of clinical and forensic samples there exists a need for highly sensitive, rapid and specific DNA detection methods. Though conventional DNA amplification using PCR can provide fast results, it is not widely practised in diagnostic laboratories partially because it requires skilled personnel and expensive equipment. To overcome these limitations nanoparticles have been explored as signalling probes for ultrasensitive DNA detection that can be used in field applications. Among the nanomaterials, gold nanoparticles (AuNPs) have been extensively used mainly because of its optical property and ability to get functionalized with a variety of biomolecules.

Results: We report a protocol for the use of gold nanoparticles functionalized with single stranded oligonucleotide (AuNP- oligo probe) as visual detection probes for rapid and specific detection of Escherichia coli. The AuNP- oligo probe on hybridization with target DNA containing complementary sequences remains red whereas test samples without complementary DNA sequences to the probe turns purple due to acid induced aggregation of AuNP- oligo probes. The color change of the solution is observed visually by naked eye demonstrating direct and rapid detection of the pathogenic Escherichia coli from its genomic DNA without the need for PCR amplification. The limit of detection was ~54 ng for unamplified genomic DNA. The method requires less than 30 minutes to complete after genomic DNA extraction. However, by using unamplified enzymatic digested genomic DNA, the detection limit of 11.4 ng was attained. Results of UV-Vis spectroscopic measurement and AFM imaging further support the hypothesis of aggregation based visual discrimination. To elucidate its utility in medical diagnostic, the assay was validated on clinical strains of pathogenic Escherichia coli obtained from local hospitals and spiked urine samples. It was found to be 100% sensitive and proves to be highly specific without any cross reaction with non-Escherichia coli strains.

Conclusion: This work gives entry into a new class of DNA/gold nanoparticles hybrid materials which might have optical property that can be controlled for application in diagnostics. We note that it should be possible to extend this strategy easily for developing new types of DNA biosensor for point of care detection. The salient feature of this approach includes low-cost, robust reagents and simple colorimetric detection of pathogen.

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Related in: MedlinePlus

Representative snap shot of AuNP- oligo probe assay on DNA samples of indicated bacterial species. Escherichia coli sample exhibit red color confirming the presence of target DNA. All other samples exhibit purple color suggesting the absence target DNA.
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Figure 5: Representative snap shot of AuNP- oligo probe assay on DNA samples of indicated bacterial species. Escherichia coli sample exhibit red color confirming the presence of target DNA. All other samples exhibit purple color suggesting the absence target DNA.

Mentions: In order to obtain an indication of the methods performance on clinical samples, we applied the optimized assay on DNA isolated from the bacterial species listed in Table 1. In total 31 Escherichia coli clinical samples were tested in the assay and were found to be true positives. During the evaluation we found that all true negatives in the AuNP- oligo probe assay was negative controls confirmed in our laboratory by PCR. Figure 5 gives representative snap-shot of visual observations of various negative controls compared with Escherichia coli strain exhibiting visible color change of the solution from red to purple after acid addition. Thus the discrimination of Escherichia coli and non- Escherichia coli strains were accomplished visually without the need of any sophisticated instrument. The application of the proposed method on the DNA isolated from the clinical isolates produced exactly the same results every time (n = 5) when the assay was repeated. Hence the assay defines to have 100% specificity and repeatability thus enabling reliable and highly specific detection without any cross reaction with related bacteria. An interesting feature of this assay is that the positive samples when preserved overnight at room temperature continues to retain its color suggesting the long-time stability of AuNP- oligo probe hybridization with the target sequence. This feature is particularly useful for prolonged read-out capability required for high-throughput applications.


A direct detection of Escherichia coli genomic DNA using gold nanoprobes.

Padmavathy B, Vinoth Kumar R, Jaffar Ali BM - J Nanobiotechnology (2012)

Representative snap shot of AuNP- oligo probe assay on DNA samples of indicated bacterial species. Escherichia coli sample exhibit red color confirming the presence of target DNA. All other samples exhibit purple color suggesting the absence target DNA.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3306828&req=5

Figure 5: Representative snap shot of AuNP- oligo probe assay on DNA samples of indicated bacterial species. Escherichia coli sample exhibit red color confirming the presence of target DNA. All other samples exhibit purple color suggesting the absence target DNA.
Mentions: In order to obtain an indication of the methods performance on clinical samples, we applied the optimized assay on DNA isolated from the bacterial species listed in Table 1. In total 31 Escherichia coli clinical samples were tested in the assay and were found to be true positives. During the evaluation we found that all true negatives in the AuNP- oligo probe assay was negative controls confirmed in our laboratory by PCR. Figure 5 gives representative snap-shot of visual observations of various negative controls compared with Escherichia coli strain exhibiting visible color change of the solution from red to purple after acid addition. Thus the discrimination of Escherichia coli and non- Escherichia coli strains were accomplished visually without the need of any sophisticated instrument. The application of the proposed method on the DNA isolated from the clinical isolates produced exactly the same results every time (n = 5) when the assay was repeated. Hence the assay defines to have 100% specificity and repeatability thus enabling reliable and highly specific detection without any cross reaction with related bacteria. An interesting feature of this assay is that the positive samples when preserved overnight at room temperature continues to retain its color suggesting the long-time stability of AuNP- oligo probe hybridization with the target sequence. This feature is particularly useful for prolonged read-out capability required for high-throughput applications.

Bottom Line: Though conventional DNA amplification using PCR can provide fast results, it is not widely practised in diagnostic laboratories partially because it requires skilled personnel and expensive equipment.It was found to be 100% sensitive and proves to be highly specific without any cross reaction with non-Escherichia coli strains.The salient feature of this approach includes low-cost, robust reagents and simple colorimetric detection of pathogen.

View Article: PubMed Central - HTML - PubMed

Affiliation: AU-KBC Research Centre, M.I.T.Campus of Anna University, Tamil Nadu, India.

ABSTRACT

Background: In situation like diagnosis of clinical and forensic samples there exists a need for highly sensitive, rapid and specific DNA detection methods. Though conventional DNA amplification using PCR can provide fast results, it is not widely practised in diagnostic laboratories partially because it requires skilled personnel and expensive equipment. To overcome these limitations nanoparticles have been explored as signalling probes for ultrasensitive DNA detection that can be used in field applications. Among the nanomaterials, gold nanoparticles (AuNPs) have been extensively used mainly because of its optical property and ability to get functionalized with a variety of biomolecules.

Results: We report a protocol for the use of gold nanoparticles functionalized with single stranded oligonucleotide (AuNP- oligo probe) as visual detection probes for rapid and specific detection of Escherichia coli. The AuNP- oligo probe on hybridization with target DNA containing complementary sequences remains red whereas test samples without complementary DNA sequences to the probe turns purple due to acid induced aggregation of AuNP- oligo probes. The color change of the solution is observed visually by naked eye demonstrating direct and rapid detection of the pathogenic Escherichia coli from its genomic DNA without the need for PCR amplification. The limit of detection was ~54 ng for unamplified genomic DNA. The method requires less than 30 minutes to complete after genomic DNA extraction. However, by using unamplified enzymatic digested genomic DNA, the detection limit of 11.4 ng was attained. Results of UV-Vis spectroscopic measurement and AFM imaging further support the hypothesis of aggregation based visual discrimination. To elucidate its utility in medical diagnostic, the assay was validated on clinical strains of pathogenic Escherichia coli obtained from local hospitals and spiked urine samples. It was found to be 100% sensitive and proves to be highly specific without any cross reaction with non-Escherichia coli strains.

Conclusion: This work gives entry into a new class of DNA/gold nanoparticles hybrid materials which might have optical property that can be controlled for application in diagnostics. We note that it should be possible to extend this strategy easily for developing new types of DNA biosensor for point of care detection. The salient feature of this approach includes low-cost, robust reagents and simple colorimetric detection of pathogen.

Show MeSH
Related in: MedlinePlus