Time-Gated Luminescent In Situ Hybridization (LISH): Highly Sensitive Detection of Pathogenic <i>Staphylococcus aureus</i>
We describe simple direct conjugation of a single TEGylated Europium chelate to DNA that binds to intracellular rRNA and is then detected using a homogeneous luminescent in situ hybridisation (LISH) technique. As a proof-of-principle, <i>Staphylococcus aureus (S. aureus)</i> was selected...
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MDPI AG
2019-05-01
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| Series: | Molecules |
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| Online Access: | https://www.mdpi.com/1420-3049/24/11/2083 |
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| author | Nima Sayyadi Russell E. Connally Thomas S. Lawson Jingli Yuan Nicolle H. Packer James A. Piper |
| author_facet | Nima Sayyadi Russell E. Connally Thomas S. Lawson Jingli Yuan Nicolle H. Packer James A. Piper |
| author_sort | Nima Sayyadi |
| collection | DOAJ |
| description | We describe simple direct conjugation of a single TEGylated Europium chelate to DNA that binds to intracellular rRNA and is then detected using a homogeneous luminescent in situ hybridisation (LISH) technique. As a proof-of-principle, <i>Staphylococcus aureus (S. aureus)</i> was selected as a model for our study to show the ability of this probe to bind to intracellular 16S ribosomal rRNA. A highly purified Europium chelate conjugated oligonucleotide probe complementary to an rRNA sequence-specific <i>S. aureus</i> was prepared and found to be soluble and stable in aqueous solution. The probe was able to bind specifically to <i>S. aureus</i> via in situ hybridisation to differentiate <i>S. aureus</i> from a closely related but less pathogenic <i>Staphylococcus</i> species (<i>S. epidermidis</i>). A time-gated luminescent (TGL) microscope system was used to generate the high signal-to-noise ratio (SNR) images of the <i>S. aureus</i>. After excitation (365 nm, Chelate λ<sub>max</sub> = 335 nm), the long-lived (Eu<sup>3+</sup>) luminescent emission from the probe was detected without interference from natural background autofluorescence typically seen in biological samples. The luminescent images were found to have 6 times higher SNR or sensitivity compared to the fluorescent images using conventional fluorophore Alexa Fluor 488. The TEGylated Europium chelate -oligo probe stained <i>S. aureus</i> with mean signal intensity 3.5 times higher than the threshold level of signal from <i>S. epidermidis</i> (with SNR 8 times higher). A positive control probe (EUB338−BHHTEGST−Eu<sup>3+</sup>) has mean signal intensity for <i>S. aureus</i> and <i>S. epidermidis</i> equally 3.2 times higher than the threshold of signal for a negative NON-EUB338 control probe. The direct conjugation of a single Europium chelate to DNA provides simplicity and improvement over existing bovine serum albumin (BSA)/streptavidin/biotinylated DNA platforms for multi-attachment of Europium chelate per DNA and more importantly makes it feasible for hybridisation to intracellular RNA targets. This probe has great potential for highly sensitive homogeneous in situ hybridisation detection of the vast range of intracellular DNA targets. |
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| issn | 1420-3049 |
| language | English |
| last_indexed | 2024-12-21T08:09:05Z |
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| spelling | doaj.art-6bb8f2c9f9984873a90a4e74f9d1b3c02022-12-21T19:10:43ZengMDPI AGMolecules1420-30492019-05-012411208310.3390/molecules24112083molecules24112083Time-Gated Luminescent In Situ Hybridization (LISH): Highly Sensitive Detection of Pathogenic <i>Staphylococcus aureus</i>Nima Sayyadi0Russell E. Connally1Thomas S. Lawson2Jingli Yuan3Nicolle H. Packer4James A. Piper5Department of Molecular Sciences, Macquarie University, Sydney, NSW 2109, AustraliaDepartment of Physics and Astronomy, Macquarie University, Sydney, NSW 2109, AustraliaARC Centre of Excellence for Nanoscale Biophotonics (CNBP), Macquarie University, Sydney, NSW 2109, AustraliaState Key Laboratory of Fine Chemicals, School of Chemistry, Dalian University of Technology, Dalian 116023, ChinaDepartment of Molecular Sciences, Macquarie University, Sydney, NSW 2109, AustraliaARC Centre of Excellence for Nanoscale Biophotonics (CNBP), Macquarie University, Sydney, NSW 2109, AustraliaWe describe simple direct conjugation of a single TEGylated Europium chelate to DNA that binds to intracellular rRNA and is then detected using a homogeneous luminescent in situ hybridisation (LISH) technique. As a proof-of-principle, <i>Staphylococcus aureus (S. aureus)</i> was selected as a model for our study to show the ability of this probe to bind to intracellular 16S ribosomal rRNA. A highly purified Europium chelate conjugated oligonucleotide probe complementary to an rRNA sequence-specific <i>S. aureus</i> was prepared and found to be soluble and stable in aqueous solution. The probe was able to bind specifically to <i>S. aureus</i> via in situ hybridisation to differentiate <i>S. aureus</i> from a closely related but less pathogenic <i>Staphylococcus</i> species (<i>S. epidermidis</i>). A time-gated luminescent (TGL) microscope system was used to generate the high signal-to-noise ratio (SNR) images of the <i>S. aureus</i>. After excitation (365 nm, Chelate λ<sub>max</sub> = 335 nm), the long-lived (Eu<sup>3+</sup>) luminescent emission from the probe was detected without interference from natural background autofluorescence typically seen in biological samples. The luminescent images were found to have 6 times higher SNR or sensitivity compared to the fluorescent images using conventional fluorophore Alexa Fluor 488. The TEGylated Europium chelate -oligo probe stained <i>S. aureus</i> with mean signal intensity 3.5 times higher than the threshold level of signal from <i>S. epidermidis</i> (with SNR 8 times higher). A positive control probe (EUB338−BHHTEGST−Eu<sup>3+</sup>) has mean signal intensity for <i>S. aureus</i> and <i>S. epidermidis</i> equally 3.2 times higher than the threshold of signal for a negative NON-EUB338 control probe. The direct conjugation of a single Europium chelate to DNA provides simplicity and improvement over existing bovine serum albumin (BSA)/streptavidin/biotinylated DNA platforms for multi-attachment of Europium chelate per DNA and more importantly makes it feasible for hybridisation to intracellular RNA targets. This probe has great potential for highly sensitive homogeneous in situ hybridisation detection of the vast range of intracellular DNA targets.https://www.mdpi.com/1420-3049/24/11/2083homogeneous luminescent in situ hybridisationTEGylated Europium chelatetime-gated luminescent microscopy<i>Staphylococcus aureus</i> (<i>S. aureus</i>) detection |
| spellingShingle | Nima Sayyadi Russell E. Connally Thomas S. Lawson Jingli Yuan Nicolle H. Packer James A. Piper Time-Gated Luminescent In Situ Hybridization (LISH): Highly Sensitive Detection of Pathogenic <i>Staphylococcus aureus</i> Molecules homogeneous luminescent in situ hybridisation TEGylated Europium chelate time-gated luminescent microscopy <i>Staphylococcus aureus</i> (<i>S. aureus</i>) detection |
| title | Time-Gated Luminescent In Situ Hybridization (LISH): Highly Sensitive Detection of Pathogenic <i>Staphylococcus aureus</i> |
| title_full | Time-Gated Luminescent In Situ Hybridization (LISH): Highly Sensitive Detection of Pathogenic <i>Staphylococcus aureus</i> |
| title_fullStr | Time-Gated Luminescent In Situ Hybridization (LISH): Highly Sensitive Detection of Pathogenic <i>Staphylococcus aureus</i> |
| title_full_unstemmed | Time-Gated Luminescent In Situ Hybridization (LISH): Highly Sensitive Detection of Pathogenic <i>Staphylococcus aureus</i> |
| title_short | Time-Gated Luminescent In Situ Hybridization (LISH): Highly Sensitive Detection of Pathogenic <i>Staphylococcus aureus</i> |
| title_sort | time gated luminescent in situ hybridization lish highly sensitive detection of pathogenic i staphylococcus aureus i |
| topic | homogeneous luminescent in situ hybridisation TEGylated Europium chelate time-gated luminescent microscopy <i>Staphylococcus aureus</i> (<i>S. aureus</i>) detection |
| url | https://www.mdpi.com/1420-3049/24/11/2083 |
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