3D Printed Monolithic Microreactors for Real-Time Detection of <em>Klebsiella pneumoniae</em> and the Resistance Gene <em>bla</em><sub>NDM-1</sub> by Recombinase Polymerase Amplification
We investigate the compatibility of three 3D printing materials towards real-time recombinase polymerase amplification (rtRPA). Both the general ability of the rtRPA reaction to occur while in contact with the cured 3D printing materials as well as the residual autofluorescence and fluorescence drif...
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| Format: | Article |
| Language: | English |
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MDPI AG
2020-06-01
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| Series: | Micromachines |
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| Online Access: | https://www.mdpi.com/2072-666X/11/6/595 |
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| author | Ole Behrmann Matthias Hügle Franz Eckardt Iris Bachmann Cecilia Heller Marina Schramm Carrie Turner Frank T. Hufert Gregory Dame |
| author_facet | Ole Behrmann Matthias Hügle Franz Eckardt Iris Bachmann Cecilia Heller Marina Schramm Carrie Turner Frank T. Hufert Gregory Dame |
| author_sort | Ole Behrmann |
| collection | DOAJ |
| description | We investigate the compatibility of three 3D printing materials towards real-time recombinase polymerase amplification (rtRPA). Both the general ability of the rtRPA reaction to occur while in contact with the cured 3D printing materials as well as the residual autofluorescence and fluorescence drift in dependence on post curing of the materials is characterized. We 3D printed monolithic rtRPA microreactors and subjected the devices to different post curing protocols. Residual autofluorescence and drift, as well as rtRPA kinetics, were then measured in a custom-made mobile temperature-controlled fluorescence reader (mTFR). Furthermore, we investigated the effects of storage on the devices over a 30-day period. Finally, we present the single- and duplex rtRPA detection of both the organism-specific <i>Klebsiella</i> haemolysin (<i>khe)</i> gene and the New Delhi metallo-β-lactamase 1 (<i>bla</i><sub>NDM-1</sub>) gene from <i>Klebsiella pneumoniae</i>. Results: No combination of 3D printing resin and post curing protocol completely inhibited the rtRPA reaction. The autofluorescence and fluorescence drift measured were found to be highly dependent on printing material and wavelength. Storage had the effect of decreasing the autofluorescence of the investigated materials. Both <i>khe</i> and <i>bla</i><sub>NDM-1</sub> were successfully detected by single- and duplex-rtRPA inside monolithic rtRPA microreactors printed from NextDent Ortho Clear (NXOC). The reaction kinetics were found to be close to those observed for rtRPA performed in a microcentrifuge tube without the need for mixing during amplification. Singleplex assays for both <i>khe</i> and <i>bla</i><sub>NDM-1</sub> achieved a limit of detection of 2.5 × 10<sup>1</sup> DNA copies while the duplex assay achieved 2.5 × 10<sup>1</sup> DNA copies for <i>khe</i> and 2.5 × 10<sup>2</sup> DNA copies for <i>bla</i><sub>NDM-1</sub>. Impact: We expand on the state of the art by demonstrating a technology that can manufacture monolithic microfluidic devices that are readily suitable for rtRPA. The devices exhibit very low autofluorescence and fluorescence drift and are compatible with RPA chemistry without the need for any surface pre-treatment such as blocking with, e.g., BSA or PEG. |
| first_indexed | 2024-03-10T19:06:43Z |
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| language | English |
| last_indexed | 2024-03-10T19:06:43Z |
| publishDate | 2020-06-01 |
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| spelling | doaj.art-0fdc8a26845941e188d493e4e710f9b82023-11-20T04:03:37ZengMDPI AGMicromachines2072-666X2020-06-0111659510.3390/mi110605953D Printed Monolithic Microreactors for Real-Time Detection of <em>Klebsiella pneumoniae</em> and the Resistance Gene <em>bla</em><sub>NDM-1</sub> by Recombinase Polymerase AmplificationOle Behrmann0Matthias Hügle1Franz Eckardt2Iris Bachmann3Cecilia Heller4Marina Schramm5Carrie Turner6Frank T. Hufert7Gregory Dame8Institute of Microbiology and Virology, Brandenburg Medical School Theodor Fontane, 16816 Neuruppin, GermanyInstitute of Microbiology and Virology, Brandenburg Medical School Theodor Fontane, 16816 Neuruppin, GermanyInstitute of Microbiology and Virology, Brandenburg Medical School Theodor Fontane, 16816 Neuruppin, GermanyInstitute of Microbiology and Virology, Brandenburg Medical School Theodor Fontane, 16816 Neuruppin, GermanyInstitute of Microbiology and Virology, Brandenburg Medical School Theodor Fontane, 16816 Neuruppin, GermanyInstitute of Microbiology and Virology, Brandenburg Medical School Theodor Fontane, 16816 Neuruppin, GermanyNational Infections Service, Public Health England, Porton Down SP4 0JG, UKInstitute of Microbiology and Virology, Brandenburg Medical School Theodor Fontane, 16816 Neuruppin, GermanyInstitute of Microbiology and Virology, Brandenburg Medical School Theodor Fontane, 16816 Neuruppin, GermanyWe investigate the compatibility of three 3D printing materials towards real-time recombinase polymerase amplification (rtRPA). Both the general ability of the rtRPA reaction to occur while in contact with the cured 3D printing materials as well as the residual autofluorescence and fluorescence drift in dependence on post curing of the materials is characterized. We 3D printed monolithic rtRPA microreactors and subjected the devices to different post curing protocols. Residual autofluorescence and drift, as well as rtRPA kinetics, were then measured in a custom-made mobile temperature-controlled fluorescence reader (mTFR). Furthermore, we investigated the effects of storage on the devices over a 30-day period. Finally, we present the single- and duplex rtRPA detection of both the organism-specific <i>Klebsiella</i> haemolysin (<i>khe)</i> gene and the New Delhi metallo-β-lactamase 1 (<i>bla</i><sub>NDM-1</sub>) gene from <i>Klebsiella pneumoniae</i>. Results: No combination of 3D printing resin and post curing protocol completely inhibited the rtRPA reaction. The autofluorescence and fluorescence drift measured were found to be highly dependent on printing material and wavelength. Storage had the effect of decreasing the autofluorescence of the investigated materials. Both <i>khe</i> and <i>bla</i><sub>NDM-1</sub> were successfully detected by single- and duplex-rtRPA inside monolithic rtRPA microreactors printed from NextDent Ortho Clear (NXOC). The reaction kinetics were found to be close to those observed for rtRPA performed in a microcentrifuge tube without the need for mixing during amplification. Singleplex assays for both <i>khe</i> and <i>bla</i><sub>NDM-1</sub> achieved a limit of detection of 2.5 × 10<sup>1</sup> DNA copies while the duplex assay achieved 2.5 × 10<sup>1</sup> DNA copies for <i>khe</i> and 2.5 × 10<sup>2</sup> DNA copies for <i>bla</i><sub>NDM-1</sub>. Impact: We expand on the state of the art by demonstrating a technology that can manufacture monolithic microfluidic devices that are readily suitable for rtRPA. The devices exhibit very low autofluorescence and fluorescence drift and are compatible with RPA chemistry without the need for any surface pre-treatment such as blocking with, e.g., BSA or PEG.https://www.mdpi.com/2072-666X/11/6/595recombinase polymerase amplification (RPA)antibiotic resistance3D printing |
| spellingShingle | Ole Behrmann Matthias Hügle Franz Eckardt Iris Bachmann Cecilia Heller Marina Schramm Carrie Turner Frank T. Hufert Gregory Dame 3D Printed Monolithic Microreactors for Real-Time Detection of <em>Klebsiella pneumoniae</em> and the Resistance Gene <em>bla</em><sub>NDM-1</sub> by Recombinase Polymerase Amplification Micromachines recombinase polymerase amplification (RPA) antibiotic resistance 3D printing |
| title | 3D Printed Monolithic Microreactors for Real-Time Detection of <em>Klebsiella pneumoniae</em> and the Resistance Gene <em>bla</em><sub>NDM-1</sub> by Recombinase Polymerase Amplification |
| title_full | 3D Printed Monolithic Microreactors for Real-Time Detection of <em>Klebsiella pneumoniae</em> and the Resistance Gene <em>bla</em><sub>NDM-1</sub> by Recombinase Polymerase Amplification |
| title_fullStr | 3D Printed Monolithic Microreactors for Real-Time Detection of <em>Klebsiella pneumoniae</em> and the Resistance Gene <em>bla</em><sub>NDM-1</sub> by Recombinase Polymerase Amplification |
| title_full_unstemmed | 3D Printed Monolithic Microreactors for Real-Time Detection of <em>Klebsiella pneumoniae</em> and the Resistance Gene <em>bla</em><sub>NDM-1</sub> by Recombinase Polymerase Amplification |
| title_short | 3D Printed Monolithic Microreactors for Real-Time Detection of <em>Klebsiella pneumoniae</em> and the Resistance Gene <em>bla</em><sub>NDM-1</sub> by Recombinase Polymerase Amplification |
| title_sort | 3d printed monolithic microreactors for real time detection of em klebsiella pneumoniae em and the resistance gene em bla em sub ndm 1 sub by recombinase polymerase amplification |
| topic | recombinase polymerase amplification (RPA) antibiotic resistance 3D printing |
| url | https://www.mdpi.com/2072-666X/11/6/595 |
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