LoopTag FRET Probe System for Multiplex qPCR Detection of Borrelia Species
Abstract
:1. Introduction
2. Materials and Methods
2.1. qPCR Reaction and Melting Curve Analysis
2.2. Borrelia Strains and DNA Extraction and qPCR Conditions
Data Analysis
3. Results
3.1. Mechanism of the LoopTag System
3.2. Amplification of the Flagellin Gene
3.3. Differentiation of Borrelia Species Based on Melting Curve Analysis
3.4. Internal Control for the Detection and Differentiation of Borrelia Species
3.5. Sensitivity and Efficiency
4. Discussion
5. Conclusions
6. Patents
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
Abbreviations
A.U. | Arbitrary units |
BHQ2 | Black Hole Quencher 2 |
Cq | Cycle of quantification by SDM method |
estA1 | Heat-stable toxin I gene |
FRET | Foerster Resonance Energy Transfer |
qPCR | Quantitative PCR |
RFU | Relative fluorescence units [Arbitrary Unit] |
rMFI | Reference mean fluorescence value (see [47]) |
SDM | Second derivative maximum (see [42,48]) |
stx1 | Shigatoxin 1 gene |
Tm | Melting temperature |
Appendix A. Melting Point Analysis of Borrelia Specific Probes
Comparison | Difference (°C) | Lower | Upper | p |
---|---|---|---|---|
B. bavariensis vs. B. afzelii | −1.55 | −1.98 | −1.12 | <0.001 |
B. bissettii vs. B. afzelii | −2.70 | −3.13 | −2.27 | <0.001 |
B. burgdorferi vs. B. afzelii | 0.46 | 0.02 | 0.89 | 0.03 |
B. lusitaniae vs. B. afzelii | −3.48 | −3.91 | −3.04 | <0.001 |
B. spielmanii vs. B. afzelii | −4.24 | −4.67 | −3.81 | <0.001 |
B. bissettii vs. B. bavariensis | −1.15 | −1.58 | −0.72 | <0.001 |
B. burgdorferi vs. B. bavariensis | 2.00 | 1.57 | 2.44 | <0.001 |
B. garinii vs. B. bavariensis | 1.56 | 1.12 | 1.99 | <0.001 |
B. kurtenbachii vs. B. bavariensis | 1.52 | 1.09 | 1.96 | <0.001 |
B. lusitaniae vs. B. bavariensis | −1.93 | −2.36 | −1.50 | <0.001 |
B. spielmanii vs. B. bavariensis | −2.69 | −3.12 | −2.26 | <0.001 |
B. valaisiana vs. B. bavariensis | 1.79 | 1.35 | 2.22 | <0.001 |
B. burgdorferi vs. B. bissettii | 3.15 | 2.72 | 3.59 | <0.001 |
B. garinii vs. B. bissettii | 2.71 | 2.27 | 3.14 | <0.001 |
B. kurtenbachii vs. B. bissettii | 2.68 | 2.24 | 3.11 | <0.001 |
B. lusitaniae vs. B. bissettii | −0.78 | −1.21 | −0.35 | <0.001 |
B. spielmanii vs. B. bissettii | −1.54 | −1.97 | −1.11 | <0.001 |
B. valaisiana vs. B. bissettii | 2.94 | 2.50 | 3.37 | <0.001 |
B. garinii vs. B. burgdorferi | −0.45 | −0.88 | −0.01 | 0.04 |
B. kurtenbachii vs. B. burgdorferi | −0.48 | −0.91 | −0.05 | 0.02 |
B. lusitaniae vs. B. burgdorferi | −3.93 | −4.37 | −3.50 | <0.001 |
B. spielmanii vs. B. burgdorferi | −4.69 | −5.13 | −4.26 | <0.001 |
B. lusitaniae vs. B. garinii | −3.49 | −3.92 | −3.05 | <0.001 |
B. spielmanii vs. B. garinii | −4.25 | −4.68 | −3.81 | <0.001 |
B. lusitaniae vs. B. kurtenbachii | −3.45 | −3.89 | −3.02 | <0.001 |
B. spielmanii vs. B. kurtenbachii | −4.22 | −4.65 | −3.78 | <0.001 |
B. spielmanii vs. B. lusitaniae | −0.76 | −1.20 | −0.33 | <0.001 |
B. valaisiana vs. B. lusitaniae | 3.71 | 3.28 | 4.15 | <0.001 |
B. valaisiana vs. B. spielmanii | 4.48 | 4.04 | 4.91 | <0.001 |
Appendix B. Duplex LoopTag qPCR on a Planar Microbead Assays
- Base primer sequences were taken from [49] and transformed into a LoopTag probe system as described above. An important difference is that a part of the probes are bound to the surface of microbeads, so that they can function as catcher molecules (Table A2). The gene estA1 (length of amplification product: 158 bp) and stx1 (length of amplification product: 211 bp) were used in this proof-of-concept study.
- Colonies of E. coli strains containing genes estA1 and stx1 were inoculated in 1.5 mL LB medium. After incubation at 37 °C overnight cells were separated, re-suspended in 300 L of water, and lysed by heating at 99 °C for 10 min. Cell debris were removed by centrifugation and supernatant was stored at −20 °C. Thawed lysates were directly used as templates for PCR.
Function | Sequence |
---|---|
estA1 fw | BHQ2-ATCTACCAACTGAATCACTTGACTCTT (GC: 37%, 27 nt, Tm: 62.45 °C) |
estA1 rv | TTAATAACATCCAGCACAGG (GC: 40%, 20 nt, Tm: 55.57 °C) |
estA1 probe | 2B-AGTCTCTAATGTAATTTTCTCTTTTGGTAGAT-Atto647N (GC: 28%, 32 nt, Tm: 61.51 °C) |
stx1 fw | BHQ2-ATGTATGTTGCAGGGATCAGTCGT (GC: 45%, 24 nt, Tm: 64.57 °C) |
stx1 rv | AGAACGCCCACTGAGATCATC (GC: 52%, 21 nt, Tm: 62.41 °C) |
stx1 probe | 2B-GTCAACGAATGGCGATTTATCTGCATCCCGTACAT-Atto647N (GC: 45%, 35 nt, Tm: 62.41 °C) |
- Briefly, 300,000 microbeads were re-suspended in 100 L of 100 mM methylimidazole (MeIm, Sigma, Milwaukee, WI, USA) buffer (pH 7.0) containing 25 mg/mL N-(3-dimethy-laminopropyl)-N-ethylcarbodiimide hydrochloride (EDC, Sigma). Activated microbeads were incubated with streptavidin solution (300 g/mL in MeIm-buffer for 5 h at 50 °C) with continuous agitation for covalent cross-linking. After washing three times with TBST buffer, streptavidin-coated microbeads were ready for loading with biotinylated oligonucleotides. Microbeads were mixed with 100 L TBST buffer containing 100 nM of 5-bis-biotinylated and 3-Atto647N-labeled oligonucleotides (biomers.net, Ulm, Germany). After 15 min incubation at room temperature, unbound oligonucleotides were removed by washing microbeads three times with 200 L TBST. Finally, microbeads were exposed to 95 °C for 10 min.
- All PCR reactions were conducted within a volume of 20 L Biotherm polymerase buffer (Genecraft, Colone, Germany) containing 5 mM MgCl, 200 M of each dNTP, 250 M of BHQ2 labeled fw primer, 250 M of rv primer, 20–200 streptavidin-coupled microbeads with bound bis-biotinylated capture probes, 1 U Biotherm DNA-polymerase (Genecraft), and 1 L of E. coli lysate as template. The mixture was transferred into a cavity of a nucleolink TopYield strip (Nunc, Roskilde, Denmark), covered with 30 L of mineral oil, and the strip was placed into sockets of the heating and cooling unit. Here, a PCR was performed using the following program: 4 min, 94 °C, 40× (60 s 94 °C, 60 s 55 °C, 90 s 72 °C), 5 min 72 °C. We monitored microbead surface fluorescence while the temperature was increased at 1 °C/min starting from 35 °C to 90 °C. By use of the VideoScan technology fluorescence images of microbeads on the bottom of the well were taken after every cycle (55 °C). Images were evaluated with imaging processing software allowing the recognition of microbeads, the assignment to a population and the determination of their surface fluorescence.
- The mean fluorescence value for every population (rMFI → RFU) was plotted against the cycle number leading to the shown real-time kinetic curve. The amplification curve data were pre-processed and analyzed using the qpcR (v. 1.4.1) [50], MBmca, and chipPCR packages. After finishing real-time duplex PCR, a melting curve analysis was done as described in [39,43].
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Species | Melting Temperature (°C) |
---|---|
B. afzelii | 62.4 ± 0.1 |
B. bavariensis | 61.1 ± 0.4 |
B. bissettii | 60.2 ± 0.8 |
B. burgdorferii | 63.7 ± 0.7 |
B. garinii | 62.3 ± 0.3 |
B. kurtenbachii | 63.6 ± 0.3 |
B. lusitaniae | 59.7 ± 1 |
B. spielmanii | 59.1 ± 0.5 |
B. valaisiana | 62.4 ± 0.2 |
Borrelia | Species | DMAGE |
---|---|---|
Borrelia burgdorferi s.l. complex | B. afzelii | ≥10 |
B. bavariensis | ≥10 | |
B. bissettii | ≥10 | |
B. burgdorferi s.s. | ≥10 | |
B. garinii | PBr : approx. 10 | |
PHei: ≥10 | ||
TN: approx. 700 | ||
PRef: ≥10 | ||
PLa: ≥10 | ||
PWudII: ≥10 | ||
B. kurtenbachii | 100 | |
B. lusitaniae | ≥10 | |
B. spielmanii | ≥10 | |
B. valaisiana | ≥10 | |
Relapsing fever Borrelia | B. anserina | ≥400.000 * |
B. duttonii | ≥400.000 * | |
B. miyamotoi | Not detectable | |
B. parkerii | ≥400.000 * | |
B. recurrentis | Not detectable | |
B. turicatae | ≥200.000 * | |
Negative controls (other species) | E. coli (2 strains) | Not detectable |
Leptospira (2 strains) | Not detectable | |
Treponema phagedenis (2 strains) | Not detectable |
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Hanschmann, H.; Rödiger, S.; Kramer, T.; Hanschmann, K.; Steidle, M.; Fingerle, V.; Schmidt, C.; Lehmann, W.; Schierack, P. LoopTag FRET Probe System for Multiplex qPCR Detection of Borrelia Species. Life 2021, 11, 1163. https://doi.org/10.3390/life11111163
Hanschmann H, Rödiger S, Kramer T, Hanschmann K, Steidle M, Fingerle V, Schmidt C, Lehmann W, Schierack P. LoopTag FRET Probe System for Multiplex qPCR Detection of Borrelia Species. Life. 2021; 11(11):1163. https://doi.org/10.3390/life11111163
Chicago/Turabian StyleHanschmann, Henning, Stefan Rödiger, Toni Kramer, Katrin Hanschmann, Michael Steidle, Volker Fingerle, Carsten Schmidt, Werner Lehmann, and Peter Schierack. 2021. "LoopTag FRET Probe System for Multiplex qPCR Detection of Borrelia Species" Life 11, no. 11: 1163. https://doi.org/10.3390/life11111163