<i>LoopTag</i> FRET Probe System for Multiplex qPCR Detection of <i>Borrelia</i> Species
Background: Laboratory diagnosis of Lyme borreliosis refers to some methods with known limitations. Molecular diagnostics using specific nucleic acid probes may overcome some of these limitations. Methods: We describe the novel reporter fluorescence real-time polymerase chain reaction (PCR) probe sy...
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| Main Authors: | , , , , , , , , |
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| Format: | article |
| Language: | EN |
| Published: |
MDPI AG
2021
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| Subjects: | |
| Online Access: | https://doaj.org/article/a4d9894e284b430d8a85d72e2c4f4371 |
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| Summary: | Background: Laboratory diagnosis of Lyme borreliosis refers to some methods with known limitations. Molecular diagnostics using specific nucleic acid probes may overcome some of these limitations. Methods: We describe the novel reporter fluorescence real-time polymerase chain reaction (PCR) probe system <i>LoopTag</i> for detection of <i>Borrelia</i> species. Advantages of the <i>LoopTag</i> system include having cheap conventional fluorescence dyes, easy primer design, no restrictions for PCR product lengths, robustness, high sequence specificity, applicability for multiplex real-time PCRs, melting curve analysis (single nucleotide polymorphism analysis) over a large temperature range, high sensitivity, and easy adaptation of conventional PCRs. Results: Using the <i>LoopTag</i> probe system we were able to detect all nine tested European species belonging to the <i>Borrelia burgdorferi</i> (sensu lato) complex and differentiated them from relapsing fever <i>Borrelia</i> species. As few as 10 copies of <i>Borrelia</i> in one PCR reaction were detectable. Conclusion: We established a novel multiplex probe real-time PCR system, designated <i>LoopTag</i>, that is simple, robust, and incorporates melting curve analysis for the detection and in the differentiation of European species belonging to the <i>Borrelia burgdorferi</i> s.l. complex. |
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