Evaluation of Touchdown Loop-Mediated Isothermal Amplification for the Detection of <i>Giardia duodenalis</i>
<i>Giardia duodenalis</i> is a flagellated protozoan pathogen causing parasitic enteric disease outbreaks worldwide. Among detection methods, loop-mediated isothermal amplification (LAMP) has high selectivity and sensitivity, and the detection time is lower than that of conventional mole...
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MDPI AG
2025-05-01
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| author | Márton Hartdégen András József Laki Kolos Farkasvölgyi Kristóf Iván Judit Plutzer |
| author_facet | Márton Hartdégen András József Laki Kolos Farkasvölgyi Kristóf Iván Judit Plutzer |
| author_sort | Márton Hartdégen |
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| description | <i>Giardia duodenalis</i> is a flagellated protozoan pathogen causing parasitic enteric disease outbreaks worldwide. Among detection methods, loop-mediated isothermal amplification (LAMP) has high selectivity and sensitivity, and the detection time is lower than that of conventional molecular methods. In this study, three published <i>Giardia</i> LAMP primer sets were tested and adapted to touchdown LAMP conditions. The measurement time, the volume of reagents, the effect of the denaturation step, different kinds of polymerases, and the presence or absence of betaine on the reaction were tested and evaluated. Based on the results of this study, the 66–60 °C range touchdown LAMP with the use of betaine, 90 °C denaturation step, Bst 2.0 WarmStart<sup>®</sup> DNA Polymerase, and the primer set of Momoda et al. were the optimal conditions. We increased the analytical sensitivity of the LAMP reaction to 7.8- and 8-fold higher than the previously published methods for <i>G. duodenalis</i> assemblages A and B, with detection limits of 20 and 19.5 fg/assay, respectively, instead of 156 fg/assay. The detection time was less than 49 min for <i>G. duodenalis</i> assemblage A and less than 35 min for assemblage B, compared to the previously published 60 min. Our optimized LAMP protocol can be directly applied to improve <i>Giardia</i> LAMP tests in routine testing laboratories, could be implemented in standard diagnostic or environmental monitoring workflows, and can be used for the development of <i>Giardia</i> LAMP point-of-care devices or high-throughput systems. |
| format | Article |
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| institution | Kabale University |
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| language | English |
| publishDate | 2025-05-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Parasitologia |
| spelling | doaj-art-3d1abd87cab24b9eb17f98183ceda5f72025-08-20T03:27:32ZengMDPI AGParasitologia2673-67722025-05-01522510.3390/parasitologia5020025Evaluation of Touchdown Loop-Mediated Isothermal Amplification for the Detection of <i>Giardia duodenalis</i>Márton Hartdégen0András József Laki1Kolos Farkasvölgyi2Kristóf Iván3Judit Plutzer4Biomicrofluidics Laboratory, Faculty of Information Technology and Bionics, Pázmány Péter Catholic University, Práter Str. 50/A, 1083 Budapest, HungaryBiomicrofluidics Laboratory, Faculty of Information Technology and Bionics, Pázmány Péter Catholic University, Práter Str. 50/A, 1083 Budapest, HungaryBiomicrofluidics Laboratory, Faculty of Information Technology and Bionics, Pázmány Péter Catholic University, Práter Str. 50/A, 1083 Budapest, HungaryBiomicrofluidics Laboratory, Faculty of Information Technology and Bionics, Pázmány Péter Catholic University, Práter Str. 50/A, 1083 Budapest, HungaryBiomicrofluidics Laboratory, Faculty of Information Technology and Bionics, Pázmány Péter Catholic University, Práter Str. 50/A, 1083 Budapest, Hungary<i>Giardia duodenalis</i> is a flagellated protozoan pathogen causing parasitic enteric disease outbreaks worldwide. Among detection methods, loop-mediated isothermal amplification (LAMP) has high selectivity and sensitivity, and the detection time is lower than that of conventional molecular methods. In this study, three published <i>Giardia</i> LAMP primer sets were tested and adapted to touchdown LAMP conditions. The measurement time, the volume of reagents, the effect of the denaturation step, different kinds of polymerases, and the presence or absence of betaine on the reaction were tested and evaluated. Based on the results of this study, the 66–60 °C range touchdown LAMP with the use of betaine, 90 °C denaturation step, Bst 2.0 WarmStart<sup>®</sup> DNA Polymerase, and the primer set of Momoda et al. were the optimal conditions. We increased the analytical sensitivity of the LAMP reaction to 7.8- and 8-fold higher than the previously published methods for <i>G. duodenalis</i> assemblages A and B, with detection limits of 20 and 19.5 fg/assay, respectively, instead of 156 fg/assay. The detection time was less than 49 min for <i>G. duodenalis</i> assemblage A and less than 35 min for assemblage B, compared to the previously published 60 min. Our optimized LAMP protocol can be directly applied to improve <i>Giardia</i> LAMP tests in routine testing laboratories, could be implemented in standard diagnostic or environmental monitoring workflows, and can be used for the development of <i>Giardia</i> LAMP point-of-care devices or high-throughput systems.https://www.mdpi.com/2673-6772/5/2/25loop-mediated isothermal amplification (LAMP)touchdown LAMP<i>Giardia duodenalis</i>sensitivity analysisoptimization analysis |
| spellingShingle | Márton Hartdégen András József Laki Kolos Farkasvölgyi Kristóf Iván Judit Plutzer Evaluation of Touchdown Loop-Mediated Isothermal Amplification for the Detection of <i>Giardia duodenalis</i> Parasitologia loop-mediated isothermal amplification (LAMP) touchdown LAMP <i>Giardia duodenalis</i> sensitivity analysis optimization analysis |
| title | Evaluation of Touchdown Loop-Mediated Isothermal Amplification for the Detection of <i>Giardia duodenalis</i> |
| title_full | Evaluation of Touchdown Loop-Mediated Isothermal Amplification for the Detection of <i>Giardia duodenalis</i> |
| title_fullStr | Evaluation of Touchdown Loop-Mediated Isothermal Amplification for the Detection of <i>Giardia duodenalis</i> |
| title_full_unstemmed | Evaluation of Touchdown Loop-Mediated Isothermal Amplification for the Detection of <i>Giardia duodenalis</i> |
| title_short | Evaluation of Touchdown Loop-Mediated Isothermal Amplification for the Detection of <i>Giardia duodenalis</i> |
| title_sort | evaluation of touchdown loop mediated isothermal amplification for the detection of i giardia duodenalis i |
| topic | loop-mediated isothermal amplification (LAMP) touchdown LAMP <i>Giardia duodenalis</i> sensitivity analysis optimization analysis |
| url | https://www.mdpi.com/2673-6772/5/2/25 |
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