TY - JOUR A1 - Balint, Miklos A1 - Marton, Orsolya A1 - Schatz, Marlene A1 - Düring, Rolf-Alexander A1 - Grossart, Hans-Peter T1 - Proper experimental design requires randomization/balancing of molecular ecology experiments JF - Ecology and evolution N2 - Properly designed (randomized and/or balanced) experiments are standard in ecological research. Molecular methods are increasingly used in ecology, but studies generally do not report the detailed design of sample processing in the laboratory. This may strongly influence the interpretability of results if the laboratory procedures do not account for the confounding effects of unexpected laboratory events. We demonstrate this with a simple experiment where unexpected differences in laboratory processing of samples would have biased results if randomization in DNA extraction and PCR steps do not provide safeguards. We emphasize the need for proper experimental design and reporting of the laboratory phase of molecular ecology research to ensure the reliability and interpretability of results. KW - batch effect KW - bias KW - DNA extraction KW - environmental DNA KW - laboratory practice KW - lake community KW - metabarcoding KW - nondemonic intrusions KW - PCR KW - sediment Y1 - 2018 U6 - https://doi.org/10.1002/ece3.3687 SN - 2045-7758 VL - 8 IS - 3 SP - 1786 EP - 1793 PB - Wiley CY - Hoboken ER - TY - GEN A1 - Bálint, Miklós A1 - Márton, Orsolya A1 - Schatz, Marlene A1 - Düring, Rolf-Alexander A1 - Grossart, Hans-Peter T1 - Proper experimental design requires randomization/balancing of molecular ecology experiments T2 - Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe N2 - Properly designed (randomized and/or balanced) experiments are standard in ecological research. Molecular methods are increasingly used in ecology, but studies generally do not report the detailed design of sample processing in the laboratory. This may strongly influence the interpretability of results if the laboratory procedures do not account for the confounding effects of unexpected laboratory events. We demonstrate this with a simple experiment where unexpected differences in laboratory processing of samples would have biased results if randomization in DNA extraction and PCR steps do not provide safeguards. We emphasize the need for proper experimental design and reporting of the laboratory phase of molecular ecology research to ensure the reliability and interpretability of results. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 616 KW - batch effect KW - bias KW - DNA extraction KW - environmental DNA KW - laboratory practice KW - lake community KW - metabarcoding KW - nondemonic intrusions KW - PCR KW - sediment Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-423878 SN - 1866-8372 IS - 616 ER - TY - JOUR A1 - Kersting, Sebastian A1 - Rausch, Valentina A1 - Bier, Frank Fabian A1 - von Nickisch-Rosenegk, Markus T1 - Rapid detection of Plasmodium falciparum with isothermal recombinase polymerase amplification and lateral flow analysis JF - Malaria journal N2 - Background: Nucleic acid amplification is the most sensitive and specific method to detect Plasmodium falciparum. However the polymerase chain reaction remains laboratory-based and has to be conducted by trained personnel. Furthermore, the power dependency for the thermocycling process and the costly equipment necessary for the read-out are difficult to cover in resource-limited settings. This study aims to develop and evaluate a combination of isothermal nucleic acid amplification and simple lateral flow dipstick detection of the malaria parasite for point-of-care testing. Methods: A specific fragment of the 18S rRNA gene of P. falciparum was amplified in 10 min at a constant 38 C using the isothermal recombinase polymerase amplification (RPA) method. With a unique probe system added to the reaction solution, the amplification product can be visualized on a simple lateral flow strip without further labelling. The combination of these methods was tested for sensitivity and specificity with various Plasmodium and other protozoa/bacterial strains, as well as with human DNA. Additional investigations were conducted to analyse the temperature optimum, reaction speed and robustness of this assay. Results: The lateral flow RPA (LF-RPA) assay exhibited a high sensitivity and specificity. Experiments confirmed a detection limit as low as 100 fg of genomic P. falciparum DNA, corresponding to a sensitivity of approximately four parasites per reaction. All investigated P. falciparum strains (n = 77) were positively tested while all of the total 11 non-Plasmodium samples, showed a negative test result. The enzymatic reaction can be conducted under a broad range of conditions from 30-45 degrees C with high inhibitory concentration of known PCR inhibitors. A time to result of 15 min from start of the reaction to read-out was determined. Conclusions: Combining the isothermal RPA and the lateral flow detection is an approach to improve molecular diagnostic for P. falciparum in resource-limited settings. The system requires none or only little instrumentation for the nucleic acid amplification reaction and the read-out is possible with the naked eye. Showing the same sensitivity and specificity as comparable diagnostic methods but simultaneously increasing reaction speed and dramatically reducing assay requirements, the method has potential to become a true point-of-care test for the malaria parasite. KW - Plasmodium falciparum KW - Recombinase polymerase amplification KW - RPA KW - PCR KW - Lateral flow KW - Point-of-care testing KW - Rapid test KW - Isothermal nucleic acid amplification Y1 - 2014 U6 - https://doi.org/10.1186/1475-2875-13-99 SN - 1475-2875 VL - 13 PB - BioMed Central CY - London ER - TY - GEN A1 - Kersting, Sebastian A1 - Rausch, Valentina A1 - Bier, Frank Fabian A1 - von Nickisch-Rosenegk, Markus T1 - Rapid detection of Plasmodium falciparum with isothermal recombinase polymerase amplification and lateral flow analysis T2 - Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe N2 - Background Nucleic acid amplification is the most sensitive and specific method to detect Plasmodium falciparum. However the polymerase chain reaction remains laboratory-based and has to be conducted by trained personnel. Furthermore, the power dependency for the thermocycling process and the costly equipment necessary for the read-out are difficult to cover in resource-limited settings. This study aims to develop and evaluate a combination of isothermal nucleic acid amplification and simple lateral flow dipstick detection of the malaria parasite for point-of-care testing. Methods A specific fragment of the 18S rRNA gene of P. falciparum was amplified in 10 min at a constant 38°C using the isothermal recombinase polymerase amplification (RPA) method. With a unique probe system added to the reaction solution, the amplification product can be visualized on a simple lateral flow strip without further labelling. The combination of these methods was tested for sensitivity and specificity with various Plasmodium and other protozoa/bacterial strains, as well as with human DNA. Additional investigations were conducted to analyse the temperature optimum, reaction speed and robustness of this assay. Results The lateral flow RPA (LF-RPA) assay exhibited a high sensitivity and specificity. Experiments confirmed a detection limit as low as 100 fg of genomic P. falciparum DNA, corresponding to a sensitivity of approximately four parasites per reaction. All investigated P. falciparum strains (n = 77) were positively tested while all of the total 11 non-Plasmodium samples, showed a negative test result. The enzymatic reaction can be conducted under a broad range of conditions from 30-45°C with high inhibitory concentration of known PCR inhibitors. A time to result of 15 min from start of the reaction to read-out was determined. Conclusions Combining the isothermal RPA and the lateral flow detection is an approach to improve molecular diagnostic for P. falciparum in resource-limited settings. The system requires none or only little instrumentation for the nucleic acid amplification reaction and the read-out is possible with the naked eye. Showing the same sensitivity and specificity as comparable diagnostic methods but simultaneously increasing reaction speed and dramatically reducing assay requirements, the method has potential to become a true point-of-care test for the malaria parasite. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 948 KW - Plasmodium falciparum KW - Recombinase polymerase amplification KW - RPA KW - PCR KW - lateral flow KW - point-of-care testing KW - rapid test KW - isothermal nucleic acid amplification Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-431562 SN - 1866-8372 IS - 948 ER -