Rapid detection of Plasmodium falciparum with isothermal recombinase polymerase amplification and lateral flow analysis

  • 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 otherBackground: 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.show moreshow less

Export metadata

Additional Services

Share in Twitter Search Google Scholar Statistics
Metadaten
Author:Sebastian Kersting, Valentina Rausch, Frank Fabian BierORCiDGND, Markus von Nickisch-Rosenegk
DOI:https://doi.org/10.1186/1475-2875-13-99
ISSN:1475-2875 (print)
Pubmed Id:http://www.ncbi.nlm.nih.gov/pubmed?term=24629133
Parent Title (English):Malaria journal
Publisher:BioMed Central
Place of publication:London
Document Type:Article
Language:English
Year of first Publication:2014
Year of Completion:2014
Release Date:2017/03/27
Tag:Isothermal nucleic acid amplification; Lateral flow; PCR; Plasmodium falciparum; Point-of-care testing; RPA; Rapid test; Recombinase polymerase amplification
Volume:13
Pagenumber:9
Funder:German Federal Ministry of Education and Research [03IS2201A, 03IS2201B]
Organizational units:Mathematisch-Naturwissenschaftliche Fakultät / Institut für Biochemie und Biologie
Peer Review:Referiert
Publication Way:Open Access
Notes extern:Zweitveröffentlichung in der Schriftenreihe Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe ; 948