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

Abstract

BackgroundNucleic 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.MethodsA 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.ResultsThe 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.ConclusionsCombining 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.

Quantification of HIV-1 DNA Using Real-Time Recombinase Polymerase Amplification

“…4,5 For these reasons, a number of recent reports have proposed RPA-based strategies for the detection of pathogens. [6][7][8][9][10] Although some papers demonstrate a relationship between nucleic acid concentration and onset of amplification, 10,11 to the best of our knowledge, RPA has not yet been implemented to quantify sample concentration using a standard curve. Moreover, the accuracy with which samples can be quantified with real-time RPA has not yet been characterized.…”

Loop-mediated isothermal amplification based approach as an alternative to recombinase polymerase amplification based detection of Mangalitza component in food products

“…Each technique has its pros and cons. As for sample preparation, RPA is extremely insensitive to the quality of the DNA (KERSTING et al, 2014), even homogenised, unprocessed sausage sample can serve as a template for RPA reaction (SZÁNTÓ-EGÉSZ et al, 2016). Although LAMP produced successful amplifi cation from raw Mangalitza liver paté (Fig.…”

“…Amplifi cation conditions for 50 μl RPA reaction (TwistDx, UK) were 39 °C for 30 min. RPA amplifi ed product was detected by Universal MileniaHybriDetect (MileniaBiotec, Germany) (KERSTING et al, 2014). LAMP primers were designed by LAMP Designer 1.12 (http://www.premierbiosoft.…”

Application of Isothermal Amplification Techniques for Identification of Madurella mycetomatis, the Prevalent Agent of Human Mycetoma

“…After amplification, the RPA product can be detected within as short a time as 15 to 40 min (25,26). The method has been successfully applied for the world's major infectious diseases, including HIV, malaria, and tuberculosis (26)(27)(28)(29).…”