A reliable and rapid method for molecular detection of malarial parasites using microwave irradiation and loop mediated isothermal amplification.

Julia R Port,Selorme Adukpo,Thirumalaisamy P Velavan,Christian Nguetse

doi:10.1186/1475-2875-13-454

Abstract

BackgroundImproved living conditions together with appropriate diagnosis can reduce avoidable malarial deaths substantially. Microscopy remains the gold standard in the diagnosis of malaria. However, rapid molecular diagnostic tests (RmDT) are becoming increasingly important and will, most likely, be the diagnostic techniques of choice in the next years.MethodsIn this study, a rapid and reliable nucleic acid extraction procedure from human blood and malarial parasites using microwave irradiation as a promising platform is described. In addition, a tailored loop mediated isothermal amplification (LAMP) methodology that utilizes hydroxynaphthol blue (HNB) and Bst 2.0 DNA polymerases in molecular detection of malarial parasites is described.ResultsFollowing microwave irradiation for DNA isolation, conventional PCR assays were able to detect up to five malaria parasites/μl. The LAMP methodology described here was capable to detect as low as one Plasmodium falciparum parasite/μl after DNA extraction by microwave irradiation. A turnover time of 45 minutes from nucleic acid extraction to final visual read-out was achieved.ConclusionsThe described procedure offers a cheap, simple and fast method of molecular detection of malaria parasites. This test can easily be performed in basic laboratories. The methodology has been validated as a proof of concept and has specifically be developed for use at low-resource settings. Such RmDTs may aid health providers to make timely therapeutic interventions in malaria endemic regions.

Highlights

Improved living conditions together with appropriate diagnosis can reduce avoidable malarial deaths substantially
Microscopy of blood smears is still considered the gold standard for diagnosing malaria infections
In 2006, a study described a first set of loop-mediated isothermal amplification (LAMP) primers for malaria [13] and LAMP specific primers for four Plasmodium species infecting humans were reported in 2007 [14], all targeting the 18 s rRNA region of the Plasmodium species

Summary

Introduction

Improved living conditions together with appropriate diagnosis can reduce avoidable malarial deaths substantially. A commercial kit for malaria LAMP is currently available from Eiken Chemical Co., Ltd., targeting this genetic region of plasmodia. This LAMP assay has successfully been tested under field conditions for both the18s rRNA [16] and the mtDNA primers [17,18] and been successful in clinical evaluation of symptomatic and asymptomatic infections [10]. LAMP amplification causes production of magnesium pyro-phosphate precipitates, which may be recorded as a rise in turbidity Such observations are difficult to make for untrained staff and an extra step of visualization would be advantageous. Compared to SYBR Green© and calcein, HNB is the cheapest dye and successful use of HNB LAMP has recently been demonstrated to be effective in detecting amplified DNA from malaria parasites [21]

Methods

Results

Discussion

Conclusion