Abstract
BackgroundA simple and accurate molecular diagnostic method for malaria is urgently needed due to the limitations of conventional microscopic examination. In this study, we demonstrate a new diagnostic procedure for human malaria using loop mediated isothermal amplification (LAMP) and the MinION™ nanopore sequencer.MethodsWe generated specific LAMP primers targeting the 18S–rRNA gene of all five human Plasmodium species including two P. ovale subspecies (P. falciparum, P. vivax, P. ovale wallikeri, P. ovale curtisi, P. knowlesi and P. malariae) and examined human blood samples collected from 63 malaria patients in Indonesia. Additionally, we performed amplicon sequencing of our LAMP products using MinION™ nanopore sequencer to identify each Plasmodium species.ResultsOur LAMP method allowed amplification of all targeted 18S–rRNA genes of the reference plasmids with detection limits of 10–100 copies per reaction. Among the 63 clinical samples, 54 and 55 samples were positive by nested PCR and our LAMP method, respectively. Identification of the Plasmodium species by LAMP amplicon sequencing analysis using the MinION™ was consistent with the reference plasmid sequences and the results of nested PCR.ConclusionsOur diagnostic method combined with LAMP and MinION™ could become a simple and accurate tool for the identification of human Plasmodium species, even in resource-limited situations.
Highlights
A simple and accurate molecular diagnostic method for malaria is urgently needed due to the limitations of conventional microscopic examination
The first confirmed case of human P. knowlesi infection was reported in 1965 [4], it has become clear from the results of a retrospective investigation in 2004 using polymerase chain reaction (PCR) methods that many P. knowlesi human infection cases in Malaysia have been misdiagnosed as P. malariae by microscopy [5]
Plasmid construction The templates used for the analytical loop mediated isothermal amplification (LAMP) reactions were six sets of pEX-A2J1 plasmids harboring partial sequences within the 18S–rRNA of six human Plasmodium parasites, P. falciparum, P. vivax, P. ovale curtisi, P. ovale wallikeri, P. knowlesi and P. malariae which were constructed by Eurofines Genomics Co., Ltd. (Tokyo, Japan) based on the reference sequences (GenBank accession numbers: P. falciparum, HQ283212.1; P. vivax, KF018662.1; P. ovale curtisi, KF696370.1; P. ovale wallikeri, KJ619947.1; P. knowlesi, KJ917903.1; and P. malariae, LT615260.1)
Summary
A simple and accurate molecular diagnostic method for malaria is urgently needed due to the limitations of conventional microscopic examination. Malaria is a severe public health problem prevalent in tropical and subtropical areas worldwide. It is a mosquitoborne infectious disease caused by the following five protozoan parasites: Plasmodium falciparum, P. vivax, P. malariae, P. ovale and P. knowlesi. Compared with molecular diagnostic methods such as polymerase chain reaction (PCR), only half the number of patients with malaria are correctly diagnosed via microscopy in endemic areas [1, 2]. The first confirmed case of human P. knowlesi infection was reported in 1965 [4], it has become clear from the results of a retrospective investigation in 2004 using PCR methods that many P. knowlesi human infection cases in Malaysia have been misdiagnosed as P. malariae by microscopy [5]
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