A new visually improved and sensitive loop mediated isothermal amplification (LAMP) for diagnosis of symptomatic falciparum malaria

Graphical abstract

A new 18s rDNA based primer set found to be more effective for diagnosis of symptomatic falciparum malaria. Introduction of Hydroxy Napthol Blue has improved visual detection.

Highlights

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  Introduction of Hydroxy Nepthol Blue (HNB) has lead easy and simple naked eye visualization of the LAMP amplicons.

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  The new primer set was found to be 99.1% sensitive and 99% specific in comparison with microscopy.

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  In comparison with Real Time PCR, sensitivity was 98.1% along with 100% specificity.

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  This new method can detect at least five parasite per μL of blood.

Abstract

Molecular diagnosis of malaria by nucleotide amplification requires sophisticated and expensive instruments, typically found only in well-established laboratories. Loop-mediated isothermal amplification (LAMP) has provided a new platform for an easily adaptable molecular technique for molecular diagnosis of malaria without the use of expensive instruments. A new primer set has been designed targeting the 18S rRNA gene for the detection of Plasmodium falciparum in whole blood samples. The efficacy of LAMP using the new primer set was assessed in this study in comparison to that of a previously described set of LAMP primers as well as with microscopy and real-time PCR as reference methods for detecting P. falciparum. Pre-addition of hydroxy napthol blue (HNB) in the LAMP reaction caused a distinct color change, thereby improving the visual detection system. The new LAMP assay was found to be 99.1% sensitive compared to microscopy and 98.1% when compared to real-time PCR. Meanwhile, its specificity was 99% and 100% in contrast to microscopy and real-time PCR, respectively. Moreover, the LAMP method was in very good agreement with microscopy and real-time PCR (0.94 and 0.98, respectively). This new LAMP method can detect at least 5 parasites/μL of infected blood within 35 min, while the other LAMP method tested in this study, could detect a minimum of 100 parasites/μL of human blood after 60 min of amplification. Thus, the new method is sensitive and specific, can be carried out in a very short time, and can substitute PCR in healthcare clinics and standard laboratories.