Use of Malachite Green-Loop Mediated Isothermal Amplification for Detection of Plasmodium spp. Parasites

Abstract

Malaria elimination efforts are hampered by the lack of sensitive tools to detect infections with low-level parasitemia, usually below the threshold of standard diagnostic methods, microscopy and rapid diagnostic tests. Isothermal nucleic acid amplification assays such as the loop-mediated isothermal amplification (LAMP), are well suited for field use as they do not require thermal cyclers to run the test. However, the use of specialized equipment, as described by many groups, reduces the versatility of the LAMP technique as a simple tool for use in endemic countries. In this study, the use of the malachite green (MG) dye, as a visual endpoint readout, together with a simple mini heat block was evaluated for the detection of malaria parasites. The assay was performed for 1 hour at 63°C and the results scored by 3 independent human readers. The limit of detection of the assay was determined using well-quantified Plasmodium spp. infected reference samples and its utility in testing clinical samples was determined using 190 pre-treatment specimens submitted for reference diagnosis of imported malaria in the United States. Use of a simplified boil and spin methods of DNA extraction from whole blood and filter paper was also investigated. We demonstrate the accurate and sensitive detection of malaria parasites using this assay with a detection limit ranging between 1–8 parasites/μL, supporting its applicability for the detection of infections with low parasite burden. This assay is compatible with the use of a simple boil and spin sample preparation method from both whole blood and filter papers without a loss of sensitivity. The MG-LAMP assay described here has great potential to extend the reach of molecular tools to settings where they are needed.

Evaluation of the Illumigene Malaria LAMP: A Robust Molecular Diagnostic Tool for Malaria Parasites

“…However, like many other malaria LAMP platforms reported to date, the Illumi gene Malaria LAMP assays can test a limited number of samples per run. Therefore, scenarios that require the testing of a large number of samples may require other platforms such as the recently described colorimetric LAMP assays910 with the limitation that these high-throughput colorimetric malaria LAMP assays still require sample preparation and lyophilized reagents for ease of use in the field. As technology advances, it is possible to envision the possibility of adapting the Illumi gene reader to facilitate the testing of larger sample size.…”

“…The LAMP assay has been shown to be a highly sensitive and rapid molecular method and malaria LAMP assays have been developed for the detection of Plasmodium species5678. In an effort to simplify the LAMP assay for use in resource limited countries, several different formats of LAMP have been investigated and evaluated, including the use of colorimetric high throughput assays such as hydroxynaphthol blue9 and the malachite green dyes10.…”

LAMP kit for diagnosis of non-falciparum malaria in Plasmodium ovale infected patients

“…The limit of detection of P. ovale malaria was determined between 2 and 0.8 parasites/µl of blood, even more sensitive than the results obtained in previous studies about LoDs for P. ovale , which were performed with malachite green-LAMP or with ultraviolet (UV) light as reading methods (3 and 10, respectively) [16, 18]. …”

Loop-mediated isothermal amplification (LAMP): a versatile technique for detection of micro-organisms

“…Other dyes such as malachite green dye (Sriworarat et al 2015;Lucchi et al 2016;Nzelu et al 2016), Picogreen dye (Batra et al 2015), Gelred dye (Wozniakowski et al 2013;Wassermann et al 2014), SYTO fluorescent dye (Liu et al 2013;Watts et al 2014;Mwendwa et al 2017) Zhou et al, 2014). The reaction between free calcein and magnesium ions intensified the fluorescence of LAMP product (ii.)…”