Abstract
BackgroundAs an alternative to PCR methods, LAMP is increasingly being used in the field of molecular diagnostics. Under isothermal conditions at 65 °C, the entire procedure takes approximately 30 min to complete. In this study, we establish a sensitive and visualized LAMP method in a closed-tube system for the detection of Plasmodium knowlesi.MethodsA total of 71 malaria microscopy positive blood samples collected in blood spots were obtained from the Sarawak State Health Department. Using 18s rRNA as the target gene, nested PCR and SYBR green I LAMP assay were performed following the DNA extraction. The colour changes of LAMP end products were observed by naked eyes.ResultsLAMP assay demonstrated a detection limit of 10 copies/µL in comparison with 100 copies/µL nested PCR. Of 71 P. knowlesi blood samples collected, LAMP detected 69 microscopy-positive samples. LAMP exhibited higher sensitivity than nested PCR assay. The SYBR green I LAMP assay was 97.1% sensitive (95% CI 90.2–99.7%) and 100% specific (95% CI 83.2–100%). Without opening the cap, incorporation of SYBR green I into the inner cap of the tube enabled the direct visualization of results upon completion of amplification. The positives instantaneously turned green while the negatives remained orange.ConclusionsThese results indicate that SYBR green I LAMP assay is a convenient diagnosis tool for the detection of P. knowlesi in remote settings.
Highlights
As an alternative to PCR methods, Loop-mediated isothermal amplification (LAMP) is increasingly being used in the field of molecular diagnostics
The results showed that none of these samples were amplified by SYBR green I LAMP assay
The detection limit of the SYBR green I LAMP assay was lower than nested PCR, which had a detection limit of 10–20 parasites/μL of P. knowlesi, well below the threshold of detection by microscopy with ~ 30 parasites/μL
Summary
As an alternative to PCR methods, LAMP is increasingly being used in the field of molecular diagnostics. We establish a sensitive and visualized LAMP method in a closed-tube system for the detection of Plasmodium knowlesi. Malaria is one of the global health diseases caused by the deadly parasite Plasmodium spp. which requires a rapid and reliable diagnosis tool. In Malaysia, most malaria cases are contributed by Plasmodium knowlesi which was previously thought to only infect monkeys. This parasite has been shown to infect humans as well [1, 2]. Some rapid test kits were made available as a tool for malaria diagnosis. Malaria cases may be undetected due to low parasitaemia and improper storage conditions of the kits. The results may vary depending on different types of rapid test kits used. Patients with P. knowlesi could be misdiagnosed as non-P
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