Eun-Taek Han1,2, Risa Watanabe1, Jetsumon Sattabongkot3, Benjawan Khuntirat3, Jeeraphat Sirichaisinthop4, Hideyuki Iriko5, Ling Jin5, Satoru Takeo1 and Takafumi Tsuboi1,5,*
1Cell-Free Science and Technology Research Center
5Venture Business Laboratory, Ehime University, Matsuyama, Ehime 790-8577, Japan
2Department of Parasitology, Kangwon National University College of Medicine, Chunchon 200-701, Korea
3Department of Entomology, Armed Forces Research Institute of Medical Sciences, Bangkok 10400, Thailand
4Vector Borne Disease Training Center, Pra Budhabat, Saraburi 18120, Thailand
Loop-mediated isothermal amplification (LAMP), a novel nucleic acid amplification method, was developed for the clinical detection of four species of human malaria parasites: Plasmodium falciparum, P. vivax, P. malariae, and P. ovale. We evaluated the sensitivity and specificity of LAMP in comparison with the results of microscopic examination and nested PCR. LAMP showed a detection limit (analytical sensitivity) of 10 copies of the target 18S rRNA genes for P. malariae and P. ovale and 100 copies for the genus Plasmodium, P. falciparum, and P. vivax. LAMP detected malaria parasites in 67 of 68 microscopically positive blood samples (sensitivity, 98.5%) and 3 of 53 microscopically negative samples (specificity, 94.3%), in good agreement with the results of nested PCR. The LAMP reactions yielded results within about 26 min, on average, for detection of the genus Plasmodium, 32 min for P. falciparum, 31 min for P. vivax, 35 min for P. malariae, and 36 min for P. ovale. Accordingly, in comparison to the results obtained by microscopy, LAMP had a similar sensitivity and a greater specificity and LAMP yielded results similar to those of nested PCR in a shorter turnaround time. Because it can be performed with a simple technology, i.e., with heat-treated blood as the template, reaction in a water bath, and inspection of the results by the naked eye because of the use of a fluorescent dye, LAMP may provide a simple and reliable test for routine screening for malaria parasites in both clinical laboratories and malaria clinics in areas where malaria is endemic.