Detection of four Plasmodium species by genus- and species-specific loop-mediated isothermal amplification for clinical diagnosis

Abstract

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.

FIG. 1.

Location and sequence of LAMP targets and priming sites for Plasmodium genus (A) and four Plasmodium species (B). (A) The locations of the priming sites by the Plasmodium genus-specific primer set in the reference sequence (GenBank accession no. M19173.1) are indicated by arrows. (B) Partial sequence alignment of the 18S rRNA genes of four human malaria parasites, P. falciparum (Pf; GenBank accession no. M19173.1), P. vivax (Pv; GenBank accession no. U03079), P. malariae (Pm; GenBank accession no. M54897), and P. ovale (Po; GenBank accession no. L48986), along with the species-specific primer annealing sites.

FIG. 2.

Sensitivities of Plasmodium genus- and species-specific real-time LAMP assays performed with serial dilutions of plasmid DNA (10⁶ copies to 1 copy per reaction) containing an 18S rRNA gene. (A) Amplification with a Plasmodium genus-specific primer set. One representative result of four replicates is shown. Samples contained a plasmid harboring the P. falciparum 18S rRNA gene. (B) Plot of the mean threshold time of the Plasmodium genus-specific LAMP. The error bars represent the standard errors of the mean values from four replicates. The plot of the mean threshold time against the log of the input DNA fit a linear function (r² = 0.96). (C) Amplification with the P. falciparum species-specific primer set. One representative result of four replicates tested with the plasmid harboring the P. falciparum 18S rRNA gene is shown. (D) Plot of the mean threshold time of the P. falciparum species-specific LAMP from four replicates, which fit a linear function (r² = 0.91). (E) Amplification with a P. vivax species-specific primer set. One representative result of four replicates tested with plasmid harboring P. vivax 18S rRNA gene is shown. (F) Plot of the mean threshold time of the P. vivax species-specific LAMP from four replicates, which fit a linear function (r² = 0.95). (G) Amplification with a P. malariae species-specific primer set. One representative result of four replicates tested with a plasmid harboring the P. malariae 18S rRNA gene is shown. (H) Plot of the mean threshold time of the P. malariae species-specific LAMP from four replicates, which fit a linear function (r² = 0.84). (I) Amplification with a P. ovale species-specific primer set. One representative result of four replicates tested with a plasmid harboring the P. ovale 18S rRNA gene is shown. (J) Plot of the mean threshold time of the P. ovale species-specific LAMP from four replicates, which fit a linear function (r² = 0.90). Genus, genus Plasmodium LAMP; Pf, P. falciparum LAMP; Pv, P. vivax LAMP; Pm, P. malariae LAMP; Po, P. ovale LAMP; O.D., optical density.

FIG. 3.

Restriction analysis of Plasmodium genus- and species-specific LAMP products amplified from plasmid DNA containing each target 18S rRNA gene. The digestion products were run on a 3% agarose gel. Lane M, DNA ladder marker; lane 1, genus Plasmodium LAMP product; lane 2, DdeI digestion of genus Plasmodium product (123-, 44-, and 20-bp bands were expected); lane 3, P. falciparum LAMP product; lane 4, HpyCH4V digestion of P. falciparum product (130- and 79-bp bands were expected); lane 5, P. vivax LAMP product; lane 6, HpyCH4V digestion of P. vivax product (121- and 77-bp bands were expected); lane 7, P. malariae LAMP product; lane 8, HpyCH4V digestion of P. malariae product (142- and 84-bp bands were expected); lane 9, P. ovale LAMP product; lane 10, AluI digestion of P. ovale product (152- and 69-bp bands were expected).