Article Figures & Data
Figures
- 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).
Tables
- TABLE 1.
Primer sets used for amplification of 18S rRNA genes in LAMP
Species Primer Sequence (5′→3′) Plasmodium genus F3 GTATCAATCGAGTTTCTGACC B3c CTTGTCACTACCTCTCTTCT FIP (F1c-F2) TCGAACTCTAATTCCCCGTTACCTATCAGCTTTTGATGTTAGGGT BIP (B1-B2c) CGGAGAGGGAGCCTGAGAAATAGAATTGGGTAATTTACGCG LPF CGTCATAGCCATGTTAGGCC LPB AGCTACCACATCTAAGGAAGGCAG P. falciparum F3 TGTAATTGGAATGATAGGAATTTA B3c GAAAACCTTATTTTGAACAAAGC FIP (F1c-F2) AGCTGGAATTACCGCGGCTG GGTTCCTAGAGAAACAATTGG BIP (B1-B2c) TGTTGCAGTTAAAACGTTCGTAGCCCAAACCAGTTTAAATGAAAC LPF GCACCAGACTTGCCCT LPB TTGAATATTAAAGAA P. vivax F3 GGAATGATGGGAATTTAAAACCT B3c ACGAAGTATCAGTTATGTGGAT FIP (F1c-F2) CTATTGGAGCTGGAATTACCGCTCCCAAAACTCAATTGGAGG BIP (B1-B2c) AATTGTTGCAGTTAAAACGCTCGTAAGCTAGAAGCGTTGCT LPF GCTGCTGGCACCAGACTT LPB AGTTGAATTTCAAAGAATCG P. malariae F3 CAAGGCCAAATTTTGGTT B3c CGGTTATTCTTAACGTACA FIP (F1c-F2) TATTGGAGCTGGAATTACCGCGATGATGGGAATTTAAAACCT BIP (B1-B2c) AATTGTTGCAGTTAAAACGCCTATGTTATAAATATACAAAGCATT LPF GCCCTCCAATTGCCTTCTG LPB TCGTAGTTGAATTTCAAGGAATCA P. ovale F3 GGAATGATGGGAATTTAAAACC B3c GAATGCAAAGAACAGATACGT FIP (F1c-F2) TATTGGAGCTGGAATTACCGCGTTCCCAAAATTCAATTGGAGG BIP (B1-B2c) GTTGCAGTTAAAACGCTCGTAGTGTATTGTCTAAGCATCTTATAGCA LPF TGCTGGCACCAGACTTGC LPB TGAATTTCAAAGAATCAA - TABLE 2.
Detailed comparison of microscopy, nested PCR, and LAMP for malaria parasite detection and species identification
Parasite(s) detected by each method (no. of samples)a Microscopy Nested PCR LAMPb P. falciparum (12)c P. falciparum (12) P. falciparum (12) P. falciparum + P. vivax (5) P. falciparum + P. vivax (4), P. vivax (1) P. falciparum + P. vivax (4), P. vivax (1) P. vivax (34) P. vivax (30), P. vivax (1), P. vivax (1), P. ovale (2) P. vivax (30), P. vivax + P. falciparum (1), negative (1), P. ovale (2) P. malariae (12) P. malariae (8), P. malariae + P. vivax (1), P. ovale (1), P. malariae (1), negative (1) P. malariae (8), P. malariae + P. vivax (1), P. ovale (1), Plasmodium spp.d (1), P. malariae (1) P. ovale (5) P. ovale (5) P. ovale (5) Negative (53) Negative (50), P. falciparum (1), P. vivax (2) Negative (50), P. falciparum (1), P. vivax (2) ↵ a Results that were nonconcordant between nested PCR and LAMP are underlined.
↵ b LAMP assays were run twice; in all cases the two experiments gave the same results.
↵ c Each row provides the results obtained with identical DNA samples.
↵ d Positive for the genus Plasmodium by LAMP but negative by all four species-specific LAMPs.
