Fig 1.
The PCR-targeted region of the SSU rRNA genes from 5 malaria parasite species.
Partial sequences of the SSU rRNA genes from malaria parasites are indicated. Pf-S: The sequence from P. falciparum expressed at the sexual stage [GenBank: M19173]. Pf-A: The sequence from P. falciparum expressed at the asexual stage [GenBank: M19172]. Pv-S: The sequence from P. vivax expressed at the sexual stage [GenBank: U03080]. Pv-A: The sequence from P. vivax expressed at the asexual stage [GenBank: X13926]. Poc: The sequence from P. ovale curtisi [GenBank: L48986]. Pow: The sequence from P. ovale wallikeri [GenBank: AB182491] Pm: The sequence from P. malariae [GenBank: M54897]. Pk: The sequence from P. knowlesi [GenBank: AY327550]. The universal primers for the conserved region are highlighted in yellow (P1 and P2, see Table 1). The inner-specific primers (F2, V3, M4, Oc4, Ow1, and K1 [see Table 1]) are highlighted in blue.
Table 1.
Sequences of the primers used in the present study.
Table 2.
Detailed conditions of the nested PCRs.
Fig 2.
Results of the first PCR with the universal primers P1 and P2.
The first PCR products were visualized on 2% agarose-TAE gel containing GelRed (Wako). Lane L indicates a molecular marker (100-bp ladder). The letters shown below each lane indicate the template DNA used for the first PCR reactions (lanes F, V, Oc, M, K, and Ow indicate the plasmid DNA containing each partial sequence of the SSU rRNA genes from P. falciparum, P. vivax, P. ovale curtisi, P. malariae, P. knowlesi, and P. ovale wallikeri, respectively; lane N indicates the negative control [water]). The PCR products are shown at 136–159 bp.
Fig 3.
Comparison of the performance of the classic and new primer sets in the second PCR.
(A) Comparison of the classic O2 primer and new Oc4 primer. (B) Comparison of the classic M1 primer and new M4 primer. The second PCR was performed with 30 cycles. The PCR products were visualized on 2% agarose-TAE gel containing GelRed (Wako). Lane L indicates a molecular marker (100-bp ladder). The letters shown below each lane indicate the template used for the second PCR reactions (lanes F, V, Oc, M, K, and Ow indicate diluted first PCR products amplified from the sequences of P. falciparum, P. vivax, P. ovale curtisi, P. malariae, P. knowlesi, and P. ovale wallikeri, respectively; lane N indicates the diluted first PCR product from water; lane N’, water). The primer sets are indicated below the gels.
Fig 4.
Results of the second PCR with the universal primer P1 and the inner-specific primers.
The second PCR products were visualized on 2% agarose-TAE gel containing GelRed (Wako). Lane L indicates a molecular marker (100-bp ladder). The letters shown below each lane indicate the template used for the second PCR reactions (lanes F, V, Oc, M, K, and Ow indicate diluted first PCR products amplified from the sequences of P. falciparum, P. vivax, P. ovale curtisi, P. malariae, P. knowlesi, and P. ovale wallikeri, respectively; lane N indicates the diluted first PCR product from water; lane N’, water). Arrows indicate the PCR products (100–106 bp). The primer sets are indicated below the gels.
Fig 5.
The second PCR-targeted region of the SSU rRNA genes from variant parasite isotypes.
(A) Partial sequences of the variant SSU rRNA genes from malaria parasites. Pf, Pv, Po, and Pm indicate P. falciparum, P. vivax, P. ovale and P. malariae respectively. Pf-standard: The sequence from P. falciparum [GenBank: M19172]. Pf isotype-1 and isotype-2: The identified variant sequences from P. falciparum [Genbank: KJ170099.1 and JQ627151.1]. Pv-standard: The sequence from P. vivax [GenBank: X13926]. Pv isotype-1, isotype-2 and isotype-3: The identified variant sequences from P. vivax [Genbank: U83877.1, KC750244.1 and AF145335.1]. Poc standard: The sequence from P. ovale curtisi [GenBank: L48986]. Poc isotype-1, isotype-2, isotype-3 and isotype-4: The identified variant sequences from P. ovale curtisi [Genbank: KF696376.1, KC633228.1, KJ871671.1 and KF696371.1]. Pow standard: The sequence from P. ovale wallikeri [GenBank: AB182491] Pm standard: The sequence from P. malariae [GenBank: M54897]. Pm isotype-1 and isotype-2: The identified variant sequences from P. malariae [Genbank: KJ619947.1 and KJ170106.1]. All variant sequences from each species are indicated as multiple alignment comparisons. The universal P1 primer region is highlighted in yellow (P1). The inner-specific primers (F2, V3, Oc4 and M4,) are highlighted in blue. The nucleotide changes identified in each variant are highlighted in red. (B) Results of the PCR with the universal primer P1 and the inner-specific primers using the variant DNAs as templates. The PCR reactions were performed with the universal P1 primer and each species-specific primer. The PCR conditions were same as those of the second PCR (see Materials and Methods). In each reaction mix, 0.1 ng of the synthesized DNA of each variant sequence was included as template. The products were visualized on 2% agarose-TAE gel containing GelRed (Wako). Lane L indicates a molecular marker (100-bp ladder). The letters shown below each lane indicate the specific primer used for the second PCR reactions (see Table 1). Arrows indicate the PCR products (100–106 bp). The template DNAs are indicated below the gels.
Fig 6.
Determination of the P. falciparum detection limit of the nested PCR system.
The second PCR products were visualized on 2% agarose-TAE gel containing GelRed (Wako). The primer set used for the second PCR was P1 and F2. Lane L indicates a molecular marker (100-bp ladder). The letters shown below each lane indicate the template used for the second PCR reactions (lane P indicates the first PCR products amplified from the sequences of P. falciparum; lanes 103, 102, 1, 10−1, 10−2, and 10−3 indicate the first PCR products amplified from the DNA extracted from the blood sample containing 103, 102, 1, 10−1, 10−2, and 10−3 parasites/μL of blood, respectively; lane 0 indicates the first PCR products amplified from the DNA extracted from healthy blood; lane N indicates the diluted first PCR product from water; lane N’, water). The P. falciparum-specific PCR products are indicated with an arrow on the right side.
Fig 7.
Gel electrophoresis of PCR products for the diagnosis of a clinical sample.
(A) The first PCR products were visualized on 2% agarose-TAE gel containing GelRed (Wako). Lane L indicates a molecular marker (100-bp ladder). The letters shown below each lane indicate the template DNA used for the first PCR reactions (lane S indicates a DNA sample from a patient’s blood; lanes F, V, Oc, M, K, and Ow indicate plasmid DNA containing each partial sequence of the SSU rRNA genes from P. falciparum, P. vivax, P. ovale curtisi, P. malariae, P. knowlesi, and P. ovale wallikeri, respectively; lane N, indicates the negative control [water]). The PCR products are shown at 136–159 bp. (B) The second PCR products were visualized on 2% agarose-TAE gel containing GelRed (Wako). Lane L indicates a molecular marker (100-bp ladder). The letters shown below each lane indicate the template used for the second PCR reactions (lane S indicates the diluted first PCR product amplified from a DNA sample from a patient’s blood; lanes F, V, Oc, M, K, and Ow indicate the diluted first PCR products amplified from the sequences of P. falciparum, P. vivax, P. ovale curtisi, P. malariae, P. knowlesi, and P. ovale wallikeri, respectively; lane N indicates the diluted first PCR product from water; lane N’, water). The arrow indicates the PCR product amplified from a DNA sample from the blood of a patient with P. vivax-specific primers. The primer sets are indicated below the gel.
Table 3.
Detection of the 5 species of human malaria parasite in the patient blood samples.