Direct PCR amplification and sequence analysis of extrachromosomal Plasmodium DNA from dried blood spots

ACTA TROPICA ELSEVIER

Acta Tropica 68 (1997) 105-114

Direct PCR amplification and sequence analysis of extrachromosomal Plasmodium D N A from dried blood spots T.M.C. Tan a, J.S. Nelson a, H.C. Ng a, R.C.Y. Ting a U.A.K. Kara b,, a Institute of Molecular and Cell Biology, National University of Singapore, I0 Kent Ridge Crescent, Sl19260, Singapore, Singapore b Molecular Parasitology Laboratory, School of Biological Sciences, National University of Singapore, I0 Kent Ridge Crescent, Sl19260, Singapore, Singapore Received 27 March 1997; received in revised form 4 April 1997; accepted 1 May 1997

Abstract

The Plasmodium parasite possesses two extrachromosomal genomes; the mitochondrial genetic element and the extrachromosomal plastid-like DNA. The latter has only been fully described for one culture strain of P. falciparum. In this study, a rapid procedure for amplifying plastid D N A from dried blood spots of blood infected with different malaria species was developed• PCR amplification of a 595 bp fragment within the plastid-like large subunit ribosomal-RNA (LSU-rRNA) gene was achieved using primers derived from the P. falciparum sequence. The PCR product was observed in all Plasmodium species examined• Sequence analysis of amplified products homologous to an LSU-rRNA fragment of the plastid-like extrachromosomal circle revealed extensive conservation between Plasmodium species including P. falciparum, P. vivax, P. rnalariae and P. berghei. © 1997 Elsevier Science B.V. Keywords: Malaria; PCR amplification; Extrachromosomal plastid; Blood spots

* Corresponding author: Tel.: + 65 7727834; fax: + 65 7792486; e-mail: [email protected] 0001-706X/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S0001-706X(97)00080-6

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1. Introduction

Apicomplexans are parasitic protists that can cause severe diseases such as malaria and toxoplasmosis. Organisms in this phylum harbour extrachromosomal genetic material. The malaria parasites carry two types of extrachromosomal organellar DNA; one is mitochondrial, the other one is a circular molecule resembling the remnant of an algal plastid genome (Williamson et al., 1994). Although this circular plastid-like DNA has been observed in electron micrographs of DNA obtained from organelle-containing subcellular fractions of Plasmodium lophurae, P. knowlesi, P. berghei and P. falciparum (Kilejian, 1975; Dore et al., 1983; Williamson et al., 1985; Gardner et al., 1988), there has been little sequence information about the circular DNA with the exception of that derived from P. falciparum (Wilson et al., 1996) and P. berghei (Yap et al., unpublished data). To date, only the 35 kb circle of P. falciparum has been fully mapped (Wilson et al., 1996). One striking feature of the P. falciparum circular DNA is the presence of a palindrome. Each arm of this palindrome contains one set of small subunit (SSU) and large subunit (LSU) ribosomal-RNA genes and a cluster of intervening transfer-RNA genes (Gardner et al., 1993). Enzymatic amplification of DNA by the polymerase chain reaction (PCR) is a promising tool for identifying low levels of parasite DNA. In this study, we designed a pair of primers to amplify the LSU-rRNA gene from malaria-infected blood spotted on filter paper. This has enabled us to amplify and analyse the LSU-rRNA gene from various Plasmodium species with minimal amount of infected blood required and without time and labour consuming preparations of the circular DNA. We report a PCR-based assay using clinical and field collected samples from subjects infected with three species of human Plasmodiurn and compare the results with data obtained from murine P. berghei malaria samples.

2. Materials and methods

2.1. Specimen collection Blood was collected by fingerprick (5-10/~1) or by venipuncture from subjects with Giemsa smear-positive P. faIciparum, P. vivax and P. malariae malaria as well as from non-parasitaemic controls and spotted in replicates onto Whatman filter paper. P. berghei (ANKA) infected mouse blood (5 /ll) was collected from the tail. P. berghei infections were maintained by serial blood passage of 10 7 parasites. Dried blood spots were placed individually into 200 /~1 PCR tubes and fixed with the addition of methanol for 5 min. The methanol was poured off and the blood spot was dried thoroughly prior to PCR amplification.

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2.2. PCR amplification Amplification was carried out as previously described (Long et al., 1995) with some modifications. Each 100/~1 reaction mixture contained 1 x PCR buffer (70 mM Tris, pH 8.8, 20 mM (NH4)2804, 1 mM DTT, 0.1 /~g//~l BSA), 2.5 mM MgC12, 0.4/~g of each primer, 5 units of Taq DNA polymerase (Amersham) and 0.2 mM of each dNTPs. Reaction tubes were overlaid with one drop of mineral oil. The reaction was soaked at 95°C for 5 min then held at 80°C prior to the addition of Taq DNA polymerase and dNTPs. Amplification involved 40 cycles of 1 min denaturation at 90°C, 2 min annealing at 52°C and 3 rain primer extension at 72°C. A 5 min primer extension at 72°C was included following the final cycle.

2.3. Sequences of primers Three sets of primers were used in this study. The primers used for amplifying the LSU-rRNA gene were L1 5'GAC CTG CAT GAA AGA TG3' and L2 5'GTA TCG CTT TAA TAG GCG3'. A second set of primers DHFR1 5'GCA ATA TGT GCA TGT TGT AAA3' and DHFR2 5'ATT CTT TAT AAA CAG ACG3' was designed to amplify the dihydrofolate reductase-thymidylate synthase (DHFR-TS) gene from P. berghei genomic DNA. The primers used for amplifying the human fl-actin gene were AC1 5'GGG CGA CGA GGC CCA GAG C3' and AC2 5'GCA TCC TGT CGG CAA TGC C3'.

2.4. Agarose gel electrophoresis Ten microliters of each PCR product was resolved in 1% agarose gels with TAE electrophoresis buffer (40 mM Tris-acetate and 1 mM EDTA, pH 8.0). Electrophoresis was carried out at 100 V for 1.5 h and the fragments were visualised under UV.

2.5. DNA sequencing protocol The PCR products were loaded onto a 1% low-melting point agarose gel and extracted by the freeze-thaw method (Shoemaker and Salyers, 1990). They were then cloned into the pGEM-T vector (Promega). The clones were sequenced in both directions using the ABI PRISM Dye terminator cycle sequencing kit (Perkin Elmer) on the 373 DNA sequencer from Applied Biosystems. Multiple sequence alignment using the cluster method was carried out with the DNASTAR programme.

3. Results

3.1. Detection of P. berghei infection in blood spots Conditions for the PCR amplification of P. berghei-infected mouse blood SPotted on filter paper were optimised using DHFR1 and DHFR2 primers. Once {hese

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conditions were established, the L S U - r R N A primer set was also tested. The L S U - r R N A primer set was designed to amplify a 594 bp fragment from the P. berghei circular D N A while the D H F R - T S primer set amplified a 511 bp fragment from P. berghei genomic D N A . Blood spots were prepared daily for 5 days from a mouse which was initially infected with 5 x 10 4 parasites. Giemsa staining of thin blood films from the same animal was done daily. The amplified L S U - r R N A fragment was detectable by ethidium bromide staining one day after infection (see Fig. 1) while the D H F R - T S P C R product was only visible 2 days post-infection. At these two time points, no parasite was detected on the corresponding Giemsastained blood films. Parasites were only observed on the film 3 days post-infection.

3.2. PCR amplification of blood spots from malaria infected patients The above P C R amplification protocol was also applied to blood spots from 31 malaria-infected patients. Fifteen of these samples were obtained from patients admitted to the National University Hospital in Singapore. O f these, seven had P. falciparum infection, one had P. malariae and the remaining had P. vivax as determined by Giemsa and Quantitative Buffy Coat (QBC) diagnosis. All samples

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Fig. 1. PCR amplification products generated with the primer sets (a) L1/L2 and (b) DHFR1/DHFR2. Blood was drawn daily for 5 days from a mouse initially infected with 5 x 1 0 4 parasites. Lanes 1-5 show the PCR fragments from blood spots 1 5 days post-infection correspondingly. Lane 6 is the negative control with blood from an uninfected mouse and lane 7 is the positive control using 50 ng of purified P.berghei total DNA as template. M indicates the 100 bp DNA ladder (Promega) used as markers.

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Fig. 2. PCR amplification of blood spots from Laotian patients. All patients had been diagnosed by Giemsa microscopy and the ParaF dipstick with P. falciparum malaria with the exception of one which was infected with P. vivax (lane 11). The LSU-rRNA product (LSU) was observed for all specimens (Ianes 1 11). Lane 12 is the negative control with a blood spot from a non-parasitaemic person and lane 13 is the positive control using 50 ng of purified P, falciparum (FC2? strain) total DNA as a template. M indicates the 100 bp DNA ladder (Promega) used as markers,

were positive for amplification with L S U primers (data not shown). The other 16 samples were from patients in Laos with 15 P. falciparum infections and one P. vivax malaria infection as determined by Giemsa and P a r a F dipstick diagnosis. L S U - r R N A P C R amplifications were positive for all 16 specimens. Fig. 2 shows the P C R products from 11 of the 16 Laotian specimens. Eight non-parasitaemic persons and total D N A from two h u m a n carcinoma cell lines, CaSki and H e L a were used as controls. These were all negative when using the L S U - r R N A primer set for P C R amplification but were all positive for h u m a n fl-actin (data not shown).

3.3. Sequence alignment of L S U - r R N A from various Plasmodium species Eleven of the L S U - r R N A fragments amplified from the blood spots were cloned into the p G E M - T vector and sequenced. The fragments were randomly selected from the Singaporean, Laotian, Indian and Pakistani samples. These clones contained P C R products generated from P. falciparum, P. vivax and P. malariae infected blood. The published P. falciparum sequence (C10 strain) was used as the basis for all alignments and comparisons. Comparison of the Plasmodium species used in this study showed that this region of the L S U - r R N A gene is highly conserved and the similarity between P. falciparum, P. vivax, P. malariae and P. berghei is greater than 92% (Table 1). The similarity between the C10 and other P. falciparum sequences ranged from 98.3 to 99.8%, while that between the C10 and the P. vivax sequences ranged from 92.9 to 99.7%. The greatest divergence in the sequence was observed for the two P. vivax specimens from Pakistan. In all cases, divergence in sequence was due to one or two base changes in isolated regions within the L S U - r R N A fragment (see Fig. 3).

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4. Discussion

Amplification of malaria genomic DNA from blood spots on filter paper has been described previously (Long et al., 1995). In this study, we have shown that it is possible to likewise amplify the extrachromosomal circular plastid-like DNA found in the malaria parasite. This has allowed us to proceed with characterising the LSU-rRNA gene from the circular DNA of malaria-infected patients using only a small volume of blood spotted on filter paper. The LSU-rRNA genes on the circular DNA of P. falciparum and P. berghei are highly conserved (Yap et al., unpublished). We have designed a pair of primers based on the sequences from P. faleiparum and P. berghei such that the primers are completely homologous for both species. Using these primers, we have been able to amplify the corresponding LSU-rRNA fragment from P. falciparum, P. vivax, P. malariae and P. berghei infected blood. Sequence analysis of these fragments indicates that this region of the LSU-rRNA is highly conserved between different species of Plasmodium. In addition, regionally different isolates of P. falciparum and P. vivax from Asia do not show distinct variations for the LSU-rRNA fragment. GenBank searches indicate that this fragment sequence is unique. The high homology between the various Plasmodium species has led us to examine if the LSU-rRNA specific primers are useful for the detection of malaria infections. The LSU-rRNA primer set was able to detect P. berghei-infected mouse blood spots. All 31 patient blood spots tested were positive regardless of the Plasmodium species involved while none from non-parasitaemic persons was Table 1 Percent homology of LSU-rRNA sequences with the P. falciparum (C10 strain) sequence Name of sequencea

Similarity index with the P.faleiparum (C10 strain) b

Pfl0/P Pfl 1/P Pfl9/I Pf20/L Pfl8/S Pvl2/P Pv13/P Pvl5/I Pvl6/L Pvl7/S Pml/S Pb(ANKA)

98.3 98.5 99.7 99.7 99.8 93.4 92.9 99.5 99.7 93.4 93.2 94.2

pf denotes P. falciparum, Pv denotes P.vivax, Pm denotes P. malariae and Pb denotes P. berghei. The alphabet at the end of each name indicates the origin of the specimen; P, Pakistan; I, India; L, Laos and S, Singapore. The GenBank accession numbers for Pf(C10) and Pb(ANKA) are X95275 and U79731, respectively. b Using the Martinez-Needleman-Wunsch DNA alignment programme. a

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positive. These results indicate that the LSU-rRNA primers may be useful for the diagnosis of malaria infection. More extensive testing will be needed to ensure that these primers are indeed Plasmodiurn specific only. The ease of direct PCR amplification of extrachromosomal Plasrnodiurn circular DNA from dried blood spots has provided us with the means to study and characterise the genes present on this DNA molecule. To date, none of the genes on the circular DNA of P. vivax and P. malariae have been described. This is the first description of an analysis of the LSU-rRNA gene from different field isolates of P. vivax, P. malariae and P. falciparum. More investigations will have to be carried out to determine how extensively the sequence conservation is maintained and what the arrangement of the genes on the circular DNA from different Plasmodium species is.

Acknowledgements The authors would like to acknowledge the technical expertise provided by Melvyn Yap, Esther Wong, Alice Tay and Cheryl Lee. We would also like to thank Professor V. Zaman, Aga Khan University, Karachi, Drs K. Pholsena, H. Bouasy and H. Viengxay, Institute of Malaria, Parasitology and Entomology, Vientiane and the National University Hospital, Singapore for helping with the patient materials. This work was supported by a grant from the National Science and Technology Board, Singapore and the research grant RP940361 from the National University of Singapore.

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Williamson, D.H., Gardner, M.J., Preiser, P., Moore, D.J., Rangachari, K., Wilson, R.J.M., 1994. The evolutionary origin of the 35 kb circular DNA of Plasmodium falciparum-new evidence supports a possible rhodophyte ancestry. Mol. Gen. Genet. 243, 249-252. Wilson, R.J.M., Denny, P.W., Presier, P.R., Rangachari, K., Roberts, K., Roy, A., Whyte, A., Strath, M., Moore, D.J., Moore, P.W., Williamson, D.H., 1996. Complete gene map of the plastid-like DNA of the malaria parasite Plasmodiumfalciparum. J. Mol. Biol. 261, 155 172.