EXPERIMENTALPARASITOLOGY75207-212 (1992)
Plasmodium
falciparum: A Simple Polymerase Chain Reaction Method for Differentiating Strains
JASON WOODEN,ELIZABETH ENARIGOULD,ANNETERRY AND CAROL HOPKINS SIBLEY
PAULL,
Department of Genetics @K-50), University of Washington, Seattle, Washington 98195, U.S.A. WOODEN, J., GOULD, E. E., PAULL, A. T., AND SIBLEY, C. H. 1992.Plasmodium falciparum: A simple polymerase chain reaction method for differentiating strains. Experimental Parasitology 75, 207-212. Laboratory studies of the protozoan parasite Plasmodium falciparum have been hampered by difftculties in defining differences between isolates. We have developed a method based on the polymerase chain reaction (PCR) that allowed us to identify quickly the various strains with which we routinely work. We also adapted methods for easily purifying enough DNA to produce a PCR product from a small volume of culture: 100 ~1 of an in vitro culture infected at 1% parasitemia. The primers were chosen from conserved regions flanking the variable repeats in four cloned genes, RESA, MSA-1, MSA2, and CSP. The PCR products amplified from three of these genes differed in size and allowed us to identify particular isolates on this basis alone. The variation was between strains, and not a reflection of genetic instability during in vitro culture of one isolate. The method is sufficiently sensitive to detect a 1% contamination of one strain with another, an advantage for monitoring the integrity of strains when different isolates are grown in the same laboratory. The technical ease and speed of this assay and the small amount of culture required make it ideal for monitoring strains in the laboratory. 6 1992Academicpress,IKE. INDEX DESCRIPTORS AND ABBREVIATIONS:Circumsporozoite surface protein (CSP); Desoxyribonucleic acid (DNA); Precursor of the major merozoite surface antigen (MSA-1); Merozoite surface antigen-2 (MSA-2); Polymerase chain reaction (PCR); Ring-infected erythrocyte . _ surface antigen (RESA); Surface antigen (S-Ag); Malaria; Polymorphism; Strain identification.
INTRODUCTION Plasmodium falciparum has become increasingly amenable to laboratory study in recent years, but as more strains have become available, difficulties in defining origins and genealogies of laboratory strains have increased (Robson et al. 1992). This has particularly hampered genetic analysis. There are a number of established techniques for standardizing strains (Fenton et al. 1985), but we sought to develop a rapid method using the polymerase chain reaction (PCR). This is certainly not a new idea; PCR has been used frequently to type P. fulciparum strains for particular loci (Foote et al. 1990; Wellems et al. 1990; Zolg et al. 1990). We have simply expanded it into a routine screen. We also adapted methods for isolation of DNA from small amounts of
sample (Innes et al. 1990); DNA for 100 (~1 of an in vitro culture infected at 1% parasitemia is more than sufficient to produce a PCR product. We used published sequences of genes that have conserved 5’ and 3’ ends but contain a region with blocks of repeated sequences which vary in size and DNA sequence from strain to strain (Kemp et al. 1987). We selected highly conserved regions and chose primers that spanned the repeats in live surface antigens: S-antigen (Saint et al. 1987), ring-infected erythrocyte surface antigen (RESA; Favalaro et al. 1986), the precursor of the major merozoite surface antigen (MSA-1; Mackay et al. 1985), the major surface antigen-2 (MSA-2; Fenton et al. 1991; Smythe et al. 1991), and circumsporozoite surface protein (CSP; Dame et al. 1984).
207 0014-4894192 $5.00 Copyright8 1992by AcademicPress,Inc. AU rightsof reproductionin any form reserved.
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Although not all of these genes varied saline (PBS) (0.14 34 NaCl, 0.01 M sodium phosphate, among the eight strains we examined, even pH 7.2), and the red cells were lysed by suspension in 1% saponin for 5 min on ice. The P. falciparum cells this small set was adequate to distinguish were isolated by centrifugation for 30 set in a miour strains. It seems likely that these genes crofuge (12,OOOg)and a single washing in PBS. This and others that are identified in studies of pellet was then resuspended in a 5% (w/v) suspension P. falciparum will allow all of the laborato- of Chelex-100 in water, pH > 9.5 (Bio-Rad) and incuries using P. falciparum to define the bated in boiling water for 8 min. The Chelex was removed by centrifugation for 30 set in the microfuge, strains now in use, and perhaps to begin to and the resulting suspension was either used directly determine their relationships. This stan- or precipitated once with alcohol, dried, and resusdardization will be useful not only to genet- pended in 50 pl water, and 2 u.1used directly for PCR amplification. icists but to other workers, as well. METHODS We used four strains: FCR-8, American Type Culture Collection (Trager and Jensen 1976); 3D7, Dr. Thomas Wellems (Wellems et al. 1987); Cl0 and its clone, D2, Dr. Jean Feagin (Hempelmann et al. 1981). In addition, we surveyed DNA isolated from a number of other strains: Sierra Leone, Tanzania 1, Honduras
RESA 5’ primer 3’ primer MSA-1 5’ primer 3’ primer MSA-2 5’ primer 3’ primer CSP 5’ primer 3’ primer SAG 5’ primer 3’ primer 1 3’ primer 2
The PCR protocols were all adaptations of standard procedures described in Higuchi (1989) and Saiki et al. (1985). The reactions contained a total volume of 100 ~1, with 0.2 mM dNTPs and 1.25 U of Replitherm (Epicentre). The reactions were run in a Biosycler with the following parameters: initial denaturation for 2 min at 94°C and 29 cycles with 20 set at 94”C, 20 set at 55°C and 20 set at 72°C. The primers used for each reaction are listed below:
5’-GATCAAGGAGGAGAGAACC-3’ S’CAGCATTAACACCAACACC-3’ S’GAAGATGCAGTATTGACAGG-3’ 5’-GAGTTCTTTAATAGTGAACAAG-3’ 5’-GAGTATAAGGAGAAGTATGG-3’ 5’-CCTGTACCTTTATTCTCTGG-3’ 5’-ATAGTAGATCACTTGGAGA-3’ 5’-GCATATTGTGACCTTGTCCA-3’ 5’-CAAGCAGAAGGAGAAGGT-3’ 5’-TCCATGTCCTTCAGCTCCTCC-3’ 5’-TCCACCACTTCCTGGTCCTCC-3’
1, Kl (courtesy of Dr. Randy Howard), and PLF 3Bll (courtesy of Dr. Jean Feagin). Cells were grown in vitro with a modification of the method of Trager (Zolg et al. 1982). Briefly, they were grown in RPM1 1640 supplemented with 5% (v/v) washed human red cells, 10% fresh human serum, 5 p&ml hypoxanthine, and 600 pg/ml reduced glutathione and maintained in an atmosphere of 5% oxygen 5% carbon dioxide, and 90% nitrogen. Initial isolation of DNA was done with a modification of the standard method of Higuchi (1989). In later experiments, a modiication of the method of Walsh and colleagues (1991) proved much easier. About 200 pl of culture was washed once in phosphate-buffered
PCR products were analyzed on minigels with 2% agarose, run in 0.5~ TBE (4.25 mM Tris, 4.45 mM borate, 1.25 mM EDTA, pH 8.2) for 1 hr at 100 mV. The double-stranded PCR products were separated from the primers by centrifugation through a Centricon-30 membrane, and the sequencing reactions were performed with a Sequenase kit (U.S. Biochemical), according to the manufacturer’s instructions.
RESULTS
In order to easily distinguish various strains of P. falciparum, we chose genes with blocks of repeats that we expected to
PCR FOR DIFFERENTIATING
Plasmodiumfalciparum
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Specificity was determined by direct sevary in size. Among the published sequences, the variation appeared to be be- quencing of the products of some of the tween strains and not a reflection of ex- PCR reactions. In addition, we tested the treme instability over short-term culture. primers on DNA derived from a variety of We chose four genes for our initial analysis: species, Mus musculus, Drosophila melaS-Ag (Saint et al. 1987), RESA (Favalaro et nogaster, Saccharomyces cerevisiae, Eschal. 1986), MSA-1 (Mackay et al. 1985), erichia coli, and A phage. No product was MSA-2 (Fenton et al. 1991; Smythe et al. observed in any of these reactions or when 1991), and CSP (Dame et al. 1984). The se- the primers were tested on human DNA quences of these primers are listed under (data not shown). We tested all of the primer sets on DNA Methods. All of the primers except those for S-Ag gave consistent products in the derived from the strains listed under MethPCR reaction for all of the strains tested. In ods. Figure 1A shows typical results from fact, we tried several different downstream these experiments. Although there are seprimers for S-Ag, but none gave a consis- quence differences among these strains, tent product, even at low stringency. RESA did not show a size polymorphism The basic conditions for the amplification (Fig. 1A). In contrast, amplification of reactions were optimized for each primer DNA from all strains using primers for CSP set, paying particular attention to the tem- (Fig. lB), MSA-2 (Fig. lC), and MSA-2 perature of hybridization and the magne- (Fig. 1D) yielded fragments whose sizes sium concentration. All of the primers ex- were easily distinguished on 2% agarose cept S-Ag can be used with one standard gels. Comparison of these three gels allows protocol detailed under Methods. the six strains shown here to be uniquely REBA
MSA-1
FIG. 1. The PCR products from three genes vary in size. The PCR reactions were carried out under standard conditions using primer pairs to amplify 50 ng of DNA isolated from the Sierra Leone, Honduras 1, Kl , Tanzania 1, PLF 3B11, and 3D7 lines as template in each reaction. PCR products were analyzed on 2% agarose gels as described under Methods. The markers are the I-kb ladder (BRL). (A) RESA; (B) CSP; (C) MSA-I; (D) MSA-2.
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determined. This suggests that this approach may be generally useful for identification of a wide variety of strains. In choosing the primers, we aimed for sequences of about 20 bp, with as close to 50% G/C as possible. All of the primers are similar enough that we use identical conditions for the reaction mix and amplification protocol, a real convenience when one has many reactions to perform! This also affords the possibility that the three primers can be used in one reaction tube (multiplexed), greatly reducing the expense and manipulation for one analysis. Figure 2A shows that the RESA, MSA-1, and MSA-2 primers can be multiplexed. The fact that amplification of RESA produces fragments of the same length from all of these strains provides a convenient control to ensure that the lanes are all running in an equivalent manner. To test the stability of the size differences, we compared the amplified fragments generated from DNA isolated from Cl0 and D2, a subline derived from C 10 12 months before (J. Feagin, personal communication). All primers amplified fragments of identical size from both sources of DNA (data not shown). In addition, we have tested DNA isolated from strain 3D7 derived from two different laboratories, and these gave identical profiles (data not shown). Thus, it seems that these size differences are sufIiciently stable under laboratory conditions to make useful markers of particular strains. When several strains are grown simultaneously in the laboratory, it is important to be able to distinguish the different strains so that any inadvertent contamination or substitution of one strain with another can be easily detected. To determine the sensitivity of the PCR polymorphisms, we made a series of mixtures of cultures of 3D7 and D2 in various ratios from 1:l to 1:104, prepared the DNA, and subjected each sample to amplification with the primers specific
RJISA + MSA-1 + MSA-2
FEWiXl
D2 DNA
10
1
1
1
1
0
3Dl DNA
1
1
10
102
103
1
I0.3 Lb-
FIG. 2. (A) The PCR reactions can be multiplexed. The PCR reactions were carried out under standard conditions using DNA isolated from Tanzania, Kl, Honduras, or PLF 3B 11 and primer pairs to amplify RESA, MSA-I, and MSA-2 in the same tube. PCR products were analyzed on 2% gels as described under Methods. The markers are the 1-kb ladder (BRL). (B) A minor contribution from a contaminating strain can be recognized. Separate cultures of the two strains, D2 and 3D7, were mixed in the ratios indicated, the DNA was prepared by the Chelex-100 method, and 50 ng of the resulting mixed preparations of DNA was used as template for the CSP primers. PCR products were analyzed on 2% agarose gels described under Methods. The markers are the 1-kb ladder (BRL).
for CSP and MSA-I. Figure 2B shows the results for the CSP products. Both the larger D2 and the smaller 3D7 product were detected in lanes 1,2, and 3; a 10% contamination with either DNA was easily detected. Lane 4 shows the PCR amplified from a culture that contained only 1% D2, and even there, a clear product was observed on the original gels.
PCR FOR DIFFERENTIATING
Plasmodium fakiparum
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211
neously in the same laboratory. Even the most careful culturing techniques can We have outlined a simple method for sometimes prove inadequate. For example, identifying size polymorphisms in the PCR in situations where selection for a rare phenotype like drug resistance is the goal, it is products from three genes in P. falciparum. The approach is not a new one; PCR poly- clear that contamination can be a particular morphisms have been used in many studies problem (Tanaka ef al. 1990). This method to identify particular genes. However, we is sufficiently sensitive to detect even a 1% have used several different sets of primers, contamination, and thus is a useful check in and it has allowed us to distinguish eight these circumstances. All of our work has been performed on different strains. This suggests that the approach may provide an extremely easy way laboratory populations. However, it seems likely that the method might be adapted of characterizing strains in the laboratory. The same primer pairs were not equally fairly easily to samples collected in the efficient on DNA derived from all of the field. A drop of blood dried onto filter paper strains. For example, in Fig. 2B we show would be stable for long periods and could the amplification products from various be processed in the laboratory with a modmixtures of DNA using the CSP primers. ification of the Chelex-100 preparation described here. A recent publication provides The smaller 3D7 product predominated even when the initial mix contained a lo- a basis for such a method (Warhurst et al. fold excess of D2 DNA. When the reactions 1991). It would be particularly useful when are carried to 30 cycles, the ratio of the final the temporal or spatial heterogeneity of P. products is not a measure of the starting falciparum is to be studied, or when the amounts of DNA, so no quantitative esti- possibility of mixed infections in one humate of relative amounts of the starting man is important to ascertain (Willet et al. DNA can be made with this protocol. How- 1991). Whether this approach proves useful ever, this does increase the sensitivity of the in the field or not, it clearly can solve some problems faced by workers interested in the method in detecting minor contamination. The difficulty of identifying the various genetics of P. falciparum. We hope that it P. falciparum strains commonly used in will be as useful to others as it has been to us. laboratories around the world has often ACKNOWLEDGMENTS been an impediment to understanding their origin and interrelationships (Robson ef al. We thank Drs. Jean Feagin, Randy Howard, and 1992). It seems likely that with cooperation Thomas Wellems for P. fulciparum strains and DNA. In addition, the help of Dr. Feagin and Erica Werner in among the various laboratories using P. falinstructing us in the arcane art of P. fulciparum culture ciparum strains it would be fairly easy to was critical to our completion of this project. Drs. Pedefine a set of primers that could uniquely ter Myler, Jean Feagin, and Randy Howard, the other identify each of the commonly used strains. members of the “Malaria Group,” and Dr. Keith MarThe technical ease and speed of this assay tin all provided important advice and information at and the small amount of culture required several stages of the project. This investigation refinancial support from the UNDPiWorld Bank/ make it ideal for monitoring strains in the ceived WHO Special Programme for Research and Training laboratory. Even clones available in very in Tropical Diseases (TDR). small volumes can be assayed, and the anREFERENCES swer is available within a day. In addition, it allows careful monitoring of the integrity DAME, J. B., WILLIAMS, J. L., MCCUTCHAN, T. F., of populations. This is a particular concern WEBER, J. L., AND WIRTZ, R. A., et al. 1984. Structure of the gene encoding the immunodominant surwhen several strains are cultured simultaDISCUSSION
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