Plasmodium falciparum:Parasite Typing by Using a Multicopy Microsatellite Marker, PfRRM

EXPERIMENTAL PARASITOLOGY ARTICLE NO.

89, 262–265 (1998)

PR984299

RESEARCH BRIEF Plasmodium falciparum: Parasite Typing by Using a Multicopy Microsatellite Marker, PfRRM

Xin-Zhuan Su,*,1 Daniel J. Carucci,†,2 and Thomas E. Wellems* *Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20982-0425; and †Naval Medical Research Institute, Rockville, Maryland 20852

Su, X.-Z., Carucci, D. J., and Wellems, T. E. 1998. Plasmodium falciparum: Parasite typing by using a multicopy microsatellite marker, PfRRM. Experimental Parasitology 89, 262–265. Index Descriptors and Abbreviations: Malaria, genetic diversity; simple sequence length polymorphisms; interspersed repetitive elements; genetic fingerprinting.

It is time consuming for laboratories maintaining multiple clones of Plasmodium falciparum to routinely verify parasite identity and test for cross-contamination. Independent laboratories that maintain reference stocks of parasite clones also require standard means of confirmation. For these purposes, two methods based on polymorphic genetic markers are frequently used to type or fingerprint malaria parasites. The first method is based on DNA restriction fragment length polymorphism (RFLP) analysis. Parasite DNAs are digested with restriction enzymes, separated on agarose gels, blotted to nitrocellulose or nylon membrane, and probed with polymorphic or repetitive DNA sequences (Dolan et al. 1990, 1993; Limpaiboon et al. 1991). The second method relies on length polymorphisms of DNA repeats (microsatellites or minisatellites) amplified by the polymerase chain reaction (PCR) with primers from flanking sequences. The PCR-based typing has several advantages over the 1 To whom correspondence should be addressed at Building 4, Room 126, LPD, NIAID, NIH Campus, Bethesda, MD 20892-0425. Fax: (301) 402-0079. E-mail: [email protected]. 2 Disclaimer: The opinions and assertions contained herein are the private ones of the author and are not to be construed as official or as reflecting the views of the Department of the Navy or the Naval Service at large.

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RFLP method: PCR typing is rapid and requires a small amount of DNA; parasite DNA samples can be prepared quickly from a few drops of blood or cultures and used directly in the PCR; and PCR products may be directly visualized without blotting and hybridization (Wooden et al. 1993; Carcy et al. 1995; Su and Wellems 1997). PCR typing can involve single-copy or multicopy elements from the genome. As examples of single-copy elements, polymorphic repeat regions of the major merozoite surface protein 1 and 2 (MSP1, MSP2) and circumsporozoite surface protein (CSP) genes may be amplified to produce bands that distinguish different parasites (Kimura et al. 1990; Reeder and Marshall 1994; Wooden et al. 1993). These polymorphisms have been useful for field studies of heterogeneous parasite populations and mixed infections. Although potentially limited only by the number of markers available, practical applications generally incorporate at most a few markers because of the multiple reactions involved. The limited number of polymorphic regions thus tested may miss clones that have certain alleles in common but vary elsewhere in the genome. In such cases, the use of polymorphic, multicopy repetitive elements distributed widely in the parasite genome has the advantage of incorporating large numbers of loci in a single analysis. Amplification of these repetitive sequences distinguishes parasite isolates in multiple bands (Carcy et al. 1995; Su and Wellems, 1997). In this report, we describe the typing of parasite isolates based on a polymorphic microsatellite (PfRRM) within a known multicopy PfRR, or rif, repetitive element of P. falciparum (Weber 1988; de Bruin et al. 1994). The advantage of this typing method over our previously described typing marker PJ3 is that DNA bands from the PfRRM are less sensitive to the changes in PCR cycling conditions. Identical patterns of DNA bands amplified with PfRRM primers can be reproduced despite variations in annealing temperatures (40–508C) in PCR. Figure 1 shows a typical autoradiograph

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FIG. 1. Microsatellite PfRRM fingerprinting of 16 P. falciparum field isolates. Primers (58-TACGTTACATTATGTTTTA-38 and 58-ATATGTATTGCGCTTTTA-38) flanking a multicopy simple repeat sequence were synthesized with Milipore Expedite 8909 DNA synthesizer. One of the PCR primers was labeled with 32P by polynucleotide kinase (Boehringer Mannheim, Germany). PCR mixtures contained 1.5 ml 10 3 PCR buffer, 0.3 ml 10 mM dNTPs, 0.5ml of each primer (10 pM/ml), 0.1 ml Taq polymerase (0.5 U), 1 ml DNA (10 ng), and H2O to 15 ml. The mixtures were denatured at 948C for 2 min and cycled at 948C for 20 s, 458C and 408C for 10 s each; and 608C for 30, s, for 30 cycles. For typing cultured parasites, DNA from 100 ml parasite culture with 0.5 – 1.0 % parasitemia was quickly prepared by saponin lysis and boiling (Su and Wellems, 1997). Five microliters of PCR products was mixed with 4 ml sequencing dye, heat denatured, and separated on 6% polyacrylamine gel (60 W in 1X TBE for about 2 h). The gels were then dried and exposed to Kodak Bio-Max films.

of PCR products from 16 P. falciparum isolates (Bayoumi et al. 1993; Dolan et al. 1993; Su et al. 1997). Multiple bands are evident from all the parasite DNAs, with a unique band pattern for each parasite. PfRRM was also used to type and verify the identity of four 3D7 clones maintained in different laboratories. The 3D7 isolate has been selected as a model parasite in genome mapping (Dame et al. 1996) and genome sequencing efforts, underscoring the necessity of verifying that the parasite clones used in different sequencing centers are actually the same parasite. Fingerprinting of the 3D7 clones with the PfRRM marker produced an identical pattern of 10 DNA bands from all of the four 3D7 clones, whereas other P. falciparum isolates showed variations in the number and sizes of bands (Fig. 2). This result confirms an identical clonal origin for all the 3D7 stocks. (Results from PJ3 typing and other microsatellite markers also were the same, data not shown). The pattern also demonstrates the mitotic stability of the PfRRM repeats through numerous generations of cultivation in different laboratories.

To avoid using radioactive materials and to take advantage of the high throughput of the automatic DNA sequencer, we also labeled the PfRRM PCR products with fluorescent dye and detected the DNA products with an ABI 377 automatic DNA sequencer. As shown in Fig. 3, unique band patterns were detected from different P. falciparum isolates. Comparison of the band patterns of 16 parasite isolates by radioactive labeling and [F]dUTP labeling methods (Figs. 1 and 3) showed similar results, although the relative intensity of bands and spatial separation differed between the two methods. These differences were presumably due to longer separation in automatic sequencing gel and the software treatment of labeled DNA signals. The P. falciparum fingerprinting method described here is rapid and reliable. Consistent band patterns, or “fingerprints” of different parasite isolates are reproducible and can be obtained in a single day. With the PfRRM reactions, exactly the same band patterns were reproduced despite changes of a few degrees in annealing temperature, in contrast with the sensitivity of band patterns from

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FIG. 2. PfRRM typing of 3D7 clones from different laboratories. The patterns from all four 3D7 clones are identical, indicating the same parasite or the same origin. 3D7/3A was obtained from Dr. Imogene Snyder, WRAIR (1995); 3D7/OX was from Dr. Alister Craig, Oxford; 3D7/NIH was from NIH stocks of the original clone (Walliker et al. 1987), and 3D7/G1 was from Dr. Imogene Snyder, WRAIR (1997). FVO was from Dr. William E. Collins at the CDC, Atlanta.

PJ3 primers to such variation. From our experience, labeling the PCR product with [F]dUTP is a simple way to detect PfRRM polymorphism if an automatic sequencer is available and provides a useful nonradioactive means of identifying individual parasite clones.

(We thank Dr. Mike Ferdig for critical reading of the manuscript. We also thank Mrs. Laura Kirkmen, Mrs. Anna Liu, and Ms. Kathy Moch for assistance in parasite cultures and DNA sample preparations.)

SU, CARUCCI, AND WELLEMS

FIG. 3. Fluorescent PfRRM fingerprints of 16 parasite isolates. Each typing reaction contained 3 ml 10 3 PCR buffer, 0.6 ml 10 mM dNTPs, 0.5 ml of each primer (100 pM/ml), 0.1 ml Taq polymerase (0.5 U), 0.1 ml of 10 mM [F]dUTPs (R6G, Perkin-Elmer, Foster City, CA), 1 ml DNA (10 ng), and H2O to 30 ml. The reaction was cycled 30 times at 948C for 20 s, 528C and 458C for 10 s each, and 608C for 30 s. After 70% ethanol/0.5 mM MgCl2 precipitation at R.T. for 15 min, the PCR products were centrifuged at 3000g for 15 min and dissolved in 6 ml loading dye. Two microliters of each DNA sample was loaded onto a 5% Long Ranger gel (FMC BioProducts, Rockland, ME) and subjected to electrophoresis for 5 h (R 23A module and ABI 377 DNA sequencer, PE Applied Biosystem, CA). Gel images were printed from a computer screen in a color printer. The original green display was converted into a black and white image for photographic reproduction.

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Received 18 December 1997; accepted with revision 23 February 1998