1921
Volume 75, Issue 2
  • ISSN: 0002-9637
  • E-ISSN: 1476-1645

Abstract

Antigenic variation in erythrocyte membrane protein 1, caused by a switch in transcription of the encoding gene, is an important feature of malaria. In this study, we quantified the relative abundance of gene transcripts present in parasite clones using real-time reverse transcription-polymerase chain reaction (RT-PCR) and conventional RT-PCR combined with cloning and sequencing, with the aim of directly comparing the results obtained. When there was sufficient abundance of RNA for the real-time RT-PCR assay to be operating within the region of good reproducibility, RT-PCR and real-time RT-PCR tended to identify the same dominant transcript, although some transcript-specific issues were identified. When there were differences in the estimated relative amounts of minor transcripts, the RT-PCR assay tended to produce higher estimates than real-time RT-PCR. These results provide valuable information comparing RT-PCR and real-time RT-PCR analysis of samples with small quantities of RNA as might be expected in the analysis of field or clinical samples.

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2006-08-01
2017-11-19
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References

  1. Saul A, 1999. The role of variant surface antigens on malaria-infected red blood cells. Parasitol Today 15 : 455–457.
  2. Gatton ML, Martin LB, Cheng Q, 2004. The evolution of resistance to sulfadoxine-pyrimethamine in Plasmodium falciparum parasites. Antimicrob Agents Chemother 48 : 2116–2123.
  3. Bull PC, Lowe BS, Kortok M, Molyneux CS, Newbold CI, Marsh K, 1998. Parasite antigens on the infected red cell surface are targets for naturally acquired immunity to malaria. Nat Med 4 : 358–360.
  4. Ofori MF, Dodoo D, Staalsoe T, Kurtzhals JA, Koram K, Theander TG, Akanmori BD, Hviid L, 2002. Malaria-induced acquisition of antibodies to Plasmodium falciparum variant surface antigens. Infect Immun 70 : 2982–2988.
  5. Gatton ML, Cheng Q, 2004. Modelling the development of acquired clinical immunity to falciparum malaria. Infect Immun 72 : 6538–6545.
  6. Kyes S, Horrocks P, Newbold C, 2001. Antigenic variation at the infected red cell surface in malaria. Annu Rev Microbiol 55 : 673–707.
  7. Horrocks P, Pinches R, Christodoulou Z, Kyes SA, Newbold CI, 2004. Variable var transition ratse underlie antigenic variation in malaria. Proc Natl Acad Sci USA 101 : 11129–11134.
  8. Taylor HM, Kyes SA, Harris D, Kriek N, Newbold CI, 2000. A study of var gene transcription in vitro using universal var gene primers. Mol Biochem Parasitol 105 : 13–23.
  9. Peters J, Fowler E, Gatton M, Chen N, Saul A, Cheng Q, 2002. High diversity and rapid changeover of expressed var genes during acute phase of Plasmodium falciparum infections in human volunteers. Proc Natl Acad Sci USA 99 : 10689–10694.
  10. Wunderlich G, Alves P, Gölnitz U, Tada MS, de-Camargo EFP, Pereira-da-Silva LH, 2005. Rapid turnover of Plasmodium falciparum var gene transcripts and genotypes during natural non-symptomatic infections. Rev Inst Med Trop Sao Paulo 47 : 195–201.
  11. Kaestli M, Cortes A, Lagog M, Ott M, Beck HP, 2004. Longitudinal assessment of Plasmodium falciparum var gene transcription in naturally infected asymptomatic children in Papua New Guinea. J Infect Dis 189 : 1942–1951.
  12. Salanti A, Staalsoe T, Lavstsen T, Jensen AT, Sowa MP, Arnot DE, Hviid L, Theander TG, 2003. Selective upregulation of a single distinctly structured var gene in chondroitin sulphate A-adhering Plasmodium falciparum involved in pregnancy-associated malaria. Mol Microbiol 49 : 179–191.
  13. Jensen AT, Magistrado P, Sharp S, Joergensen L, Lavstsen T, Chiucchiuini A, Salanti A, Vestergaard LS, Lusingu JP, Hermsen R, Sauerwein R, Christensen J, Nielsen MA, Hviid L, Sutherland C, Staalsoe T, Theander TG, 2004. Plasmodium falciparum associated with severe childhood malaria preferentially expresses PfEMP1 encoded by Group A var genes. J Exp Med 199 : 1179–1190.
  14. Lavstsen T, Magistrado P, Hermsen CC, Salanti A, Jensen AT, Sauerwein R, Hviid L, Theander TG, Staalsoe T, 2005. Expression of Plasmodium falciparum erythrocyte membrane protein 1 in experimentally infected humans. Malar J 4 : 21.
  15. Duffy MF, Byrne TJ, Elliott SR, Wilson DW, Rogerson SJ, Beeson JG, Noviyanti R, Brown GV, 2005. Broad analysis reveals a consistent pattern of var gene transcription in Plasmodium falciparum repeatedly selected for a defined adhesion phenotype. Mol Microbiol 56 : 774–788.
  16. Fowler EV, Peters JM, Gatton ML, Chen N, Cheng Q, 2002. Genetic diversity of the DBLalpha region in Plasmodium falciparum var genes among Asia-Pacific isolates. Mol Biochem Parasitol 120 : 117–126.
  17. Roberts DJ, Craig AG, Berendt AR, Pinches R, Nash G, Marsh K, Newbold CI, 1992. Rapid switching to multiple antigenic and adhesive phenotypes in malaria. Nature 357 : 689–692.
  18. Cheng Q, Lawrence G, Reed C, Stowers A, Ranford CL, Creasey A, Carter R, Saul A, 1997. Measurement of Plasmodium falciparum growth rates in vivo: a test of malaria vaccines. Am J Trop Med Hyg 57 : 495–500.
  19. Bozdech Z, Llinás M, Pulliam BL, Wong ED, Zhu J, DeRisi JL, 2003. The transcriptome of the intraerythrocytic development cycle of Plasmodium falciparum. PLoS Biol 1 : 85–100.
  20. Livak KJ, Schmittgen TD, 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔcT method. Methods 25 : 402–408.
  21. Soong R, Ladányi A, 2003. Improved indicators for assessing the reliability of detection and quantification by kinetic PCR. Clin Chem 49 : 973–976.
  22. Gardner MJ, Hall N, Fung E, White O, Berriman M, Hyman RW, Carlton JM, Pain A, Nelson KE, Bowman S, Paulsen IT, James K, Eisen JA, Rutherford K, Salzberg SL, Craig A, Kyes S, Chan M, Nene V, Shallom SJ, Suh B, Peterson J, Angiuoli S, Pertea M, Allen J, Selengut J, Haft D, Mather MW, Vaidya AB, Martin DMA, Fairlamb AH, Fraunholz MJ, Roos DS, Ralph SA, Mc-Fadden GI, Cummings LM, Subramanian GM, Mungall C, Venter JC, Carucci DJ, Hoffman SL, Newbold C, Davis RW, Fraser CM, Barrell B, 2002. Genome sequence of the human malaria parasite Plasmodium falciparum. Nature 419 : 498–511.
  23. Wang X, Rikihisa Y, Lai T, Kumagai Y, Zhi N, Reed SM, 2004. Rapid sequential changeover of expressed p44 genes during the acute phase of Anaplasma phagocytophilum infection in horses. Infect Immun 72 : 6852–6859.
  24. Nash TE, 2002. Surface antigenic variation in Giardia lamblia. Mol Microbiol 45 : 585–590.
  25. Ropolo AS, Saura A, Carranza PG, Lujan HD, 2005. Identification of variant-specific surface proteins in Giardia muris trophozoites. Infect Immun 73 : 5208–5211.
  26. Tu ZC, Gaudreau C, Blaser MJ, 2005. Mechanisms underlying Campylobacter fetus pathogenesis in humans: surface-layer protein variation in relapsing infections. J Infect Dis 191 : 2082–2089.
  27. Kissinger JC, Brunk BP, Crabtree J, Fraunholz MJ, Gajria B, Milgram AJ, Pearson DS, Schug J, Bahl A, Diskin SJ, Ginsburg H, Grant GR, Gupta D, Labo P, Li L, Mailman MD, McWeeney SK, Whetzel P, Stoeckert CJ, Roos DS, 2002. The Plasmodium genome database. Nature 419 : 490–492.
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Supplementary Data

Supplemental data

  • Received : 08 Jan 2006
  • Accepted : 01 Apr 2006

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