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DETECTION SENSITIVITY AND QUANTITATION OF PLASMODIUM FALCIPARUM VAR GENE TRANSCRIPTS BY REAL-TIME RT-PCR IN COMPARISON WITH CONVENTIONAL RT-PCR

Antigenic variation in Plasmodium falciparum erythrocyte membrane protein 1, caused by a switch in transcription of the encoding var gene, is an important feature of malaria. In this study, we quantified the relative abundance of var gene transcripts present in P. falciparum 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.

Received January 8, 2006. Accepted for publication April 1, 2006.

Acknowledgments: We thank the Australian Red Cross Blood Service for supply of human blood and serum for the in vitro cultivation of the parasites used in this study, and Dr. Dennis Kyle for support of the real-time PCR component of this project. The opinions expressed herein are those of the authors and do not necessarily reflect those of the Defence Health Service or any extant policy of Department of Defence, Australia.

Note: Supplemental data for this manuscript can be found online at www.ajtmh.org.

Financial support: This work was supported by NIH grants 2RO1 AI-47500-04, -05. Michelle Gatton was supported in part by a University of Queensland Postdoctoral Research Fellowship.

^* Address correspondence to Michelle Gatton, Malaria Drug Resistance and Chemotherapy Laboratory, Queensland Institute of Medical Research, PO Royal Brisbane Hospital, Queensland 4029, Australia. E-mail: michelle.gatton{at}qimr.edu.au

Authors’ addresses: Michelle L. Gatton, Jennifer M. Peters, Karryn Gresty, and Elizabeth V. Fowler, Malaria Drug Resistance and Chemotherapy Laboratory, Queensland Institute of Medical Research, PO Royal Brisbane Hospital, Herston, Queensland, Australia 4029, Emails: michelle.gatton{at}qimr.edu.au, jennifer.peters{at}defence.gov.au, and beth.fowler{at}qimr.edu.au. Nanhua Chen and Qin Cheng, Australian Army Malaria Institute, Weary Dunlop Drive, Gallipoli Barracks, Enoggera, Queensland, Australia, 4051, Emails: nanhua.chen{at}defence.gov.au and qin.cheng{at}defence.gov.au.