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Real-time quantitative reverse transcription PCR for monitoring of blood-stage Plasmodium falciparum infections in malaria human challenge trials.
Title | Real-time quantitative reverse transcription PCR for monitoring of blood-stage Plasmodium falciparum infections in malaria human challenge trials. |
Publication Type | Journal Article |
Year of Publication | 2012 |
Authors | Murphy, SC, Prentice, JL, Williamson, K, Wallis, CK, Fang, FC, Fried, M, Pinzon, C, Wang, R, Talley, AK, Kappe, SHI, Duffy, PE, Cookson, BT |
Journal | Am J Trop Med Hyg |
Volume | 86 |
Issue | 3 |
Pagination | 383-94 |
Date Published | 2012 Mar |
ISSN | 1476-1645 |
Keywords | Adult, Base Sequence, Clinical Trials as Topic, Humans, Malaria, Molecular Diagnostic Techniques, Molecular Sequence Data, Plasmodium falciparum, Real-Time Polymerase Chain Reaction, Reproducibility of Results, Reverse Transcriptase Polymerase Chain Reaction, RNA, Protozoan, RNA, Ribosomal, 18S, Sensitivity and Specificity, Sequence Analysis, RNA, Species Specificity |
Abstract | To detect pre-patent parasitemia, we developed a real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) for the asexual 18S ribosomal RNA (rRNAs) of Plasmodium falciparum. Total nucleic acids extracted from whole blood were combined with control RNA and tested by qRT-PCR. The assay quantified > 98.7% of parasite-containing samples to ±0.5 log(10) parasites/mL of the nominal value without false positives. The analytical sensitivity was ≥ 20 parasites/mL. The coefficient of variation was 0.6% and 1.8% within runs and 1.6% and 4.0% between runs for high and low parasitemia specimens, respectively. Using this assay, we determined that A-type 18S rRNAs are stably expressed at 1 × 10(4) copies per ring-stage parasite. When used to monitor experimental P. falciparum infection of human volunteers, the assay detected blood-stage infections 3.7 days earlier on average than thick blood smears. This validated, internally controlled qRT-PCR method also uses a small (50 μL) sample volume requiring minimal pre-analytical handling, making it useful for clinical trials. |
DOI | 10.4269/ajtmh.2012.10-0658 |
Alternate Journal | Am. J. Trop. Med. Hyg. |
PubMed ID | 22403305 |