Dataset Information

Real-time quantitative reverse transcription PCR for monitoring of blood-stage Plasmodium falciparum infections in malaria human challenge trials.

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 ?L) sample volume requiring minimal pre-analytical handling, making it useful for clinical trials.

SUBMITTER: Murphy SC

PROVIDER: S-EPMC3284350 | biostudies-literature | 2012 Mar

REPOSITORIES: biostudies-literature

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Real-time quantitative reverse transcription PCR for monitoring of blood-stage Plasmodium falciparum infections in malaria human challenge trials.

Murphy Sean C SC Prentice Jennifer L JL Williamson Kathryn K Wallis Carolyn K CK Fang Ferric C FC Fried Michal M Pinzon Cris C Wang Ruobing R Talley Angela K AK Kappe Stefan H I SH Duffy Patrick E PE Cookson Brad T BT

The American journal of tropical medicine and hygiene 20120301 3

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 ...[more]

PMID: 22403305

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Dataset Information

Real-time quantitative reverse transcription PCR for monitoring of blood-stage Plasmodium falciparum infections in malaria human challenge trials.

Publications

Real-time quantitative reverse transcription PCR for monitoring of blood-stage Plasmodium falciparum infections in malaria human challenge trials.

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