期刊
AMERICAN JOURNAL OF TROPICAL MEDICINE AND HYGIENE
卷 86, 期 3, 页码 383-394出版社
AMER SOC TROP MED & HYGIENE
DOI: 10.4269/ajtmh.2012.10-0658
关键词
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资金
- PATH Malaria Vaccine Initiative
- U.S. Department of Defense [W81XWH-09-1-0531]
- U.S. Army Medical Research Acquisition Activity
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 x 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 mu L) sample volume requiring minimal pre-analytical handling, making it useful for clinical trials.
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