Plasmodium vivax: parasitemia determination by real-time quantitative PCR in Aotus monkeys.
Plasmodium vivax and Plasmodium falciparum are the two prevalent human malaria species. A Colombian P. vivax wild strain has been adapted in Aotus nancymaae monkeys for use in further biological and immunological studies. We present data validating a real-time PCR assay quantifying P. vivax parasitemia, using the small subunit ribosomal RNA genes as an amplification target. P. vivax species-specific primers were designed on the 18S ribosomal gene V8 region, for amplifying both asexual and sporozoite ssrRNA genes. The assay detects amplification products bound to fluorescent SYBR-Green I dye using Perkin-Elmer GeneAmp-5700-SDS. Linear range standard curves from 6 DNA concentration logs (+0.99 correlation coefficients) were obtained. Standard curves were constructed using a plasmid containing target gene for real-time PCR amplification. This P. vivax specific assay is very sensitive, having a three parasite detection limit, and is reproducible and accurate. It involves a "closed-tube" PCR, avoids time-consuming post-PCR manipulation, and decreases potential PCR contamination.
Polanco
Juan Carlos
JC
Fundación Instituto de Inmunología de Colombia (FIDIC), Departamento de Biología Molecular, Carrera 50 No. 26-00 Bogotá, Colombia.
Rodríguez
Josefa Antonia
JA
Corredor
Vladimir
V
Patarroyo
Manuel Alfonso
MA
D000818:Animals; D016645:Aotidae; D016054:DNA, Protozoan / Q000097:blood; D004275:DNA, Ribosomal / Q000097:blood*; D016780:Malaria, Vivax / Q000175:diagnosis*; D018512:Parasitemia / Q000175:diagnosis*; D010966:Plasmodium vivax / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012335:RNA, Ribosomal / Q000235:genetics; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D016054:DNA, Protozoan; D004275:DNA, Ribosomal; D012335:RNA, Ribosomal