Real-time loop-mediated isothermal amplification (RealAmp) for the species-specific identification of Plasmodium vivax

PLoS One. 2013;8(1):e54986. doi: 10.1371/journal.pone.0054986. Epub 2013 Jan 22.

Abstract

Plasmodium vivax infections remain a major source of malaria-related morbidity and mortality. Early and accurate diagnosis is an integral component of effective malaria control programs. Conventional molecular diagnostic methods provide accurate results but are often resource-intensive, expensive, have a long turnaround time and are beyond the capacity of most malaria-endemic countries. Our laboratory has recently developed a new platform called RealAmp, which combines loop-mediated isothermal amplification (LAMP) with a portable tube scanner real-time isothermal instrument for the rapid detection of malaria parasites. Here we describe new primers for the detection of P. vivax using the RealAmp method. Three pairs of amplification primers required for this method were derived from a conserved DNA sequence unique to the P. vivax genome. The amplification was carried out at 64°C using SYBR Green or SYTO-9 intercalating dyes for 90 minutes with the tube scanner set to collect fluorescence signals at 1-minute intervals. Clinical samples of P. vivax and other human-infecting malaria parasite species were used to determine the sensitivity and specificity of the primers by comparing with an 18S ribosomal RNA-based nested PCR as the gold standard. The new set of primers consistently detected laboratory-maintained isolates of P. vivax from different parts of the world. The primers detected P. vivax in the clinical samples with 94.59% sensitivity (95% CI: 87.48-98.26%) and 100% specificity (95% CI: 90.40-100%) compared to the gold standard nested-PCR method. The new primers also proved to be more sensitive than the published species-specific primers specifically developed for the LAMP method in detecting P. vivax.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Humans
  • Malaria, Vivax / diagnosis*
  • Nucleic Acid Amplification Techniques / instrumentation
  • Nucleic Acid Amplification Techniques / methods*
  • Plasmodium vivax / genetics*
  • Plasmodium vivax / isolation & purification*
  • Plasmodium vivax / pathogenicity
  • RNA, Ribosomal, 18S / genetics
  • Sensitivity and Specificity
  • Species Specificity

Substances

  • RNA, Ribosomal, 18S

Grants and funding

This work was supported by a CDC-UGA seed grant (OPHR #8212) awarded to JCK and VU. This study was supported in part by resources and technical expertise from the University of Georgia Research Computing Center, a partnership between the Office of the Vice President for Research and the Office of the Chief Information Officer and CDC. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.