Validation of the degenerate primer pairs used in quantitative real-time PCR.

Abstract

<p>Primer pairs targeting the multi-copy gene families <i>var</i>, <i>rif-A</i>, <i>rif-B</i>, <i>stevor</i> and <i>pfmc-2tm</i> were designed to amplify a broad repertoire of different genes present in the 3D7 genome (first bar, 1), as indicated by <i>in silico</i> PCR results (second bar, 2). Experimental validation revealed similar numbers of genes amplified by the indicated primer pairs in every <i>P. falciparum</i> genotype relative to single-copy <i>fructose-bisphosphate aldolase</i> (RELATNO) (third to seventh bar, 3–7). <i>In silico</i> PCR results were experimentally confirmed for the <i>var</i> (red), <i>stevor</i> (green) and <i>pfmc-2tm</i> (yellow) primer pairs; however, the <i>rif-A</i> (dark blue) and <i>rif-B</i> (light blue) primer pairs only partially covered the genomic repertoire. Shown are mean values and standard deviations obtained by analysing two biological samples of 3D7 and <i>ex vivo</i> isolated gDNA of the clinical isolates in quadruplicates. 1: Number of genes present in the 3D7 genome (set as 100%); 2: number and percentage of genes amplified <i>in silico</i> using the one mismatch configuration (<a href="http://insilico.ehu.es" target="_blank">http://insilico.ehu.es</a>); 3–7: experimentally calculated RELATNOs of amplified genes of the indicated multi-copy gene families using gDNA from the 3D7 laboratory strain (3) (including percentages) and from clinical isolates #1 (4), #2 (5), #3 (6) and #4 (7).</p