Spurious amplification of a Plasmodium vivax small-subunit RNA gene by use of primers currently used to detect P. knowlesi

Abstract

The PCR primers commonly used to detect Plasmodium knowlesi infections in humans were found to cross-react stochastically with P. vivax genomic DNA. A nested primer set that targets one of the P. knowlesi small-subunit rRNA genes was validated for specificity and for sensitivity of detection of <10 parasite genomes.

FIG. 1.

(A) Alignments of the target sequences for Pmk8 and Pmkr9 primers in the ssrRNA-S genes of P. vivax (n = 2; accession numbers U03080 and U07368) and P. knowlesi (n = 8; accession numbers DQ350256 to DQ350262) and schematic representation of the Plasmodium ssrRNA-S gene. The Pmkr9 sequence differs from the published sequences by a single base close to the 3′ end of the primer. (B) Alignments of the target sequences for the PkF1060, PkF1140, and PkR1550 primers in the ssrRNA-A genes of P. vivax (n = 2; accession numbers U03079 and U07367) and P. knowlesi (n = 11; accession numbers L07560AY580317 and AY327549 to AY327557) and schematic representation of the Plasmodium ssrRNA-A gene. The relative positions of the different primers employed for nested PCR amplification, Topo cloning, and screening and those of the amplified fragments are indicated above (dotted lines, primary reaction) and below (solid lines, secondary reaction) the gene representations. It should be noted that the reverse primers (indicated by the arrows pointing to the left below the sequences) are presented as their reverse complemented sequences.