Cost-Effective PCR-Based Identification of (Siphonaptera) Larvae Extracted from Soil Samples Containing PCR Inhibitor-Rich Material.

Tungiasis is a neglected tropical disease caused by skin-penetrating female fleas. Although tungiasis causes severe health problems, its ecology is poorly understood and morphological descriptions of the larvae are unavailable. To identify immature stages and sites where they develop, diagnostic PCRs are required. However, flea larvae feed on soil organic matter rich in PCR inhibitors. Here, three DNA preparation methods, including a soil DNA kit that removes inhibitors, a simple ammonium acetate precipitation approach (AmAcet) and a crude lysate of larvae (CL), were combined with amplification by the highly processive FIREPol Taq or the inhibitor-resistant Phusion polymerase. Independent of the polymerase used, the frequency of successful amplification, C values and PCR efficacies for the low-cost CL and AmAcet methods were superior to the commercial kit for amplification of a 278 bp partial internal transcribed spacer-2 (ITS-2) and a 730 bp pan-Siphonaptera cytochrome oxidase II PCR. For the CL method combined with Phusion polymerase, the costs were approximately 20-fold lower than for the methods based on the soil DNA kit, which is a considerable advantage in resource-poor settings. The ITS-2 PCR did not amplify genomic or ITS-2 plasmid DNA, meaning it can be used to specifically identify .