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

Abstract:

Tungiasis is a neglected tropical disease caused by skin-penetrating female Tunga penetrans fleas. Although tungiasis causes severe health problems, its ecology is poorly understood and morphological descriptions of the larvae are unavailable. To identify T. penetrans 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, Cq 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 Ctenocephalides felis genomic or Tunga trimammilata ITS-2 plasmid DNA, meaning it can be used to specifically identify T. penetrans.
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APA

Lung'aho, A & Lung'aho, A (2024). Cost-Effective PCR-Based Identification of Tunga penetrans (Siphonaptera) Larvae Extracted from Soil Samples Containing PCR Inhibitor-Rich Material. Afribary. Retrieved from https://afribary.com/works/cost-effective-pcr-based-identification-of-tunga-penetrans-siphonaptera-larvae-extracted-from-soil-samples-containing-pcr-inhibitor-rich-material

MLA 8th

Lung'aho, Amugune and Amugune Lung'aho "Cost-Effective PCR-Based Identification of Tunga penetrans (Siphonaptera) Larvae Extracted from Soil Samples Containing PCR Inhibitor-Rich Material" Afribary. Afribary, 10 Mar. 2024, https://afribary.com/works/cost-effective-pcr-based-identification-of-tunga-penetrans-siphonaptera-larvae-extracted-from-soil-samples-containing-pcr-inhibitor-rich-material. Accessed 21 May. 2024.

MLA7

Lung'aho, Amugune, Amugune Lung'aho . "Cost-Effective PCR-Based Identification of Tunga penetrans (Siphonaptera) Larvae Extracted from Soil Samples Containing PCR Inhibitor-Rich Material". Afribary, Afribary, 10 Mar. 2024. Web. 21 May. 2024. < https://afribary.com/works/cost-effective-pcr-based-identification-of-tunga-penetrans-siphonaptera-larvae-extracted-from-soil-samples-containing-pcr-inhibitor-rich-material >.

Chicago

Lung'aho, Amugune and Lung'aho, Amugune . "Cost-Effective PCR-Based Identification of Tunga penetrans (Siphonaptera) Larvae Extracted from Soil Samples Containing PCR Inhibitor-Rich Material" Afribary (2024). Accessed May 21, 2024. https://afribary.com/works/cost-effective-pcr-based-identification-of-tunga-penetrans-siphonaptera-larvae-extracted-from-soil-samples-containing-pcr-inhibitor-rich-material