Establishment of a Simple and Rapid Nucleic Acid Detection Method for Hookworm Identification

Abstract

Hookworm infection is one of the most common neglected tropical diseases and is mainly found in tropical and subtropical areas. Two species of human hookworm are distributed in China, i.e., Ancylostoma duodenale (AD) and Necator americanus (NA). Background: Traditional microscopic technology such as the Kato-Katz method is not suitable for hookworm diagnosis due to the rapid degeneration of fragile hookworm eggs or for species identification of hookworm infection. The aim of the present study was to establish and evaluate a novel nucleic acid detection method based on recombinase-aided isothermal amplification (RAA) for the detection of hookworm infections and species identification. Methods: Based on the specific target gene sequences of hookworms (5.8S rRNA for AD and ITS2 for NA, respectively), we designed and synthesized amplification primers and fluorescence probes referring to the principle of the fluorescence recombinase-aided amplification (RAA) technique. Results: Each assay provided specific amplification of larval DNA from AD and NA by fluorescence RAA, and the detection limits in plasmids reached 102 copies and 10 copies, respectively. Genomic DNA of two hookworm species was successfully detected at a concentration of 0.1 pg/μL, revealing a high detection sensitivity. No positive amplification occurred for genomic DNA from crossed hookworm species and genomic DNA from Cryptosporidium, Giardia lamblia, Strongyloides stercoralis, Schistosoma japonicum, Ascaris lumbricoides, and Clonorchis sinensis, revealing a satisfactory specificity. Fecal sample detection results demonstrated a similar efficacy to the Kato-Katz method; however, it had a greater sensitivity than the larvae culture method. Conclusion: A simple and rapid nucleic acid method was successfully established based on RAA, which improved the detection efficacy and species identification for human hookworm infections.