Environmental detection of Fasciola hepatica by loop-mediated isothermal amplification

Abstract

Fasciola hepatica, commonly referred to as liver flukes, is a substantial zoonotic parasitic disease of humans and livestock globally. While infection is readily controlled by anthelmintics, namely triclabendazole, the heavy reliance on triclabendazole has resulted in drug resistance appearing worldwide. Due to drug resistance, it is imperative to adopt an integrated parasite management program to preserve the efficacy of currently available anthelmintics. A integrated liver fluke management plan would benefit from a simple rapid, field-deployable diagnostic for detection of F. hepatica in environment and the host. Therefore, a rapid DNA test using loop-mediated isothermal amplification was developed and optimised for the detection of F. hepatica from faecal and water samples to enable the detection of parasites both within the host and from the environment. The assay presented here is fast, with amplification in ≤20 min, and highly sensitive, with a detection limit of 5 × 10^-4 ng/µL. The workflow presented here provides a time to result of ≤60 min without requiring a commercial kit for the extraction of DNA from faecal and water samples, and pending further validation from field-samples, could potentially be used to enable real-time decision making to mitigate parasite prevalence on a farming property and with no requirement for sample transportation.

Keywords: Detection; Environment; Fasciola hepatica; Field; Fluke; LAMP; Parasite.

Figure 1. Specificity panel used to determine FhLAMP specificity using common livestock helminths and snail species.

(A, B) Specificity panels for FhLAMP using commonly found helminths; (C) snails important in F. hepatica ecology and/or lifecycle; (D) assessed non-target amplification from F. hepatica free cattle faecal samples. No T_p values were observed for all non-target specimens used.

Figure 2. Average time-to-positive (Tp, represented in minutes:seconds) values of cattle faecal samples spiked with varying quantities of F. hepatica gDNA by LAMP.

Multiple dilutions (1/20, 1/50, 1/100, and 1/200) of Fasciola hepatica genomic DNA spiked in faecal samples were detected by FhLAMP. Dilution factors producing the lowest T_p’s were assessed with a 1/50 dilution factor chosen producing the lowest and most consistent T_p values. Error bars represent standard deviation of T_p’s. ^† denotes n = 1/2 replicates amplifying, hence no standard deviation values.

Figure 3. (A–B) Average time-to-positive (Tp, represented in minutes:seconds) values of cattle faecal samples spiked with varying quantities of F. hepatica eggs, detected by FhLAMP.

Average Tp values and standard deviations of F. hepatica negative cattle faecal samples spiked with varying quantities of F. hepatica eggs, assessed using different lysis buffers for use in FhLAMP using minimal processing methods for DNA extraction. Samples were diluted 1/50 and the most consistent T_p values for each lysis buffer assessed, with SET buffer chosen producing the most consistent amplification with lowest T_p standard deviation relative to other lysis buffers used. Error bars represent standard deviation of T_p values. ^†denotes n = 1/2 replicates amplifying, hence no standard deviation values.