A PCR Test Using the Mini-PCR Platform and Simplified Product Detection Methods Is Highly Sensitive and Specific to Detect Fasciola hepatica DNA Mixed in Human Stool, Snail Tissue, and Water DNA Specimens

Abstract

Fasciola hepatica has a complex lifecycle with multiple intermediate and definitive hosts and influenced by environmental factors. The disease causes significant morbidity in children and its prevalent worldwide. There is lack of data about distribution and burden of the disease in endemic regions, owing to poor efficacy of the different diagnostic methods used. A novel PCR-based test was developed by using a portable mini-PCR^® platform to detect Fasciola sp. DNA and interpret the results via a fluorescence viewer and smartphone image analyzer application. Human stool, snail tissue, and water samples were used to extract DNA. Primers targeting the ITS-1 of the 18S rDNA gene of Fasciola sp. were used. The limit of detection of the mini-PCR test was 1 fg/μL for DNA samples diluted in water, 10 fg/μL for Fasciola/snail DNA scramble, and 100 fg/μL for Fasciola/stool DNA scramble. The product detection by agarose gel, direct visualization, and image analyses showed the same sensitivity. The Fh mini-PCR had a sensitivity and specificity equivalent to real-time PCR using the same specimens. Testing was also done on infected human stool and snail tissue successfully. These experiments demonstrated that Fh mini-PCR is as sensitive and specific as real time PCR but without the use of expensive equipment and laboratory facilities. Further testing of multiple specimens with natural infection will provide evidence for feasibility of deployment to resource constrained laboratories.

Keywords: Fasciola hepatica; ITS-1 gene; RT-PCR; mini-PCR; molecular diagnostics.

Figure 1

“Schematics of miniPCR^® standardization as a proof of concept” (Illustration created with BioRender.com, accessed on 21 May 2024).

Figure 2

“Amplification of Fasciola DNA with the mini-PCR”. Purified DNA [1 ng/µL] from F. hepatica was used as template to amplify a ~300 bp sequence in the ITS-1 region of the Fasciola 18S rDNA gene using the mini-PCR. (A) A ~300 bp amplicon was detected in agarose gels in all the positive samples. (B) The color difference between positive and negative samples was visualized in the P51 molecular fluorescence viewer. (C) The colorimetric analysis using the Prismo Mirage application determined a difference in relative fluorescence units in positive (n = 4) and non-template or negative control (n = 4) samples.

Figure 3

“Limit of detection of SYBR Green based real time PCR assay targeting the ITS-1 region of the Fasciola 18S rDNA gene using serial dilutions of Fasciola DNA in water or scrambled with DNA extracted from human stool or snail as template.” The LOD of Fasciola DNA against the reaction cycle threshold (CT) values in water (triangle), stool (empty circle) and snail tissue (black circle) specimens are shown in the graph.

Figure 4

“LOD mini-PCR assay to detect Fasciola DNA in water”. Serial dilutions of Fasciola DNA at concentrations between 10 pg/μL–1 fg/μL amplified ~300 bp products in mini-PCR. Amplicons were detected by agarose gel electrophoresis (A) and by direct visualization using the P51 molecular fluorescence viewer (B) with a LOD of 1 fg/μL. Colorimetric analysis with the Prismo Mirage application (C) showed a difference in relative fluorescence units between the no template control and the 1 fg/μL concentration.

Figure 5

“LOD of mini-PCR assay for the detection of Fasciola DNA scrambled with stool DNA”. Serial dilutions of the scrambled DNA equivalent to a Fasciola DNA concentrations between 1 ng/μL–1 fg/μL were used to determine if stool DNA interfered with the reaction. A ~300 bp amplicon was detected in agarose gel electrophoresis (A) and by direct visualization using the P51 molecular fluorescence viewer with a LOD of 100 fg/μL (B). The colorimetric analysis with Prismo Mirage application (C) showed a difference in relative fluorescence units between the 100 fg/μL sample and the no template control.

Figure 6

“LOD of mini-PCR assay for the detection of Fasciola DNA scrambled with snail DNA”. Serial dilutions of the scrambled DNA equivalent to Fasciola DNA concentrations between 1 ng/μL−1 fg/μL were used to determine if snail DNA interfered with the reaction. A ~300 bp amplicon was detected by agarose gel electrophoresis (A) and by direct visualization using the P51 molecular fluorescence viewer with a LOD of 10 fg/μL (B). The colorimetric analysis with Prismo Mirage application (C) showed a difference in relative fluorescence units between the sample with 10 fg/μL and the no template control.

Figure 7

“Specificity of mini-PCR assay”. Fasciola and other helminths DNA at a concentration of 1 ng/μL was used as template in the mini-PCR reactions. A ~300 bp product was detected in agarose gel electrophoresis (A) only in the Fasciola DNA containing sample and in none of the other helminth DNA samples. The naked eye visualization with the P51 molecular fluorescence viewer (B) and image analysis using the Prismo Mirage application (C) showed positive results only in the sample containing Fasciola DNA. Fasciola hepatica (Fh), Hymenolepis nana (Hn), Trichuris trichiura (Tt), hookworm (Ac), Ascaris lumbricoides (Al), Calicophoron microbothrioides (Cm), Taenia solium (Ts), Diphyllobothrium latum (Dl), and Echinococcus granulosus (Em), no template (NT).