Specific Detection of Fasciola hepatica and F. gigantica in Infected Domesticated Animals Using High-Resolution Melting Analysis (HRM)

Abstract

Background: It is difficult to make an exact morphological distinction between Fasciola hepatica and Fasciola gigantica. We used High Resolution Melting analysis (HRM) method to differentiate the F. hepatica species from F. gigantica in order to differentiate them. Methods: Overall, 80 adult liver flukes were collected from infected slaughtered animals including cattle, sheep and goats from Lorestan Province, western Iran from Sep 2015 to Aug 2017. Genomic DNA was extracted using commercial DNA extraction kit. The multilocus sequences of mDNA including COX1, COX3 and ND6 were amplified employing real-time PCR & HRM analysis. Specific and universal primer pairs were designed for differentiation Fasciola spp. Results: Universal primers cannot be used to distinguish between these two species, but in the contrary, specific primer pairs of each species could differentiate them properly. Molecular identification using specific primer pairs were consistent. Conclusion: HRM is a simple, fast and reliable method for detecting and differentiating F. hepatica from F. gigantica and can be used for diagnostic and epidemiological purposes.

Rapid Detection and Identification of Fasciola spp. and Dicrocoelium spp. Isolated from the Ruminant Livestock of Northwest Iran Using High-Resolution Melting Analysis (HRM)

“…The HRM technique by targeting Cox1 is as a powerful, rapid and sensitive method to differentiate into Fasciola hepatica and F. gigantica in the northwest (Ardabil) and southeast (Zahedan) of Iran. Another study was conducted to detect F. hepatica and F. gigantica by HRM analysis of mDNA including Cox1 , Cox3 , and ND6 in western Iran and concluded that the method is simple, fast and reliable ( 26 ).…”

“…As this method is generally restricted to be used in the diagnosis of helminths species, it was specifically used in two studies carried out in northwest and west of the country to survey Fasciola spp. ( 13 , 26 ). Moreover, another study used HRM to diagnose Trichostrongylus spp.…”

Towards the comprehension of fasciolosis (re-)emergence: an integrative overview

“…Lastly, the expansion of molecular techniques in the 2000s added a new dimension to the screening of fasciolosis transmission. Molecular tools solve the parasitological method's drawbacks of sensitivity and specificity, allow to discriminate both Fasciola species and the hybrid forms in a reliable way and to characterize genetically the different parasite and host populations (Martínez-Valladares and Rojo-Vázquez, 2016; Amor et al ., 2020; Moghadamizad et al ., 2020).…”

“…Different techniques within each type of non-classical detection method have been applied indistinctively in the monitoring of infection within definitive hosts such as ELISAs (Espino et al ., 1998), rapid immunochromatographic tests (Xifeng et al ., 2019), conventional PCR, loop-mediated isothermal amplification (Martínez-Valladares and Rojo-Vázquez, 2016) and high-resolution melting analysis (Moghadamizad et al ., 2020). In addition, detection of snail infections also evolved from the classical parasitological dissection to immunoenzymatic assays (Alba et al ., 2015 a ) and molecular methods (multiplex PCR, Alba et al ., 2015 b ; real-time PCR, Schweizer et al ., 2007; etc.).…”