Development and Evaluation of a Single-Step Duplex PCR for Simultaneous Detection of Fasciola hepatica and Fasciola gigantica (Family Fasciolidae, Class Trematoda, Phylum Platyhelminthes)

Abstract

A single-step multiplex PCR (here referred to as a duplex PCR) has been developed for simultaneous detection and diagnosis of Fasciola hepatica and F. gigantica. These species overlap in distribution in many countries of North and East Africa and Central and Southeast Asia and are similar in egg morphology, making identification from fecal samples difficult. Based on a comparative alignment of mitochondrial DNA (mtDNA) spanning the region of cox1-trnT-rrnL, two species-specific forward primers were designed, FHF (for F. hepatica) and FGF (for F. gigantica), and a single reverse primer, FHGR (common for both species). Conventional PCR followed by sequencing was applied using species-specific primer pairs to verify the specificity of primers and the identity of Fasciola DNA templates. Duplex PCR (using three primers) was used for testing with the DNA extracted from adult worms, miracidia, and eggs, producing amplicons of 1,031 bp for F. hepatica and 615 bp for F. gigantica. The duplex PCR failed to amplify from DNA of other common liver and intestinal trematodes, including two opisthorchiids, three heterophyids, an echinostomid, another fasciolid, and a taeniid cestode. The sensitivity assay showed that the duplex PCR limit of detection for each Fasciola species was between 0.012 ng and 0.006 ng DNA. Evaluation using DNA templates from 32 Fasciola samples (28 adults and 4 eggs) and from 25 field-collected stools of ruminants and humans revealed specific bands of the correct size and the presence of Fasciola species. This novel mtDNA duplex PCR is a sensitive and fast tool for accurate identification of Fasciola species in areas of distributional and zonal overlap.

Development of SNAP markers based on nucleotide variability of WRKY genes in coconut and their validation using multiplex PCR

“…Such selection is necessary to avoid overlapping among PCR amplicons (Culley et al 2013) which result in ambiguous scoring. Agarose gel electrophoresis can only differentiate 30 bp differences in fragments of less than 300 bp while larger DNA fragment requires larger size differences to be able to score the amplicons unambiguously (Le et al 2012;Liu and Wu 2012;Culley et al 2013). Although multiplex PCR has advantages, the potential disadvantages should also be considered before applying the approach such as selection among different sets of primers and low amplification efficiency because of primer competition (Xu et al 2012).…”

Development of Two Antibody Detection Enzyme-Linked Immunosorbent Assays for Serodiagnosis of Human Chronic Fascioliasis

“…To date, various diagnostic techniques have been developed, including molecular techniques such as PCR, facilitating the identification and discrimination of Fasciola spp. in areas in which F. gigantica and F. hepatica coexist (7,8). Recently, evaluation of field-collected stools samples from ruminants and humans by duplex PCR revealed that this method is sensitive and is able to identify Fasciola spp.…”

Bacciger bacciger (Trematoda: Fellodistomidae) infection effects on wedge clam Donax trunculus condition

“…Knowledge of these genomic sequences would be of great interest in future studies to determine new detection and quantification methods for this trematode in bivalves, using techniques such as real-time quantitative PCR. No such tools currently exist for the genus Bacciger, although PCR detection is available for various other parasites such as Perkinsus species (Park et al 2005, Abollo et al 2006 or other trematodes (Baba et al 2012, Le et al 2012. Phylogenetic analysis based upon the V4 regions of the 18S gene revealed that species belonging to the Fellodistomidae could be separated into 2 distinct groups.…”