A fluorescence-based polymerase chain reaction-linked
single-strand conformation polymorphism (F-PCR-SSCP)
assay for the identification of Fasciola spp.
Samer Alasaad & Ramón C. Soriguer & Marawan Abu-Madi & Ahmed El Behairy &
Pablo Díez Baños & Ana Píriz & Joerns Fickel & Xing-Quan Zhu
Abstract The present study aimed to establish a fluorescencebased polymerase chain reaction-linked single-strand
conformation polymorphism (F-PCR-SSCP) assay for
the identification of Fasciola spp. Based on the sequences
of the second internal transcribed spacer (ITS-2) of the
nuclear ribosomal DNA, we designed a set of genusspecific primers for the amplification of Fasciola ITS-2,
with an estimated size of 140 bp. These primers were
labelled by fluorescence dyes, and the PCR products were
analyzed by capillary electrophoresis under nondenaturing conditions (F-PCR-SSCP). Capillary electrophoresis analysis of the fluorescence-labelled DNA fragments
displayed three different peak profiles that allowed the
accurate identification of Fasciola species: one single
peak specific for either Fasciola hepatica or Fasciola
gigantica and a doublet peak corresponding to the
“intermediate” Fasciola. Validation of our novel method
was performed using Fasciola specimens from different
host animals from China, Spain, Nigeria, and Egypt. This
F-PCR-SSCP assay provides a rapid, simple, and robust
tool for the identification and differentiation between
Fasciola spp.
S. Alasaad (*) : R. C. Soriguer : A. Píriz
Estación Biológica de Doñana, Consejo Superior de
Investigaciones Científicas (CSIC),
Avda. Américo Vespucio s/n,
41092 Seville, Spain
e-mail: samer@ebd.csic.es
J. Fickel
Research Group Evolutionary Genetics, Leibniz-Institute for Zoo
and Wildlife Research,
Alfred-Kowalke-Str. 17,
10315 Berlin, Germany
Introduction
The genus Fasciola (Platyhelminthes: Trematoda: Digenea)
is the common liver fluke of a wide range species of
animals with a global geographical distribution (Spithill and
Dalton 1998). Human infection with Fasciola spp. has been
M. Abu-Madi
Department of Health Sciences,
College of Arts and Sciences, Qatar University,
Doha, Qatar
A. El Behairy
Parasitology Department, Faculty of Veterinary Medicine,
Cairo University,
Cairo, Arab Republic of Egypt
P. D. Baños
Departamento de Patología Animal, Parasitología y Enfermedades
Parasitarias, Facultad de Veterinaria,
Universidad de Santiago de Compostela,
27071 Lugo, Spain
X.-Q. Zhu (*)
State Key Laboratory of Veterinary Etiological Biology, Key
Laboratory of Veterinary Parasitology of Gansu Province,
Lanzhou Veterinary Research Institute,
CAAS,
Lanzhou, Gansu Province 730046, People’s Republic of China
e-mail: xingquanzhu1@hotmail.com
reported to occur in a number of countries, with millions of
people estimated to be infected and hundreds of millions
being at risk worldwide (Mas-Coma et al. 1999, 2005,
2009; Haseeb et al. 2002; Ishii et al. 2002). Fasciola
hepatica is the main cause of fascioliasis because of its very
wide distribution in Europe, Africa, Asia, Oceania, and the
Americas. Fasciola gigantica appears to be of less
importance because it is restricted to the Old World (MasComa and Bargues 1997; Krämer and Schnieder 1998;
Mas-Coma et al. 2005; Alasaad et al. 2008). Moreover,
several molecular studies, covering many countries, have
identified a so-called “intermediate” Fasciola type between
F. hepatica and F. gigantica (Itagaki and Tsutsumi 1998;
Agatsuma et al. 2000; Huang et al. 2004; Ashrafi et al.
2006; Lin et al. 2007; Periago et al. 2008). Identification
based on morphological features has proven to be of limited
use, especially for identification of the “intermediate”
Fasciola type (Kendall 1965; Lin et al. 2007; Le et al.
2008). Both the overlap of geographical distribution of
Fasciola species and the presence of the “intermediate”
Fasciola required molecular tools for the accurate identification of Fasciola spp. (Marcilla et al. 2002; Velusamy
et al. 2004; Cucher et al. 2006; Gunasekar et al. 2008;
Magalhães et al. 2008; Ai et al. 2010).
Polymerase chain reaction-linked single-strand conformation polymorphism (PCR-SSCP) has significant
advantages over many other nucleic acid techniques for
the accurate analysis of allelic and mutational sequence
variation. It has proven a useful and cost-effective
alternative for the direct analysis of genetic variation
(Gasser 2006; Gasser et al. 2006), particularly when a
large number of samples needs to be analyzed. This
technique relies on the differing physical properties (here,
movement through a gel matrix) of molecules of the same
or very similar size (i.e., differing by only one or a few
nucleotides). Given its technical simplicity and high
mutation detection capacity (Gasser 1997; Gasser and
Chilton 2001), PCR-SSCP has been widely used for
parasitological investigations (e.g., Zhu et al. 2002; Li
et al. 2005).
In automated fluorescence-based polymerase chain
reaction-linked single-strand conformation polymorphism
(F-PCR-SSCP) analysis, primers are labeled with fluorescence dyes, and PCR products are analyzed by
capillary electrophoresis under non-denaturing conditions.
The method is economical, rapid, simple, robust, specific, and
with no ambiguity in interpretation for the detection of welldefined SNPs, when compared with gel-based and direct
sequencing PCR assays (Bernat et al. 2002; Jones et al.
2009). The objective of the present study was to develop an
F-PCR-SSCP assay for the identification of Fasciola spp.
using ITS-2 sequences as genetic marker.
Materials and methods
Parasite samples and DNA extraction
A total of 40 samples of adult Fasciola were collected from
naturally infected horses, sheep, cattle, and Egyptian water
buffalo in China, Spain (mainland and islands), Nigeria, and
Egypt (Table 1). Adult Fasciola were washed extensively in
physiological saline before being tentatively identified to
species according to their predilection site and morphological
features, using existing classification keys and descriptions
(Yamaguti 1958). The flukes were then fixed in 70% ethanol
until extraction of their genomic DNA. Heterologous
samples of Fascioloides magna (Italy), Clonorchis sinensis
(China), Schistosoma mansoni (Puerto Rico), and Schistosoma japonicum (China) were used as controls. The DNA
was extracted from tissue samples following the standard
phenol/chloroform procedures (Sambrook et al. 1989).
Extractions were carried out in a laboratory exclusively used
for low DNA concentration samples. Two blanks (reagents
only) were included in each extraction to monitor for
contamination (Handt et al. 1994).
Table 1 Fasciola samples used in the post-optimization evaluation of the PCR-SSCP assay for Fasciola species identification
Fasciola spp.
Geographical origin
Host species
F. hepatica
F. hepatica
F. hepatica
F. hepatica
F. hepatica
“Intermediate” Fasciola
F. gigantica
F. gigantica
F. gigantica
Spain (Valencia)
Spain (País Vasco)
Spain (Lugo)
Spain (Mallorca, Balearic Islands)
Spain (Tenerife, Canary Islands)
China (Heilongjiang)
China (Guangxi)
Niger (Tera)
Egypt (Giza)
Horse (Equus caballus)
Ovine (Ovis aries)
Bovine (Bos taurus)
Bovine (Bos taurus)
Bovine (Bos taurus)
Bovine (Bos taurus)
Bovine (Bos taurus)
Ovine (Ovis aries)
Egyptian water buffalo (Bubalus bubalis)
No. of samples
4
5
2
5
4
4
2
2
12
300
200
200
100
bp
Fig. 2 Representative ITS-2
PCR-SSCP plots of F. gigantica
(a), F. hepatica (b), and the
“intermediate” Fasciola (c)
100
bp
147
144
5400
150
153
156
159
156
159
156
159
(A) Fasciola gigantica
4800
152.4 bp
4200
3600
3000
2400
1800
1200
600
0
144
147
150
153
(B) Fasciola hepatica
5400
153.8 bp
4800
4200
3600
3000
2400
1800
1200
600
0
144
5400
147
150
153
(C) Intermediate Fasciola
4800
4200
153.8 bp
3600
3000
2400
1800
1200
600
0
Size marker
No Template Control
Schistosoma japonicum
Schistosoma mansoni
Clonorchis sinensis
Fasciola gigantica
Fasciola gigantica
Fasciola gigantica
Fasciola hepatica
Fasciola hepatica
Fasciola hepatica
‘intermediate’ Fasciola
300
‘intermediate’ Fasciola
Size marker
Fig. 1 Negative agarose gel
(2%) showing the representative
amplicons, using the Fasciola
primers SSCPFaF and SSCPFaR
152.4 bp
Design of primers for F-PCR-SSCP
Based on comparison of the known ITS-2 sequences of
Fasciola species (Huang et al. 2004; Alasaad et al. 2007),
we designed a set of genus-specific primers using Primer3
(v. 0.4.0) (Rozen and Skaletsky 2000): SSCPFaF: 5′TTGGTACTCAGTTGTCAGTGTG-3′, and SSCPFaR: 5′AGCATCAGACACATGACCAAG-3′. SSCPFaF was
labeled with 6-FAM at the 5′ end. Successful amplification
will yield 140 bp long fragments.
F-PCR-SSCP procedure
Each 30 μL PCR reaction mixture consisted of 2 μL of
gDNA, together with the PCR mixture containing 0.25 μM
of the novel primers SSCPFaF and SSCPFaR, 0.12 mM
dNTPs, 3 μL of 1X kit-supplied PCR buffer (Bioline),
1.5 mM MgCl2, 0.4% BSA, 1.5 μL DMSO, and 0.2 μL
(0.2 U/reaction) Taq polymerase (Bioline). Samples were
subjected to the following thermal profile for amplification
in a 2720 thermal cycler PTC-0200 (Bio-Rad); 4 min at 94°C
(initial denaturing), followed by 30 cycles of three steps of
1 min at 94°C (denaturation), 1 min at 55°C (annealing), and
50 s at 72°C (extension), before a final elongation of 5 min at
72°C. PCR blanks (reagents only) were included with each
PCR experiment.
Aliquots of 19.3 μL of formamide with 0.7 μL LIZ size
standard (Applied Biosystems) and 2 μL of each PCR
product were analyzed on an automated A3130xl genetic
analyzer (Applied Biosystems). Allele sizes and genotypes
were determined using GeneMapper 4.0 (Applied Biosystems) followed by manual proofreading.
To verify the correct amplification of the targeted ITS-2
segment, we sequenced the 140-bp PCR products of the
normal PCR. Sequencing reactions were carried out using
the Big Dye Terminator v1.1 cycle sequencing kit technology following the manufacturer's instructions (Applied
Biosystems). Fragments were likewise resolved on an
automated (3130xl genetic analyzer (Applied Biosystems)),
and DNA sequences were aligned and edited using the
software BioEdit (Hall 1999).
No false positive was detected from F. magna, C. sinensis, S.
mansoni, and S. japonicum samples. Sequencing analyses of
the resulting amplicons demonstrated that Fasciola sequences were identical to those reported by Huang et al. (2004).
The small difference between F. hepatica and F.
gigantica specific peaks (1.4 bp) requires the use of
positive controls when performing PCR-SSCP amplifications to overcome the possible lack of consistency in peak
sizes due to the use of different machines and running
conditions in different laboratories (Pasqualotto et al.
2007). The use of positive controls is not needed in the
case of the “intermediate” Fasciola because the presence of
the doublet peak is a diagnostic feature.
The future of F-PCR-SSCP technique remains bright as
the technology permits (1) the possibility to automate
electrophoresis of the same sample at different temperatures, which maximizes the chances of detecting DNA
alterations, (2) the elimination of radioactivity, (3) the
possibility to perform rapid separations (fragments that are
300 bp or less in length can be separated in under 30 min),
and thus (4) the ability to analyze a large number of
samples (Gasser and Chilton 2001; Jianping et al. 2001;
Bernat et al. 2002; Kane et al. 2002; Susca et al. 2007).
Using selected samples representing F. hepatica, F.
gigantic, and the “intermediate” Fasciola, this paper highlights the applicability of automated fluorescence-based
PCR-SSCP for the rapid, simple, robust, and specific
Fasciola species identification; hence, this new molecular
tool could be of pivotal interest for future molecular and
epidemiological studies of these neglected parasites.
Acknowledgements The authors are indebted to Ana L. GarcíaPérez and Ramón A. Juste (Sanidad Animal, Instituto Vasco de
Investigación y Desarrollo Agrario, NEIKER), and Margarita Buades
(Head of the Sección de Mataderos, Islas Baleares) for providing
Fasciola samples and for their helpful comments. This project was
funded by the programme “Ayudas a grupos de investigación” to
RNM118 investigation group (Spain). XQZ is supported by the State
Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary
Research Institute, Chinese Academy of Agricultural Sciences. The
experiments comply with the current laws of the countries in which
the experiments were performed.
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