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A TaqMan real-time PCR-based assay for the identiﬁcation of Fasciola
Samer Alasaad a,∗ , Ramón C. Soriguer a , Marawan Abu-Madi b , Ahmed El Behairy c , Michael J. Jowers a , Pablo Díez Ban˜ os d , Ana Píriz a , Joerns Fickel e , Xing-Quan Zhu f,∗∗
a Estación Biológica de Do˜nana, Consejo Superior de Investigaciones Cientíﬁcas (CSIC), Avda. Américo Vespucio s/n, 41092 Sevilla, Spain
b College of Arts and Sciences, Department of Health Sciences, Qatar University, Doha, Qatar
c Parasitology Department, Faculty of Veterinary Medicine, Cairo University, Cairo, Egypt
d Departamento de Patología Animal, Parasitología y Enfermedades Parasitarias, Facultad de Veterinaria, Universidad de Santiago de Compostela, 27071
e Research Group Evolutionary Genetics, Leibniz-Institute for Zoo and Wildlife Research, Alfred-Kowalke-Str. 17, D-10315 Berlin, Germany
f State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, CAAS, Lanzhou, Gansu Province 730046, PR China
a b s t r a c t
Real time qPCRTaqMan probe
Parasite identiﬁcation Fasciola hepatica Fasciola gigantica
The ‘intermediate’ Fasciola
Real time quantitative PCR (qPCR) is one of the key technologies of the post-genome era, with clear advantages compared to normal end-point PCR. In this paper, we report the ﬁrst qPCR-based assay for the identiﬁcation of Fasciola spp. Based on sequences of the second internal transcribed spacers (ITS-2) of the ribosomal rRNA gene, we used a set of genus- speciﬁc primers for Fasciola ITS-2 ampliﬁcation, and we designed species-speciﬁc internal TaqMan probes to identify F. hepatica and F. gigantica, as well as the hybrid ‘intermediate’ Fasciola. These primers and probes were used for the highly speciﬁc, sensitive, and simple identiﬁcation of Fasciola species collected from different animal host from China, Spain, Niger and Egypt. The novel qPCR-based technique for the identiﬁcation of Fasciola spp. may provide a useful tool for the epidemiological investigation of Fasciola infection, including their intermediate snail hosts.
Digenean trematodes of the genus Fasciola (Digenea: Fasciolidae) are the common liver ﬂukes of a range of animals (especially sheep and cattle) with global geograph- ical distribution (Spithill and Dalton, 1998). Fasciolosis caused by Fasciola spp. is a signiﬁcant animal health prob- lem, which causes substantial economic losses worldwide (Spithill and Dalton, 1998). Human infection with Fasciola spp. has been reported in a number of countries and mil-
∗ Corresponding author. Tel.: +34 954466700; fax: +34 954621125.
∗∗ Corresponding author. Tel.: +86 931 8342837; fax: +86 931 8340977.
E-mail addresses: firstname.lastname@example.org (S. Alasaad), email@example.com (X.-Q. Zhu).
lions of people are estimated to be infected, and hundreds of millions of people are at risk throughout the world with Bolivia, Peru, Egypt, the eastern Mediterranean, Vietnam and China being the hyper-endemic areas (Mas-Coma et al.,
1999, 2005, 2009).
Of the several species which have been described within the Fasciola genus, only Fasciola hepatica and Fasciola gigantica are commonly recognized as taxonomically valid (Huang et al., 2004; Mas-Coma et al., 2005). While F. hepat- ica mainly occurs in temperate areas, F. gigantica occurs in tropical zones, but both species can overlapp in subtropical areas (Krämer and Schnieder, 1998; Mas-Coma et al., 2005; Alasaad et al., 2008). Based on the use of ﬁrst and/or second internal transcribed spacers (ITS-1 and ITS-2) of riboso- mal RNA (rRNA) gene, an intermediate Fasciola between F. hepatica and F. gigantica has been identiﬁed in many coun-
Fasciola samples used in the post-optimisation evaluation of the TaqMan qPCR assay for Fasciola species identiﬁcation. Samples were collected from the livers of the infected hosts.
tries, such as China, Vietnam, Korea, Japan, Iran and Egypt (Itagaki and Tsutsumi, 1998; Huang et al., 2004; Ashraﬁ et al., 2006; Lin et al., 2007; Periago et al., 2008).
Morphological identiﬁcation of Fasciola species requires signiﬁcant parasitological expertise and is not a deﬁnitive method of characterization, especially for the ‘intermedi- ate’ form (Kendall, 1965; Lin et al., 2007; Le et al., 2008). Hence, different molecular tools have been developed dur- ing the last decade for the accurate identiﬁcation of Fasciola spp. (Marcilla et al., 2002; Velusamy et al., 2004; Cucher et al., 2006; Magalhães et al., 2008; Ai et al., 2010; Alasaad et al., in press). All these methods were based on end-point PCR.
Real time quantitative PCR (qPCR) is considered one of the most important molecular tools of the new genetic era (Syvanen et al., 1988). There are numerous applications of this technique in different molecular ﬁelds where the use of qPCR has nearly supplanted other approaches (Weksberg et al., 2005; VanGuilder et al., 2008).
Originally designed for gene expression assays (Bustin,
2000), TaqMan probe-based assays found wider applica- tions in other molecular studies (Papli et al., 2010). TaqMan qPCR is characterized by its high speciﬁcity and sensitiv- ity in comparison with the normal PCR. This technique requires no sample post-PCR manipulation, and is not only used for PCR ampliﬁcation but also quantiﬁcation (Livak et al., 1995). This technique was used by Schweizer et al. (2007) to estimate the prevalence of F. hepatica in the intermediate host Lymnaea truncatula. The objective of the present study was to develop and validate a new method based on TaqMan qPCR for the identiﬁcation of Fasciola spp.
2. Materials and methods
2.1. Sample collection
Forty samples of adult Fasciola were collected from nat- urally infected horse, sheep, cattle and Egyptian water buffalo from China, Spain (mainland and islands), Nigeria, and Egypt (Table 1). Adult Fasciola specimens were washed extensively in a physiological saline buffer before being tentatively assigned to species according to its predilection site and morphological features, using the available keys and descriptions (Yamaguti, 1958). The ﬂukes were then ﬁxed in 70% ethanol until extraction of their genomic DNA. DNA samples representing heterologous species of Fasci- oloides magna (from chamois in Italy), Schistosoma mansoni
(from mouse in Puerto Rico), Schistosoma japonicum (from cattle in Yunnan, China) and Clonorchis sinensis (from cat in Guangzhou, China), and DNA samples extracted from cattle and buffalo livers were used as negative control.
2.2. DNA extraction
Genomic DNA (gDNA) was extracted from tissue samples (∼1 mm3 ) following standard phenol/chloroform procedures (Sambrook et al., 1989). Two blanks (reagents only) were included in each extraction to monitor for con- tamination.
2.3. Fasciola generic-primers and TaqMan species-speciﬁc probes
We used the set of genus-speciﬁc primers for Fas- ciola ITS-2 ampliﬁcation reported by Alasaad et al. (in press) SSCPFaF: 5t -TTGGTACTCAGTTGTCAGTGTG-3t and SSCPFaR: 5t -AGCATCAGACACATGACCAAG-3t (generating
140 bp amplicons), and based on comparison of the known ITS-2 sequences of Fasciola species (Huang et al., 2004; Alasaad et al., 2007), we designed novel species-speciﬁc TaqMan probes for the identiﬁcation of F. hepatica (ProFh:
5t -ACCAGGCACGTTCCGTCACTGTCACTTT-3t ) and F. gigan-
tica (ProFg: 5t -ACCAGGCACGTTCCGTTACTGTTACTTTGTC-
3t ). Probes were designed using Primer3 (v. 0.4.0) (Rozen and Skaletsky, 2000), according to the parameters required for the qPCR applications. Both TaqMan probes were labelled with a BHQ quencher dye (Kapa Biosystems) at their 3t -end, but at the 5t -end ProFh was labelled with an FAM reporter dye and ProFg with an HEX reporter dye.
Ampliﬁcation reactions contained 0.3 jiM of each primer (SSCPFaF and SSCPFaR), 0.1 jiM of each probe (ProFh and ProFg), 1× Master Mix (Kapa Probe Fast qPCR Kit), 1 jiL of DNA solution (replaced by water in No Template Con- trols) and nuclease free-water in a ﬁnal volume of 20 jiL. Cycling conditions for the PCR consisted of a 2 min start-up denaturation step at 95 ◦ C, followed by 45 cycles of ampli- ﬁcation for 3 s at 95 ◦ C and 30 s at 60 ◦ C. PCR efﬁciency was considered and tested by the standard curve during primer selection. In all reactions, it lay between 90% and 110%. DNA-extracts were ampliﬁed in duplicate assays, and neg- ative control samples and qPCR blanks were added in all assays.
Fig. 1. Representative ampliﬁcation plots of TaqMan qPCR from F. hepatica (A), F. gigantica (B) and the ‘intermediate’ Fasciola (C). CT is the cycle at which the ﬂuorescence exceeds a predetermined threshold level. The threshold level is an arbitrary level of ﬂuorescence chosen on the basis of the baseline variability.
Fig. 2. Agarose gel (2%) showing the normal PCR amplicons produced, using the generic Fasciola primers SSCPFaF and SSCPFaR.
The qPCR was carried out in an Mx-3005P cycler (Strata- gene). Data were analysed with the software package MxPro v4.00 (Stratagene).
2.4. Assessment of speciﬁcity and sensitivity of the
TaqMan qPCR assay
The speciﬁcity of the generic primers and TaqMan probes for Fasciola identiﬁcation were evaluated using the reference samples of F. hepatica, F. gigantica and the ‘inter- mediate’ Fasciola, as well as the heterologous samples of F. magna, S. mansoni, S. japonicum and C. sinensis, and DNA samples extracted from cattle and buffalo livers, as neg- ative control. Speciﬁcity was veriﬁed by DNA sequencing of the PCR product (using SSCPFaF and SSCPFaR primers) of all the 40 samples used in the present study. Normal PCR components, their concentrations, and thermal proﬁle were similar to that reported by Alasaad et al. (2007), with the exception of the substitution the primers by SSCPFaF and SSCPFaR.
The sensitivity of our assay was assessed using a ten-fold dilution series (between 50 ng/jiL and 0.5 ng/jiL) and two- fold dilution series (between 50 ng/jiL and 0.244 pg/jiL) of F. hepatica gDNA. The limit of detection was based on the
ﬁnal dilution at which the signal of the TaqMan probes was still exponentially ampliﬁed. After optimisation, the developed TaqMan qPCR assay was used to investigate the collected 40 samples (Table 1).
3. Results and discussion
The TaqMan qPCR technique was successful in all 40 Fas- ciola samples, evidenced by ProFh and ProFg probe-based identiﬁcation of F. hepatica and F. gigantica, respectively and by the identiﬁcation of the ‘intermediate’ Fasciola using the combined probes approach (Fig. 1). No false positives were generated by our TaqMan probes from those het- erologous samples of F. magna, S. mansoni, S. japonicum and C. sinensis, or the cattle and buffalo DNA samples. For veriﬁcation purposes we ampliﬁed all fragments in a reg- ular end-point PCR using primers SSCPFaF and SSCPFaR (Fig. 2). Subsequent sequence analysis demonstrated that the Fasciola sequences obtained by us were identical to those reported by Huang et al. (2004) (GenBank accession numbers AJ557567–AJ557571).
TaqMan qPCR is characterized by its high speciﬁcity in comparison to normal PCR. The speciﬁcity of the Taq- Man RT qPCR is guaranteed by the double complementarity
Fig. 3. TaqMan RT qPCR ampliﬁcation of gDNA from Fasciola hepatica several dilutions.
between the set of primers, the internal probes and the targets, being the ﬂuorescent signal generated only by a correct annealing between probes and the target sequence during the PCR ampliﬁcation (Livak et al., 1995); while in the case of normal PCR, the speciﬁcity is guaranteed by sin- gle complementarity between the set of primers and the targets (Saiki et al., 1985).
The sensitivity of the TaqMan qPCR method was higher
(the minimum amount of Fasciola gDNA detected was
∼1 pg/jiL) compared with end-point PCR (∼1 ng/jiL Fasci- ola gDNA detected, see Ai et al., 2010). This can be attributed to (i) the short fragment ampliﬁed (140 bp), (ii) to the fact that the detection limit in a normal PCR is based on the ﬁnal dilution at which a PCR product is still visible in agarose gels. In contrast to that the ﬂuorophore’s signal of the species-speciﬁc TaqMan probes is still detectable at much lower concentrations (Fig. 3), and/or (iii) to the used PCR mixtures/conditions of the TaqMan qPCR and end-point PCR.
An additional advantage of TaqMan qPCR is that it requires no sample post-PCR manipulation of samples. Most DNA-based techniques for parasite identiﬁcation involve multiple post-PCR manipulations of samples, such as double PCR, nested PCR, semi-nested PCR, sequencing reactions, restriction digests, and/or gel electrophoresis before results can be obtained. These manipulations add time and costs, and increase the chances of human error and contamination (Berry and Sarre, 2007; O’Reilly et al.,
In conclusion, this is the ﬁrst report of qPCR-based tech- nique for the identiﬁcation of Fasciola spp., which may prove a useful tool for the epidemiological investigation of Fasciola infection in humans and animals, including their intermediate snail hosts.
Conﬂict of interest statement
The authors declare that they have no conﬂict of inter- ests.
The authors are indebted to Ana L. García-Pérez and Ramón A. Juste (Sanidad Animal, Instituto Vasco de Investi- gación y Desarrollo Agrario, NEIKER), and Margarita Buades (Head of the Sección de Mataderos, Islas Baleares) for pro- viding Fasciola samples and for their helpful comments. This project was funded by the programme ‘Ayudas a grupos de investigación’ to RNM118 investigation group (Spain). Marawan Abu-Madi is supported by Qatar Univer- sity internal grant (Project ID: QUUG-CAS-HSD-09/10-5). XQZ is supported by the State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences.
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