Experimental Parasitology 112 (2006) 109–114 www.elsevier.com/locate/yexpr Clonorchis sinensis and Opisthorchis viverrini: Development of a mitochondrial-based multiplex PCR for their identiWcation and discrimination Thanh Hoa Le a,e,Ô, Nguyen Van De b, David Blair c, Paiboon Sithithaworn d, Donald P McManus e b a Institute of Biotechnology, Viet Nam Institute for Malariology, Parasitology and Entomology, Viet Nam c James Cook University, Australia d Khon Kaen University, Thailand e Queensland Institute of Medical Research, Australia Received July 2005; received in revised form 25 September 2005; accepted 29 September 2005 Available online 28 November 2005 Abstract We report a single, one-step PCR approach for detection and discrimination of Clonorchis sinensis and Opisthorchis viverrini in diVerent lifestage forms (adults, metacercariae, and eggs) from Wsh intermediate hosts and from infected patients Primers designed for species-speciWc PCR, amplifying portions of the mitochondrial (mt) genome, were also suitable for a multiplex PCR The latter was a single, one-step reaction under high stringency conditions, using simultaneously pairs of primers (1 pair for C sinensis—product size 612 bp, and pair for O viverrini— product size 1357 bp) Assays using serially diluted templates demonstrated that as little as 0.78 ng of genomic DNA of either species could yield amplicons Genomic DNA extracted from diVerent life-stage forms including adult worms (of both species), eggs (of O viverrini), eggs possibly of several trematode species (collected from patients infected with C sinensis in Vietnam) and mixed metacercariae of common trematodes (collected from Wshes in the C sinensis endemic areas), yielded speciWc bands of the correct size and their identity was conWrmed by sequence analysis The multiplex PCR approach described here proved to be a species-speciWc, sensitive and fast tool for accurate diagnosis of clonorchiasis and/or opisthorchiasis, permitting the detection of their metacercariae in infected Wshes or adult/eggs from patients in endemic areas 2005 Elsevier Inc All rights reserved Index descriptors: Mitochondrial DNA; Clonorchis sinensis; Opisthorchis viverrini; Metacercariae; Multiplex PCR; SpeciWcity; Sensitivity Introduction The two small liver Xukes, Clonorchis sinensis and Opisthorchis viverrini, infect >30 million people in Asia including Korea, mainland China, Taiwan island, Thailand, and Indochina (Bruckner, 1999; Chai and Lee, 2002; King and Scholz, 2001; Sithithaworn and Haswell-Elkins, 2003) These parasites also infect a number of other mammals, including dogs, cats, pigs and rodents, that can serve as reservoirs of infection In non-endemic areas (including the United States), the * Corresponding author Fax: +84 8363144 E-mail addresses: thanhL@qimr.edu.au, im-ibt@hn.vnn.vn (T.H Le) 0014-4894/$ - see front matter 2005 Elsevier Inc All rights reserved doi:10.1016/j.exppara.2005.09.012 infection is sometimes found in Asian immigrants, or following ingestion of imported, undercooked or pickled freshwater Wsh containing metacercariae (StauVer et al., 2004) The diseases caused by these zoonotic parasites can develop into a form of cholangiocarcinoma which is frequently fatal to humans (Choi et al., 2004; Mas-Coma and Bargues, 1997) Clonorchiasis and opisthorchiasis are thus important foodborne zoonotic parasitic diseases of much public concern (Fried et al., 2004; Lun et al., 2005) Clonorchis sinensis is endemic in Vietnam, mainly in the Red River Delta provinces in the North (De et al., 2003; Kino et al., 1998) Recently, we have used molecular techniques to identify O viverrini from a range of provinces in 110 T.H Le et al / Experimental Parasitology 112 (2006) 109–114 the south of Vietnam (unpublished) Both species may overlap in Central Vietnam Consequently, there is a need for accurate diagnosis/discrimination using eggs from patients or metacercariae from infected Wshes Clonorchis sinensis and O viverrini have similar lifecycles, location in the liver and pathogenicity and their morphologies, particularly in the egg and metacercariae forms, diVer only slightly These species have usually been diagnosed and distinguished from one another by detection of the eggs in feces or on examination of the reproductive organs of the adults if available (Bruckner, 1999; Fried et al., 2004) Identifying metacercariae from infected freshwater Wsh, or eggs from patients with mixed infections, is diYcult A single Wsh may host metacercariae of these liver Xukes, and also of heterophyid trematodes such as Haplorchis spp and Centrocestus spp Consequently, humans eating such Wsh undercooked often host a number of trematode species producing similar eggs Serological methods have been developed for diagnosis of clonorchiasis (Nagano et al., 2004) and common and speciWc antigens of Clonorchis and Opisthorchis have been used for immunoblot methods (Choi et al., 2003) In recent years, multiplex PCR approaches have been developed for rapid and accurate detection of organisms of particular interest, i.e., viruses (Mosquera Mdel et al., 2002), bacteria (Rivera et al., 2003), Plasmodium spp (Patsoula et al., 2003), mosquitoes (Fettene and Temu, 2003), and free-living amoebae (Pelandakis and Pernin, 2002) A mitochondrial-based multiplex PCR probe has been designed for the large liver Xuke, Fasciola hepatica in Brazil (Magalhães et al., 2004) and for the tapeworms, Taenia solium, T asiatica, and T saginata (Yamasaki et al., 2004) Molecular diagnostic methods using PCR have also been introduced to detect O viverrini in hamsters, human stool specimens, infected bithynid snails and cyprinoid Wshes (Maleewong et al., 2003; Wongratanacheewin et al., 2001, 2002) and to distinguish O felineus from other trematodes (Pauly et al., 2003) However, a multiplex PCR technique using a mix of speciWc primers, simultaneously in a single reaction under high stringency conditions, has not been developed for the discrimination of these two important species in every life-stage form In this paper, we present pairs of species-speciWc primers based on mitochondrial sequences for C sinensis and O viverrini and amplifying fragments of diVerent lengths from the diVerent species The method is accurate and sensitive and could be used to detect and identify the particular parasite(s) when present in single or mixed infections Materials and methods 2.1 Adults, metacercariae, and eggs Adult worms of C sinensis were collected from patients treated with praziquantel Metacercariae of this species were digested from tissues of infected Wshes Eggs mixed with those of other trematodes (identiWed as Haplorchis spp., Centrocestus spp., Echinostoma spp., etc by microscopy) were collected from patients in Nam Dinh province (Vietnam) Adult worms and eggs of O viverrini were from Khon Kaen, Thailand These worms were collected from golden hamsters weeks after oral infection with metacercariae The eggs were obtained by squeezing the adult worms BrieXy, they were laid on a glass and pressed with forceps to squeeze the eggs out of the body All materials were preserved in 70% ethanol and kept at ¡20 °C until use for extraction of genomic DNA 2.2 Total genomic DNA extraction Total genomic DNA was extracted from adult worms, eggs or metacercariae using the commercial QiaAmp DNA extraction kit (Qiagen) according to the manufacturer’s instructions with a slight modiWcation applied for eggs In the case of adult worms, only a single specimen was used in each DNA extraction BrieXy, a single worm was enzymatically digested in 180 l of a lysis solution (ATL buVerQiagen), then 20 l proteinase-K (50 g/ml) was added and incubated at 56 °C for 2–3 h with brief vortexing every 30 For eggs, the incubation time was set 1–2 h longer The mixture, after adding 200 l buVer AL (Qiagen) containing guanidine hydrochloride and l RNase A (100 mg/ml) and mixing by pulse-vortexing for 15 s, was further incubated at 70 °C for 10 Thereafter, 200 l of ethanol (96–100%) was added and mixed by vortexing for 15–20 s The contents were then loaded onto a QIAamp Spin Column for DNA binding and spin down for The column with the DNA bound was washed several times using solutions (AW1; AW2 buVers—Qiagen) provided according to manufacturer’s instruction Finally, the genomic DNA was eluted in 50 or 100 l elution buVer (AE) and stored at ¡20 °C until use The concentration of DNA samples was estimated using spectrophotometry (GBC UV/visible 911A spectrophotometer) The extracted genomic DNA was diluted to a working concentration of 50 ng/ l and l of this was used as template in a PCR of 25 l volume 2.3 Primer design, PCR ampliWcation, and sequencing From the complete mitochondrial DNA sequence of C sinensis and O viverrini (unpublished), and of a number of trematodes previously published (Le et al., 2002) or available in Genbank (http://www.ncbi.nlm.nih.gov/Entrez/), speciWc forward primers were designed for C sinensis on nad2; for O viverrini on nad1; and reverse primers for C sinensis on nad1 and for O viverrini on trnS1 (Fig 1) The primers were designed to produce amplicons of diVerent size for accurate discrimination between the PCR products on ethidium bromide-stained agarose gels The designed primers were: CsF (5Ј-TTAGAGGAGTTGGT GTCCCC-3Ј) and CsR (5Ј-AGCGTCACTGAACCA CACCCAC-3Ј) for C sinensis (anticipated product size 612 bp); OvF (5Ј-TACGCAGGTGGTTTGGTTG-3Ј) and T.H Le et al / Experimental Parasitology 112 (2006) 109–114 111 Fig Schematic illustration of the target regions for multiplex PCR in the mitochondrial genomes of C sinensis and O viverrini Genes and gene order of the mt genome in Xatworms are described in Le et al (2002) OvR (5Ј-AGCAGCGATAACACGACAGC-3Ј) for O viverrini (anticipated product size 1357 bp) The amplicons were cloned using TA-cloning kit (Invitrogen, USA) and recombinant plasmids were sequenced using M13 forward and reverse primers An additional reverse primer, namely OvN3R (5Ј-GCTCAATAAAGAGACCACGAAC-3Ј), which is located 539 bp from the reverse primer OvR, was designed for sequencing of the 1357 bp fragment of O viverrini Initially, PCR was carried out for each species separately, using as template total genomic DNA of either C sinensis or O viverrini PCR ampliWcation was carried out in a Wnal volume of 25 l, including 50 ng template, 10 pmol of each primer and a mix of remaining PCR components (PCR Master Mix from Promega) The PCR reactions were carried out in a MJ thermal cycler PTC-100 (MJ Research, USA) with initiation at 95 °C for min, then 35 cycles including denaturation at 95 °C for 30 s, annealing at 52 °C for 30 s, extension at 72 °C for min; and a Wnal cycle of at 72 °C to complete the ampliWcation PCR products were detected on 1% agarose gels stained with ethidium bromide in a Wealtech apparatus (Wealtech, USA) and photographs were recorded in the linked computer The PCR products were puriWed using Qiaquick PuriWcation kit (Qiagen), cloned using a TA cloning kit (Invitrogen, USA) and the selected recombinant plasmid DNAs subjected to sequencing using Big Dye Terminator CycleSequencing technology on an automated sequencer ABI 3100 Avant Genetic Analyzer The sequences were edited in SeqEd v 1.03, aligned using AssemblyLIGN v 1.9c and analysed using the MacVector 7.2.2 package (Accelrys) Identity was conWrmed by alignment to the known mtDNA sequence of the corresponding species The 612 bp sequence (nad2-V-A-D-nad1) for C sinensis and 1357 bp (nad1-N-PI-K-nad3-S1) for O viverrini were deposited in GenBank (Accession Nos.: DQ116944 for C sinensis; DQ119551 for O viverrini, respectively) (Fig 1) for O viverrini) Tests were done using diVerent combinations of template DNA (one or both species: 50 ng of template DNA from each species in every case) and primers (one or both primer pairs) The multiplex PCR conditions were exactly as those used in a single-species reaction To conWrm identity, the selected amplicons were again cloned and sequenced The sensitivity of the test was evaluated using serially diluted genomic DNA template (25, 12.5, 6.25, 3.13, 1.56, and 0.78 ng) from each species separately or combined The product obtained at the lowest template quantity (0.78 ng) was cloned and sequenced to conWrm identity 2.4 Development of multiplex PCR and testing for sensitivity The species-speciWc primer pairs, wherever used together, always generated a clear band of the expected size for C sinensis (612 bp) or O viverrini (1357 bp), depending on the template present (Fig 2) SpeciWcity of ampliWcation was conWrmed by cloning and sequencing (data not shown) Multiplex PCR was developed in a single reaction, under high stringency conditions, using simultaneously both pairs of primers reported above (1 pair for C sinensis and pair 2.5 Reaction speciWcity and sensitivity in detection of diVerent life-stage forms of C sinensis and O viverrini Total genomic DNA was extracted from samples of diVerent life-stage forms of these species, including adult worms (for both species), eggs of O viverrini (alone), eggs of mixed species (collected from patients infected with C sinensis in Vietnam and harboring those of other trematodes) and mixed metacercariae (collected from Wshes in areas endemic for C sinensis) The DNA was extracted using QiaAmp DNA extraction kit (Qiagen) and quantiWed by spectrophotometry Fifty nanograms genomic DNA of adult and metacercariae and 2.5–3.0 ng genomic DNA of eggs (equal to a quantity of approximately 2500–3000 eggs collected from 0.1 to 0.15 g feces) was used for each multiplex PCR The PCR products were detected on 1% agarose gels stained with ethidium bromide PCR products from adults, pure eggs (O viverrini), mixed eggs and mixed metacercariae (C sinensis) were cloned and sequenced to verify the mtDNA sequences Results 3.1 SpeciWcity of multiplex PCR 112 M M (Ov F-O v R) A Ov and Cs (O vFOv R Cs F O v -C sR ) (Ov and F-Ov R Ov CsF-Cs +C R) and s(O vF C Ov sF-CsR -OvR ) +C s(C sFOv Cs R) +C s(O vFOv Cs R) (Cs F-C sR ) T.H Le et al / Experimental Parasitology 112 (2006) 109–114 23.0kb 9.4kb 6.5kb 4.3kb 2.3kb 2.0kb 1357bp 23.0kb 9.4kb 6.5kb 4.3kb 0.5kb 2.3kb 2.0kb A 1357bp 0.5kb 612bp Fig Agarose gel (1%) stained with ethidium bromide showing mtDNA amplicons of C sinensis and O viverrini obtained by multiplex or single PCR (10 l/well) Cs: Clonorchis sinensis; Ov: Opisthorchis viverrini CsFCsR: primers speciWc for Cs; OvF-OvR: primers speciWc for Ov Each reaction contained 50 ng (one species) or 100 ng (both species) of template DNA Lane M, molecular marker (DNA of phage cut by HindIII) B 23.0kb 9.4kb 6.5kb 4.3kb 2.3kb 2.0kb 612bp 0.5kb 3.2 Reaction sensitivity by diluted template C The reaction assays showed high and speciWc sensitivity (Figs 3A for O viverrini and B for C sinensis, and C for the multiplex PCR of both species) Amplicons could be obtained in reactions contaning only 0.78 ng of template DNA The product generated from this dilution of template was cloned and sequenced to conWrm its identity (data not shown) In the case of C sinensis, PCR product could even be obtained when an even lower quantity of template DNA was used (Fig 3B, lane 7) 23.0kb 9.4kb 6.5kb 4.3kb 2.3kb 2.0kb 1357bp 0.5kb 612bp 3.3 Detectable ability of multiplex PCR The single, one-step multiplex PCR for C sinensis and O viverrini (using a mixture of 10 pmol of each single primer) was used to detect target DNA extracted from diVerent life-stage forms (adults, metacercariae, and eggs; Fig 4) The genomic DNA template used in each reaction was 50 ng for adult and metacercariae (about one metacercaria), and about 2.5–3.0 ng for eggs (approximately, 2500–3000 eggs) (see Wongratanacheewin et al., 2002) The multiplex PCR is able to detect the speciWc target in every template although the concentration of product from template of O viverrini eggs was somewhat lower (Fig 4, lanes 1–5) This O viverrini fragment was used, subsequently, as a template for re-ampliWcation using multiplex PCR and a distinct band was detected (data not shown) It is clear that this multiplex PCR technique can detect speciWc mtDNA of these two liver Xukes from eggs of either species alone and/or from mixed eggs and metacercariae Fig Sensitivity of the single, speciWc (A, Ov; B, Cs) and multiplex PCR for both C sinensis and O viverrini species (C) Template was genomic DNA extracted from adult worms and subjected to a dilution series for simultaneous ampliWcation for sensitivity testing Lane M, molecular marker (DNA of phage cut by HindIII); lanes 1–7, respectively, showing the amplicons obtained in the assays of the template serial dilutions equal to 25, 12.5, 6.25, 3.13, 1.56, 0.78, and 0.39 ng for each species and the sensitivity at the lowest quantity of template DNA (by arrow) Discussion Until now, microscopic examination has been the most appropriate method for detecting eggs in patients of late-stage clonorchiasis or opisthorchiasis, and for demonstrating metacercariae in Wshes Immunodiagnostic methods have also been proved to be sensitive and early techniques for early diagnosis using parasite egg antigen (Wongsaroj et al., 2001), crude speciWc antigen (common for both C sinensis and O viverrini—Choi et al., 2003); and aria g s) (e g me Cs (m Ov ixe d t ac erc de Cs (m ixe ult) (a d Ov M Cs (a d ul t) gg s e) ) T.H Le et al / Experimental Parasitology 112 (2006) 109–114 23.0kb 9.4kb 6.5kb 4.3kb 2.3kb 2.0kb 1357bp 0.5kb 612bp Fig Use of the single, one-step multiplex PCR for detecting diVerent life-stage stages of C sinensis and O viverrini In all reactions (Lanes 1–5), a mix of two primer pairs (CsF-CsR and OvF-OvR) (10 pmol of each primer) was used The amount of genomic DNA template in most reactions was approximately 50 ng except in the case of eggs (2.5–3.0 ng per reaction) (Lane 5) Lane M, molecular marker (DNA of phage cut by HindIII) Cs, C sinensis; Ov, O viverrini recombinant antigens (Nagano et al., 2004) These methods are useful for screening patients in endemic areas but cannot be used to survey the Wsh intermediate hosts A molecular technique for sensitive detection of O viverrini in Wsh, experimental snails and hamsters has been developed by a research group in Thailand (Maleewong et al., 2003; Wongratanacheewin et al., 2001, 2002) This is useful in endemic areas of O viverrini alone but may not be eYciently applied where O viverrini and C sinensis infections overlap geographically as predicted in central areas of Vietnam Here, we report a multiplex PCR probe, targeting mtDNA, for detection and discrimination of C sinensis and O viverrini The speciWcity and sensitivity of the test was conWrmed using mixed primer pairs and templates of diVerent concentrations and from diVerent life-stages Fish-borne trematodes are normally easy to diagnose with microscopy when adult samples are available Their metacercariae and/or eggs and/or cercariae, however, are notoriously diYcult to identify, especially when there are infections by multiple species in Wsh and humans It is possible that this technique may also be applied to detect cercariae collected from contaminated water or rediae/sporocysts in infected snails (see Magalhães et al., 2004) An advantage of using mtDNA targets rather than nuclear DNA is that the mitochondrial genome is present in hundreds or thousands of copies per cell (McManus et al., 2004) It has been reported that 50 T solium eggs could be detected by PCR (Chapman et al., 1995) In the case of O viverrini eggs, template extracted from fewer than 200 eggs could be ampliWed, but larger numbers yielded better 113 results (Wongratanacheewin et al., 2002) In our study, it appears that the sensitivity of multiplex PCR from template of O viverrini eggs (2.5–3.0 ng/reaction) was somewhat lower in comparison with that of mixed eggs and metacercariae of C sinensis and other trematodes (Fig 4, lanes 3–5) However, the PCR product (1357 bp for O viverrini) is clearly visible and could perhaps be improved by repeated PCR using this DNA product (1–2 l) as template (data not shown) Cloning and sequencing of such a product could overcome diYculties when sub-optimal amounts of DNA are available This multiplex PCR technique produced no cross reaction between C sinensis and O viverrini or with metacercariae of other trematodes commonly found in Wsh, or eggs from mixed infections in humans Our test for C sinensis and O viverrini is among the Wrst mtDNA-based multiplex PCR tests and the results suggest that the method developed and tested in this study is a sensitive, speciWc and suitable tool for detection of C sinensis andO viverrini infections and will be of particular value where both species co-occur Acknowledgments We thank our collaborators for their kind provision of materials used in this study This investigation received Wnancial support from Wellcome Trust, UK (Project No: 068762) to Thanh Hoa Le, Don McManus, and David Blair The authors also 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(Pelandakis and Pernin, 2002) A mitochondrial- based multiplex PCR probe has been designed for the large liver Xuke, Fasciola hepatica in Brazil (Magalhães et al., 2004) and for the tapeworms, Taenia... Vietnam and harboring those of other trematodes) and mixed metacercariae (collected from Wshes in areas endemic for C sinensis) The DNA was extracted using QiaAmp DNA extraction kit (Qiagen) and. .. Clinical and Diagnostic Laboratory Immunology 11, 411–416 Patsoula, E., Spanakos, G., SoWanatou, D., Parara, M., Vakalis, N.C., 2003 A single-step, PCR -based method for the detection and diVerentiation