doi:10.1016/j.exppara.2005.09.012 Clonorchis sinensis and Opisthorchis viverrini: Development of a mitochondrial-based multiplex PCR for their identiWcation and discrimination Thanh Ho
Trang 10014-4894/$ - see front matter 2005 Elsevier Inc All rights reserved.
doi:10.1016/j.exppara.2005.09.012
Clonorchis sinensis and Opisthorchis viverrini: Development
of a mitochondrial-based multiplex PCR for their
identiWcation and discrimination
Thanh Hoa Lea,e,¤, Nguyen Van Deb, David Blairc, Paiboon Sithithawornd,
a Institute of Biotechnology, Viet Nam
b 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 7 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
life-stage 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 2 pairs of primers (1 pair for C sinensis—product size 612 bp, and 1 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 (col-lected from Wshes in the C sinensis endemic areas), yielded speciWc bands of the correct size and their identity was conWrmed by sequence
anal-ysis 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
1 Introduction
The two small liver Xukes, Clonorchis sinensis and
Opis-thorchis viverrini, infect >30 million people in Asia including
Korea, mainland China, Taiwan island, Thailand, and
Indo-china (Bruckner, 1999; Chai and Lee, 2002; King and Scholz,
para-sites also infect a number of other mammals, including dogs,
cats, pigs and rodents, that can serve as reservoirs of
infec-tion In non-endemic areas (including the United States), the
infection is sometimes found in Asian immigrants, or follow-ing follow-ingestion of imported, undercooked or pickled freshwater Wsh containing metacercariae (StauVer et al., 2004) The dis-eases 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 food-borne 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;
tech-niques to identify O viverrini from a range of provinces in
* Corresponding author Fax: +84 4 8363144.
E-mail addresses: thanhL@qimr.edu.au , im-ibt@hn.vnn.vn (T.H Le).
Trang 2patients or metacercariae from infected Wshes.
Clonorchis sinensis and O viverrini have similar
life-cycles, 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
fresh-water 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
Haplor-chis spp and Centrocestus spp Consequently, humans
eat-ing such Wsh undercooked often host a number of
trematode species producing similar eggs Serological
meth-ods have been developed for diagnosis of clonorchiasis
Clonorchis and Opisthorchis have been used for
immuno-blot methods (Choi et al., 2003) In recent years, multiplex
PCR approaches have been developed for rapid and
accu-rate 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
PCR probe has been designed for the large liver Xuke,
tapeworms, Taenia solium, T asiatica, and T saginata
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.,
trema-todes (Pauly et al., 2003) However, a multiplex PCR
tech-nique 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
impor-tant 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
par-asite(s) when present in single or mixed infections
2 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
Khon Kaen, Thailand These worms were collected from golden hamsters 8 weeks after oral infection with metacer-cariae 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 enzymati-cally digested in 180 l of a lysis solution (ATL buVer-Qiagen), then 20 l proteinase-K (50 g/ml) was added and incubated at 56 °C for 2–3 h with brief vortexing every
30 min For eggs, the incubation time was set 1–2 h longer The mixture, after adding 200 l buVer AL (Qiagen) con-taining guanidine hydrochloride and 4 l RNase A (100 mg/ml) and mixing by pulse-vortexing for 15 s, was further incubated at 70 °C for 10 min 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 Col-umn for DNA binding and spin down for 1 min The col-umn with the DNA bound was washed several times using solutions (AW1; AW2 buVers—Qiagen) provided accord-ing 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 spectrophotom-eter) The extracted genomic DNA was diluted to a work-ing concentration of 50 ng/l and 1 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
The primers were designed to produce amplicons of diVer-ent size for accurate discrimination between the PCR prod-ucts on ethidium bromide-stained agarose gels The designed primers were: CsF (5⬘-TTAGAGGAGTTGGT GTCCCC-3⬘) and CsR (5⬘-AGCGTCACTGAACCA
CACCCAC-3⬘) for C sinensis (anticipated product size
612 bp); OvF (5⬘-TACGCAGGTGGTTTGGTTG-3⬘) and
Trang 3OvR (5⬘-AGCAGCGATAACACGACAGC-3⬘) for O
viv-errini (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 (5⬘-GCTCAATAAAGAGACCACGAAC-3⬘),
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
sepa-rately, 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 3 min, then 35 cycles
including denaturation at 95 °C for 30 s, annealing at 52 °C
for 30 s, extension at 72 °C for 2 min; and a Wnal cycle of
7 min 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
PuriW-cation kit (Qiagen), cloned using a TA cloning kit
(Invitro-gen, USA) and the selected recombinant plasmid DNAs
subjected to sequencing using Big Dye Terminator
Cycle-Sequencing 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
ana-lysed using the MacVector 7.2.2 package (Accelrys)
Iden-tity 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
(Accession Nos.: DQ116944 for C sinensis; DQ119551 for
2.4 Development of multiplex PCR and testing for
sensitivity
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 1 pair
for O viverrini) Tests were done using diVerent
combina-tions of template DNA (one or both species: 50 ng of tem-plate 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
2.5 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
trema-todes) 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 multi-plex 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 meta-cercariae (C sinensis) were cloned and sequenced to verify
the mtDNA sequences
3 Results
3.1 SpeciWcity of multiplex PCR
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)
Fig 1 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)
Trang 43.2 Reaction sensitivity by diluted template
The reaction assays showed high and speciWc sensitivity
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)
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;
was 50 ng for adult and metacercariae (about one
meta-cercaria), 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
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
meta-cercariae
4 Discussion
Until now, microscopic examination has been the most appropriate method for detecting eggs in patients of late-stage clonorchiasis or opisthorchiasis, and for demon-strating metacercariae in Wshes Immunodiagnostic meth-ods have also been proved to be sensitive and early techniques for early diagnosis using parasite egg antigen
for both C sinensis and O viverrini—Choi et al., 2003); and
Fig 2 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
CsF-CsR: 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).
7 6 5 4 3 2 1
M
0.5kb
2.0kb
4.3kb
6.5kb
23.0kb
O v
(O
C s
(C
O v
O v
C
O v
C
a C
O v
(O a C
C s
(O a C
1357bp
612bp
Fig 3 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 sensi-tivity at the lowest quantity of template DNA (by arrow).
A
0.5kb 2.0kb 4.3kb 6.5kb 9.4kb 23.0kb
1357bp
0.5kb 2.0kb 4.3kb 6.5kb 9.4kb 23.0kb
1357bp 612bp
612bp
0.5kb 2.0kb 4.3kb 6.5kb 9.4kb 23.0kb
B
C
Trang 5recombinant antigens (Nagano et al., 2004) These methods
are useful for screening patients in endemic areas but
can-not be used to survey the Wsh intermediate hosts A
molecu-lar 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;
areas of O viverrini alone but may not be eYciently applied
where O viverrini and C sinensis infections overlap
geo-graphically 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
diVer-ent concdiVer-entrations and from diVerdiVer-ent 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
possi-ble that this technique may also be applied to detect
cerca-riae 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
thou-sands 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
results (Wongratanacheewin et al., 2002) In our study, it appears that the sensitivity of multiplex PCR from
tem-plate of O viverrini eggs (2.5–3.0 ng/reaction) was
some-what 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
viv-errini) 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 prod-uct could overcome diYculties when sub-optimal amounts
of DNA are available
This multiplex PCR technique produced no cross
reac-tion between C sinensis and O viverrini or with
metacerca-riae 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 devel-oped and tested in this study is a sensitive, speciWc and
suit-able 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 thank Ms B.N Nguyen, Ms T.T
Vu (Institute of Biotechnology, Hanoi, Vietnam) for their collaboration in the laboratory work
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