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A specific PCR assay for the diagnosis of Clonorchis sinensis infection in humans, cats and fishes
Parasitology International 61 (2012) 187–190
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Parasitology International j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / p a r i n t
A specific PCR assay for the diagnosis of Clonorchis sinensis infection in humans, cats and fishes Si-Yang Huang a, Jian-Dong Tang a, b, Hui-Qun Song a, Bao-Quan Fu a, Min-Jun Xu a, Xu-Chu Hu c, Han Zhang d, Ya-Biao Weng b, Rui-Qing Lin a, b,⁎, Xing-Quan Zhu a, e, f,⁎ a 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 b College of Veterinary Medicine, South China Agricultural University, Guangzhou, Guangdong Province 510642, PR China c Department of Parasitology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong Province 510642, PR China d Guangdong Institute for Animal Disease Control and Surveillance, Guangzhou, Guangdong Province 510230, PR China e College of Animal Science and Technology, Yunnan Agricultural University, Kunming, Yunnan Province 650201, PR China f College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University, Daqing, Heilongjiang Province 163319, PR China
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Available online 14 July 2011 Keywords: Clonorchis sinensis Clonorchiasis Internal transcribed spacers (ITS) PCR assay Detection Human Cat Fish
a b s t r a c t Clonorchiasis caused by Clonorchis sinensis is a fish-borne parasitic disease which is endemic in a number of countries. Using the sequences of the internal transcribed spacers (ITS-1 and ITS-2) of nuclear ribosomal DNA (rDNA) of C. sinensis as genetic markers, a pair of C. sinensis-specific primers was designed and used to establish a specific PCR assay for the diagnosis of C. sinensis infection in humans, cats and fish. This approach allowed the specific identification of C. sinensis after optimizing amplification conditions, with no amplicons being amplified from related heterogeneous DNA samples, and sequencing of amplicons confirmed the identity of the sequences amplified. The detection limit of this assay was 1.03 pg of adult C. sinensis, 1.1 metacercariae per gram of fish filet, and a single egg in human and cat feces. The PCR assay should provide a useful tool for the diagnosis and molecular epidemiological investigation of clonorchiasis in humans and animals. © 2011 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Clonorchiasis caused by Clonorchis sinensis is a fish-borne trematode infection which is endemic in China, Korea, Japan, and other Southeast Asian countries, with approximately 35 million people being infected globally, of whom approximately 13 million were in China [1]. Snails, freshwater fishes or shrimps are intermediate hosts of C. sinensis, and humans, cats, dogs and pigs could serve as definitive hosts for C. sinensis. In mainland China, approximately 140 species of freshwater fish and four species of shrimp have been recognized as second intermediate hosts for C. sinensis. Recent analyses of epidemiological data in China suggest that clonorchiasis is having an increased human health impact due to the increased consumption of raw freshwater fish [2]. The specific identification of C. sinensis is a prerequisite for studying its epidemiology, population biology and genetic variability, and is also crucial for the diagnosis and control of clonorchiasis in humans and animals. Microscopic morphological examination of
C. sinensis adults, eggs and metacercariae is the traditional approach for the identification of C. sinensis. However, this method is timeconsuming, labor intensive and has limitation in its capacity to differentiate between closely taxa of the families Opisthorchiidae, Heterophyidae and Lecithodendriidae [1,3,4]. To overcome the diagnostic limitations associated with conventional parasitological methods, previous studies have shown that PCR-based techniques provide useful alternatives for the specific identification and diagnosis of C. sinensis [3–9]. However, prior to the present study, there had been no reports of specific PCR assay for the identification and diagnosis of C. sinensis infection in humans, cats and fishes in China. A number of previous studies have indicated that the first and/or second internal transcribed spacers (ITS-1, ITS-2) of nuclear ribosomal DNA (rDNA) provide accurate genetic markers for the identification of many parasites [9–12]. Hence, the objective of the present study was to establish, using genetic markers in the ITS-1 and ITS-2, a PCR tool for the specific diagnosis of C. sinensis infection in humans, cats, and fishes. 2. Materials and methods
⁎ Corresponding authors at: State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Parasitology of Gansu Province, 1 Xujiaping, Yanchangbu, Lanzhou Veterinary Research Institute, CAAS, Lanzhou, Gansu Province 730046, PR China. Tel.: + 86 931 8342837; fax: + 86 931 8340977. E-mail addresses: [email protected] (R.-Q. Lin), [email protected] (X.-Q. Zhu). 1383-5769/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.parint.2011.07.010
2.1. Samples Adult trematodes of C. sinensis were collected from naturally infected cats from Guangdong province, China. Fish (Pseudorasbora
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S.-Y. Huang et al. / Parasitology International 61 (2012) 187–190
Table 1 Clonorchis sinensis samples and heterologous reference samples used in the present study. Species
Sample code
Stage
Host
Geographical origin
No. of samples
Clonorchis sinensis
CsGZ4-10 CsM1-12 CsFH1-16 CsFC1-26 SJ FH FG OT TC AC TCA TS OD OV
Adult Metacercaria Egg Egg Adult Adult Adult Adult Adult Adult Adult Adult Adult Adult
Cat Fish Human Cat Rabbit Cattle Cattle Cattle Dog Dog Cat Pig Pig Hamster
Guangdong, China Guangdong, China Guangdong, China Guangdong, China Hunan, China Guizhou, China Guangxi, China Heilongjiang, China Guangdong, China Guangdong, China Guangdong, China Guangdong, China Guangdong, China Laos
7 12 16 26 1 1 1 1 1 1 1 1 1 1
Schistosoma japonicum Fasciola hepatica Fasciola gigantica Orientobilharzia turkestanicum Toxocara canis Ancylostoma caninum Toxocara cati Trichuris suis Oesophagostomum dentatum Opisthorchis viverrini
parve) samples confirmed to be infected with C. sinensis metacercariae by microscopy, and human and cat fecal samples from infected humans and cats (the presence of C. sinensis eggs were confirmed by fecal flotation and microscopic examination) were also used. The samples used in the present study, together with their sample codes, developmental stages, hosts and geographical origins are listed in Table 1. Also, some common trematodes and nematodes infecting humans and animals, namely Opisthorchis viverrini, Schistosoma japonicum, Fasciola gigantica, Fasciola hepatica, Orientobilharzia turkestanicum, Toxocara canis, Ancylostoma caninum, Toxocara cati, Trichuris suis and Oesophagostomum dentatum were included as ‘heterologous control’ samples (Table 1).
2.2. Extraction of genomic DNA and quality evaluation Total genomic DNA from individual adult worms of C. sinensis, those ‘heterologous control’ samples, as well as infected (with C. sinensis metacercariae) and non-infected (without C. sinensis metacercariae) fish muscle was extracted by sodium dodecyl sulfate/proteinase K treatment, column-purified (Wizard® SV Genomic DNA Purification System, Promega), and eluted into 60 μl H2O according to the manufacturer's recommendations [7]. The extraction of genomic DNA from human and cat fecal samples followed essentially a previous description [13]. In order to evaluate the quality of the DNA samples, for S. japonicum, F. gigantica and F. hepatica, the mitochondrial cytochrome c oxidase subunit 1 fragment was amplified by PCR using oligonucleotide primers JB3 (forward: 5′-TTTTTTGGGCATCCTGAGGTTTAT-3′) and JB4.5 (reverse: 5′-TAAAGAAAGAACATAATGAAAATG-3′) [14]. For O. turkestanicum and T. cati, the ITS fragments were amplified using primes BD1 (forward: 5′GTCGTAACAAGGTTTCCGTA-3′) and BD2 (reverse: 5′-TTATGCTTAAATTCAGCGGGT-3′) [15], while for C. sinensis, T. canis, A. caninum, T. suis, O. dentatum and O. viverrini, the ITS fragments were amplified using primers NC5 (forward: 5′-GTAGGTGAACCTGCGGAAGGATCATT-3′) and NC2 (reverse: 5′-TTAGTTTCTTTTCCTCCGCT-3′) [16].
2.3. Design of species-specific primers and optimization of specific PCR assays Based on comparison of the ITS-1 and ITS-2 sequences of C. sinensis, O. viverrini and O. felineus (GenBank™ accession numbers EU038125, AF408147 and DQ456828), a pair of primers CStjd1 (5′TGGCCTGACTGGCTGGCCGG-3′) and CStjd2 (5′-CGGCACCCCACACACATACA-3′) was designed to amplify the partial ITS of C. sinensis. The PCR conditions were optimized for the specific amplification of C. sinensis ITS fragments by varying the annealing temperatures and magnesium concentrations. Eventually, PCR reactions (in a volume of 25 μl) were performed in 10 mM Tris–HCl, pH 8.4, 50 mM KCl, 2.5 mM MgCl2, 200 μM each of dNTP, 50 pmol of each primer, and 0.625 U Taq polymerase (Takara) in a thermocycler (Biometra) under the following conditions: 94 °C for 5 min (initial denaturation), followed by 30 cycles of 94 °C for 30 s (denaturation), 61 °C for 30 s (annealing), 72 °C for 30 s (extension) and a final extension of 72 °C for 5 min. Samples with non-infected fish muscle DNA or without any genomic DNA were included in each PCR run as negative controls. The amplicons (4 μl) produced in each PCR were separated by electrophoresis in 1.0% (w/v) agarose gel, stained with ethidium bromide, and photographed using a gel documentation system (UVItec). 2.4. Assessment of specificity and sensitivity of the specific PCR assay The C. sinensis-specific primers were also evaluated for specificity using DNA samples representing those ‘heterologous control’ samples: O. viverrini, S. japonicum, F. gigantica, F. hepatica, O. turkestanicum, T. canis, A. caninum, T. cati, T. suis and O. dentatum (Table 1). For evaluation of sensitivity, the smallest amount of C. sinensis genomic DNA detectable by the specific PCR assay was estimated by amplification of 10-fold serial dilutions of one genomic DNA sample (103 ng/μl) from an adult C. sinensis specimen. To determine the minimum number of C. sinensis metacercariae in infected fish detectable by the specific PCR assay, 30 mg of non-infected (without C. sinensis metacercariae) fish filet was spiked with 1 C. sinensis
Fig. 1. Agarose gel electrophoresis of specific amplification of the internal transcribed spacers (ITS) using Clonorchis sinensis-specific primers. Lanes 1–11 represent PCR products amplified from genomic DNA extracted from C. sinensis, Opisthorchis viverrini, Schistosoma japonicum, Fasciola gigantica, Fasciola hepatica, Orientobilharzia turkestanicum, Toxocara canis, Ancylostoma caninum, Toxocara cati, Trichuris suis and Oesophagostomum dentatum, respectively. Lane 12 represents PCR products amplified from genomic DNA extracted from uninfected fish, and lane 13 represents no-DNA control. M represents a DNA size marker (ordinate values in bp).
S.-Y. Huang et al. / Parasitology International 61 (2012) 187–190
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Fig. 2. Evaluation of the sensitivity of the specific PCR assay for Clonorchis sinensis by agarose gel electrophoresis. Lane 1 represents PCR product amplified from C. sinensis DNA undiluted (103 ng/μl), and lanes 2–9 represent PCR products amplified from C. sinensis DNA diluted for 101, 102, 103, 104, 105, 106, 107, 108 times, respectively. Lane 10 represents noDNA control. M represents a DNA size marker (ordinate values in bp).
Fig. 3. Analysis of PCR products amplified from Clonorchis sinensis metacercariae in fish samples using the specific PCR assay by agarose gel electrophoresis. Lanes 1–12 represent infected fish samples, lanes 13–15 represent non-infected fish samples, lane 16 represents the “positive control”, and lane 17 represents no-DNA control. M represents a DNA size marker (ordinate values in bp).
metacercaria, and genomic DNA was extracted using the Wizard® SV Genomic DNA Purification System (Promega), and eluted into 30 μl H2O according to the manufacturer's recommendations. One microliter of the DNA was amplified by the specific PCR assay, and the amplification was repeated twice. To assess whether this PCR assay could amplify C. sinensis DNA from a single egg, single egg, 3 eggs or 5 eggs of C. sinensis were pipeted out from human and cat feces to extract genomic DNA [13], and amplified using this PCR assay. 2.5. Applications of the specific PCR assay After optimization, the utility of C. sinensis-specific PCR assay for the diagnosis and epidemiological investigation of C. sinensis infection in humans and animals was assessed by examining fecal samples of humans and cats infected with C. sinensis, as well as fish infected with metacercariae of C. sinensis, respectively. 3. Results and discussion The sizes of the cox1 or ITS PCR products amplified from DNA samples from individual specimens using the primer sets JB3/JB4.5, BD1/BD2 or NC5/NC2 varied in sizes 450–1200 bp on agarose gels, corresponding to the expected fragment lengths (not shown), thus demonstrating the integrity of DNA in each tested sample. Under the optimized PCR protocols, the C. sinensis-specific primer set Cstjd1/Cstjd2 amplified a product of approximately 400 bp solely from C. sinensis, whereas no product was amplified from heterogeneous DNA samples (see Table 1), non-infected fish DNA or no-DNA controls (Fig. 1 and Fig. 3). The amplicon produced was sequenced with the PCR primers and proved to be partial ITS of C. sinensis (data not shown), thus demonstrating its specificity. The smallest amount of C. sinensis genomic DNA detectable by the specific primers was
calculated as 1.03 pg (Fig. 2). The detection limit for C. sinensis metacercariae in infected fish was 1.1 metacercariae/g of fish filet (Fig. 3). For human and cat fecal samples, the detection limit of this specific PCR assay was a single egg of C. sinensis (Fig. 4). All of the infected samples (12 fish filet samples, 26 cat fecal samples and 16 fecal human samples, Table 1) gave positive amplifications. These results showed that the established PCR assay was specific and sensitive. Previous studies have shown that C. sinensis infection in humans, cats and fish was severe in mainland China [17,18], the traditional approach for the diagnosis of clonorchiasis in humans and animals has been based on microscopic examination of C. sinensis eggs in human and animal feces, and metacercariae in fish. But this approach can have limitation for the differentiation of closely-related species. Previous studies using PCR amplification of various target DNA regions have proven useful for the specific identification of C. sinensis [3–9,19]. The specific PCR assay established in the present study represented a useful alternative method for the rapid and accurate diagnosis of C. sinensis infection in humans, cats and fishes, which should provide a molecular tool for the diagnosis and epidemiological investigations of C. sinensis infection in humans and animals, contributing to the effective control of clonorchiasis. Acknowledgments This work is supported, in part, by the State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, the Yunnan Provincial Program for Introducing High-level Scientists (Grant No. 2009CI125), and the Program for Changjiang Scholars and Innovative Research Team in University (Grant No. IRT0723) to XQZ.
Fig. 4. Analysis of PCR products amplified from C. sinensis eggs from fecal samples using specific PCR assay by agarose gel electrophoresis. Lane 1 represents “positive control”, lanes 2 and 3 represent single egg of C. sinensis, lanes 4 and 5 represent three eggs of C. sinensis, lanes 6 and 7 represent five eggs of C. sinensis, lanes 8–14 represent representative positive fecal samples of cats, lanes 15–20 represent representative positive fecal samples of humans, lane 21 represents no-DNA control. M represents a DNA size marker (ordinate values in bp).
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References [1] Lun ZR, Gasser RB, Lai DH, Li AX, Zhu XQ, Yu XB, et al. Clonorchiasis: a key foodborne zoonosis in China. Lancet Infect Dis 2005;5:31–41. [2] Zhou P, Chen N, Zhang RL, Lin RQ, Zhu XQ. Food-borne parasitic zoonoses in China: perspective for control. Trends Parasitol 2008;24:190–6. [3] Traub RJ, Macaranas J, Mungthin M, Leelayoova S, Cribb T, Murrell KD, et al. A new PCR-based approach indicates the range of Clonorchis sinensis now extends to Central Thailand. PLoS Negl Trop Dis 2009;3:e367. [4] Parvathi A, Sanath Kumar H, Kenchanna Prakasha B, Lu J, Xu X, Hu W, et al. Clonorchis sinensis: development and evaluation of a nested polymerase chain reaction (PCR) assay. Exp Parasitol 2007;115:291–5. [5] Le TH, Van De N, Blair D, Sithithaworn P, McManus DP. Clonorchis sinensis and Opisthorchis viverrini: development of a mitochondrial-based multiplex PCR for their identification and discrimination. Exp Parasitol 2006;112:109–14. [6] Kim EM, Verweij JJ, Jalili A, van Lieshout L, Choi MH, Bae YM, et al. Detection of Clonorchis sinensis in stool samples using real-time PCR. Ann Trop Med Parasitol 2009;103:513–8. [7] Cai XQ, Xu MJ, Wang YH, Qiu DY, Liu GX, Lin A, et al. Sensitive and rapid detection of Clonorchis sinensis infection in fish by loop-mediated isothermal amplification (LAMP). Parasitol Res 2010;106:1379–83. [8] Muller B, Schmidt J, Mehlhorn H. Sensitive and species-specific detection of Clonorchis sinensis by PCR in infected snails and fishes. Parasitol Res 2007;100: 911–4. [9] Sato M, Thaenkham U, Dekumyoy P, Waikagul J. Discrimination of O. viverrini, C. sinensis, H. pumilio and H. taichui using nuclear DNA-based PCR targeting ribosomal DNA ITS regions. Acta Trop 2009;109:81–3. [10] Lin RQ, Ai L, Zou FC, Verweij JJ, Jiang Q, Li MW, et al. A multiplex PCR tool for the specific identification of Oesophagostomum spp. from pigs. Parasitol Res 2008;103: 993–7.
[11] Zhu XQ, D'Amelio S, Gasser RB, Yang TB, Paggi L, He F, et al. Practical PCR tools for the delineation of Contracaecum rudolphii A and Contracaecum rudolphii B (Ascaridoidea: Anisakidae) using genetic markers in nuclear ribosomal DNA. Mol Cell Probes 2007;21:97–102. [12] Ai L, Dong SJ, Zhang WY, Elsheikha HM, Mahmmod YS, Lin RQ, et al. Specific PCRbased assays for the identification of Fasciola species: their development, evaluation and potential usefulness in prevalence surveys. Ann Trop Med Parasitol 2010;104:65–72. [13] Müller B, Schmidt J, Mehlhorn H. PCR diagnosis of infections with different species of Opisthorchiidae using a rapid clean-up procedure for stool samples and specific primers. Parasitol Res 2007;100:905–9. [14] Bowles J, Blair D, McManus DP. Genetic variants within the genus Echinococcus identified by mitochondrial DNA sequencing. Mol Biochem Parasitol 1992;54: 165–73. [15] Ali H, Ai L, Song HQ, Ali S, Lin RQ, Zhu XQ. Genetic characterisation of Fasciola samples from different host species and geographical localities revealed the existence of F. hepatica and F. gigantica in Niger. Parasitol Res 2008;102:1021–4. [16] Zhu XQ, D'Amelio S, Palm HW, Paggi L, George-Nascimento M, Gasser RB. SSCPbased identification of members within the Pseudoterranova decipiens complex (Nematoda: Ascaridoidea: Anisakidae) using genetic markers in the internal transcribed spacers of ribosomal DNA. Parasitology 2002;124:615–23. [17] Li SL, He G, Wei MB. Epidemiological investigation of clonorchiasis in Guangxi. Chin J Parasitic Dis Cont 2002;15:214–6 (in Chinese). [18] Fang YY, Wu J, Liu Q. Investigation and analysis on epidemic status of clonorchiasis in Guangdong Province, China. J Pathog Biol 2007;2:54–6 (in Chinese). [19] Kang S, Sultana T, Loktev VB, Wongratanacheewin S, Sohn WM, Eom KS, et al. Molecular identification and phylogenetic analysis of nuclear rDNA sequences among three opisthorchid liver fluke species (Opisthorchiidae: Trematoda). Parasitol Int 2008;57:191–7.
Contents lists available at ScienceDirect
Parasitology International j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / p a r i n t
A specific PCR assay for the diagnosis of Clonorchis sinensis infection in humans, cats and fishes Si-Yang Huang a, Jian-Dong Tang a, b, Hui-Qun Song a, Bao-Quan Fu a, Min-Jun Xu a, Xu-Chu Hu c, Han Zhang d, Ya-Biao Weng b, Rui-Qing Lin a, b,⁎, Xing-Quan Zhu a, e, f,⁎ a 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 b College of Veterinary Medicine, South China Agricultural University, Guangzhou, Guangdong Province 510642, PR China c Department of Parasitology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong Province 510642, PR China d Guangdong Institute for Animal Disease Control and Surveillance, Guangzhou, Guangdong Province 510230, PR China e College of Animal Science and Technology, Yunnan Agricultural University, Kunming, Yunnan Province 650201, PR China f College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University, Daqing, Heilongjiang Province 163319, PR China
a r t i c l e
i n f o
Available online 14 July 2011 Keywords: Clonorchis sinensis Clonorchiasis Internal transcribed spacers (ITS) PCR assay Detection Human Cat Fish
a b s t r a c t Clonorchiasis caused by Clonorchis sinensis is a fish-borne parasitic disease which is endemic in a number of countries. Using the sequences of the internal transcribed spacers (ITS-1 and ITS-2) of nuclear ribosomal DNA (rDNA) of C. sinensis as genetic markers, a pair of C. sinensis-specific primers was designed and used to establish a specific PCR assay for the diagnosis of C. sinensis infection in humans, cats and fish. This approach allowed the specific identification of C. sinensis after optimizing amplification conditions, with no amplicons being amplified from related heterogeneous DNA samples, and sequencing of amplicons confirmed the identity of the sequences amplified. The detection limit of this assay was 1.03 pg of adult C. sinensis, 1.1 metacercariae per gram of fish filet, and a single egg in human and cat feces. The PCR assay should provide a useful tool for the diagnosis and molecular epidemiological investigation of clonorchiasis in humans and animals. © 2011 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Clonorchiasis caused by Clonorchis sinensis is a fish-borne trematode infection which is endemic in China, Korea, Japan, and other Southeast Asian countries, with approximately 35 million people being infected globally, of whom approximately 13 million were in China [1]. Snails, freshwater fishes or shrimps are intermediate hosts of C. sinensis, and humans, cats, dogs and pigs could serve as definitive hosts for C. sinensis. In mainland China, approximately 140 species of freshwater fish and four species of shrimp have been recognized as second intermediate hosts for C. sinensis. Recent analyses of epidemiological data in China suggest that clonorchiasis is having an increased human health impact due to the increased consumption of raw freshwater fish [2]. The specific identification of C. sinensis is a prerequisite for studying its epidemiology, population biology and genetic variability, and is also crucial for the diagnosis and control of clonorchiasis in humans and animals. Microscopic morphological examination of
C. sinensis adults, eggs and metacercariae is the traditional approach for the identification of C. sinensis. However, this method is timeconsuming, labor intensive and has limitation in its capacity to differentiate between closely taxa of the families Opisthorchiidae, Heterophyidae and Lecithodendriidae [1,3,4]. To overcome the diagnostic limitations associated with conventional parasitological methods, previous studies have shown that PCR-based techniques provide useful alternatives for the specific identification and diagnosis of C. sinensis [3–9]. However, prior to the present study, there had been no reports of specific PCR assay for the identification and diagnosis of C. sinensis infection in humans, cats and fishes in China. A number of previous studies have indicated that the first and/or second internal transcribed spacers (ITS-1, ITS-2) of nuclear ribosomal DNA (rDNA) provide accurate genetic markers for the identification of many parasites [9–12]. Hence, the objective of the present study was to establish, using genetic markers in the ITS-1 and ITS-2, a PCR tool for the specific diagnosis of C. sinensis infection in humans, cats, and fishes. 2. Materials and methods
⁎ Corresponding authors at: State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Parasitology of Gansu Province, 1 Xujiaping, Yanchangbu, Lanzhou Veterinary Research Institute, CAAS, Lanzhou, Gansu Province 730046, PR China. Tel.: + 86 931 8342837; fax: + 86 931 8340977. E-mail addresses: [email protected] (R.-Q. Lin), [email protected] (X.-Q. Zhu). 1383-5769/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.parint.2011.07.010
2.1. Samples Adult trematodes of C. sinensis were collected from naturally infected cats from Guangdong province, China. Fish (Pseudorasbora
188
S.-Y. Huang et al. / Parasitology International 61 (2012) 187–190
Table 1 Clonorchis sinensis samples and heterologous reference samples used in the present study. Species
Sample code
Stage
Host
Geographical origin
No. of samples
Clonorchis sinensis
CsGZ4-10 CsM1-12 CsFH1-16 CsFC1-26 SJ FH FG OT TC AC TCA TS OD OV
Adult Metacercaria Egg Egg Adult Adult Adult Adult Adult Adult Adult Adult Adult Adult
Cat Fish Human Cat Rabbit Cattle Cattle Cattle Dog Dog Cat Pig Pig Hamster
Guangdong, China Guangdong, China Guangdong, China Guangdong, China Hunan, China Guizhou, China Guangxi, China Heilongjiang, China Guangdong, China Guangdong, China Guangdong, China Guangdong, China Guangdong, China Laos
7 12 16 26 1 1 1 1 1 1 1 1 1 1
Schistosoma japonicum Fasciola hepatica Fasciola gigantica Orientobilharzia turkestanicum Toxocara canis Ancylostoma caninum Toxocara cati Trichuris suis Oesophagostomum dentatum Opisthorchis viverrini
parve) samples confirmed to be infected with C. sinensis metacercariae by microscopy, and human and cat fecal samples from infected humans and cats (the presence of C. sinensis eggs were confirmed by fecal flotation and microscopic examination) were also used. The samples used in the present study, together with their sample codes, developmental stages, hosts and geographical origins are listed in Table 1. Also, some common trematodes and nematodes infecting humans and animals, namely Opisthorchis viverrini, Schistosoma japonicum, Fasciola gigantica, Fasciola hepatica, Orientobilharzia turkestanicum, Toxocara canis, Ancylostoma caninum, Toxocara cati, Trichuris suis and Oesophagostomum dentatum were included as ‘heterologous control’ samples (Table 1).
2.2. Extraction of genomic DNA and quality evaluation Total genomic DNA from individual adult worms of C. sinensis, those ‘heterologous control’ samples, as well as infected (with C. sinensis metacercariae) and non-infected (without C. sinensis metacercariae) fish muscle was extracted by sodium dodecyl sulfate/proteinase K treatment, column-purified (Wizard® SV Genomic DNA Purification System, Promega), and eluted into 60 μl H2O according to the manufacturer's recommendations [7]. The extraction of genomic DNA from human and cat fecal samples followed essentially a previous description [13]. In order to evaluate the quality of the DNA samples, for S. japonicum, F. gigantica and F. hepatica, the mitochondrial cytochrome c oxidase subunit 1 fragment was amplified by PCR using oligonucleotide primers JB3 (forward: 5′-TTTTTTGGGCATCCTGAGGTTTAT-3′) and JB4.5 (reverse: 5′-TAAAGAAAGAACATAATGAAAATG-3′) [14]. For O. turkestanicum and T. cati, the ITS fragments were amplified using primes BD1 (forward: 5′GTCGTAACAAGGTTTCCGTA-3′) and BD2 (reverse: 5′-TTATGCTTAAATTCAGCGGGT-3′) [15], while for C. sinensis, T. canis, A. caninum, T. suis, O. dentatum and O. viverrini, the ITS fragments were amplified using primers NC5 (forward: 5′-GTAGGTGAACCTGCGGAAGGATCATT-3′) and NC2 (reverse: 5′-TTAGTTTCTTTTCCTCCGCT-3′) [16].
2.3. Design of species-specific primers and optimization of specific PCR assays Based on comparison of the ITS-1 and ITS-2 sequences of C. sinensis, O. viverrini and O. felineus (GenBank™ accession numbers EU038125, AF408147 and DQ456828), a pair of primers CStjd1 (5′TGGCCTGACTGGCTGGCCGG-3′) and CStjd2 (5′-CGGCACCCCACACACATACA-3′) was designed to amplify the partial ITS of C. sinensis. The PCR conditions were optimized for the specific amplification of C. sinensis ITS fragments by varying the annealing temperatures and magnesium concentrations. Eventually, PCR reactions (in a volume of 25 μl) were performed in 10 mM Tris–HCl, pH 8.4, 50 mM KCl, 2.5 mM MgCl2, 200 μM each of dNTP, 50 pmol of each primer, and 0.625 U Taq polymerase (Takara) in a thermocycler (Biometra) under the following conditions: 94 °C for 5 min (initial denaturation), followed by 30 cycles of 94 °C for 30 s (denaturation), 61 °C for 30 s (annealing), 72 °C for 30 s (extension) and a final extension of 72 °C for 5 min. Samples with non-infected fish muscle DNA or without any genomic DNA were included in each PCR run as negative controls. The amplicons (4 μl) produced in each PCR were separated by electrophoresis in 1.0% (w/v) agarose gel, stained with ethidium bromide, and photographed using a gel documentation system (UVItec). 2.4. Assessment of specificity and sensitivity of the specific PCR assay The C. sinensis-specific primers were also evaluated for specificity using DNA samples representing those ‘heterologous control’ samples: O. viverrini, S. japonicum, F. gigantica, F. hepatica, O. turkestanicum, T. canis, A. caninum, T. cati, T. suis and O. dentatum (Table 1). For evaluation of sensitivity, the smallest amount of C. sinensis genomic DNA detectable by the specific PCR assay was estimated by amplification of 10-fold serial dilutions of one genomic DNA sample (103 ng/μl) from an adult C. sinensis specimen. To determine the minimum number of C. sinensis metacercariae in infected fish detectable by the specific PCR assay, 30 mg of non-infected (without C. sinensis metacercariae) fish filet was spiked with 1 C. sinensis
Fig. 1. Agarose gel electrophoresis of specific amplification of the internal transcribed spacers (ITS) using Clonorchis sinensis-specific primers. Lanes 1–11 represent PCR products amplified from genomic DNA extracted from C. sinensis, Opisthorchis viverrini, Schistosoma japonicum, Fasciola gigantica, Fasciola hepatica, Orientobilharzia turkestanicum, Toxocara canis, Ancylostoma caninum, Toxocara cati, Trichuris suis and Oesophagostomum dentatum, respectively. Lane 12 represents PCR products amplified from genomic DNA extracted from uninfected fish, and lane 13 represents no-DNA control. M represents a DNA size marker (ordinate values in bp).
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Fig. 2. Evaluation of the sensitivity of the specific PCR assay for Clonorchis sinensis by agarose gel electrophoresis. Lane 1 represents PCR product amplified from C. sinensis DNA undiluted (103 ng/μl), and lanes 2–9 represent PCR products amplified from C. sinensis DNA diluted for 101, 102, 103, 104, 105, 106, 107, 108 times, respectively. Lane 10 represents noDNA control. M represents a DNA size marker (ordinate values in bp).
Fig. 3. Analysis of PCR products amplified from Clonorchis sinensis metacercariae in fish samples using the specific PCR assay by agarose gel electrophoresis. Lanes 1–12 represent infected fish samples, lanes 13–15 represent non-infected fish samples, lane 16 represents the “positive control”, and lane 17 represents no-DNA control. M represents a DNA size marker (ordinate values in bp).
metacercaria, and genomic DNA was extracted using the Wizard® SV Genomic DNA Purification System (Promega), and eluted into 30 μl H2O according to the manufacturer's recommendations. One microliter of the DNA was amplified by the specific PCR assay, and the amplification was repeated twice. To assess whether this PCR assay could amplify C. sinensis DNA from a single egg, single egg, 3 eggs or 5 eggs of C. sinensis were pipeted out from human and cat feces to extract genomic DNA [13], and amplified using this PCR assay. 2.5. Applications of the specific PCR assay After optimization, the utility of C. sinensis-specific PCR assay for the diagnosis and epidemiological investigation of C. sinensis infection in humans and animals was assessed by examining fecal samples of humans and cats infected with C. sinensis, as well as fish infected with metacercariae of C. sinensis, respectively. 3. Results and discussion The sizes of the cox1 or ITS PCR products amplified from DNA samples from individual specimens using the primer sets JB3/JB4.5, BD1/BD2 or NC5/NC2 varied in sizes 450–1200 bp on agarose gels, corresponding to the expected fragment lengths (not shown), thus demonstrating the integrity of DNA in each tested sample. Under the optimized PCR protocols, the C. sinensis-specific primer set Cstjd1/Cstjd2 amplified a product of approximately 400 bp solely from C. sinensis, whereas no product was amplified from heterogeneous DNA samples (see Table 1), non-infected fish DNA or no-DNA controls (Fig. 1 and Fig. 3). The amplicon produced was sequenced with the PCR primers and proved to be partial ITS of C. sinensis (data not shown), thus demonstrating its specificity. The smallest amount of C. sinensis genomic DNA detectable by the specific primers was
calculated as 1.03 pg (Fig. 2). The detection limit for C. sinensis metacercariae in infected fish was 1.1 metacercariae/g of fish filet (Fig. 3). For human and cat fecal samples, the detection limit of this specific PCR assay was a single egg of C. sinensis (Fig. 4). All of the infected samples (12 fish filet samples, 26 cat fecal samples and 16 fecal human samples, Table 1) gave positive amplifications. These results showed that the established PCR assay was specific and sensitive. Previous studies have shown that C. sinensis infection in humans, cats and fish was severe in mainland China [17,18], the traditional approach for the diagnosis of clonorchiasis in humans and animals has been based on microscopic examination of C. sinensis eggs in human and animal feces, and metacercariae in fish. But this approach can have limitation for the differentiation of closely-related species. Previous studies using PCR amplification of various target DNA regions have proven useful for the specific identification of C. sinensis [3–9,19]. The specific PCR assay established in the present study represented a useful alternative method for the rapid and accurate diagnosis of C. sinensis infection in humans, cats and fishes, which should provide a molecular tool for the diagnosis and epidemiological investigations of C. sinensis infection in humans and animals, contributing to the effective control of clonorchiasis. Acknowledgments This work is supported, in part, by the State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, the Yunnan Provincial Program for Introducing High-level Scientists (Grant No. 2009CI125), and the Program for Changjiang Scholars and Innovative Research Team in University (Grant No. IRT0723) to XQZ.
Fig. 4. Analysis of PCR products amplified from C. sinensis eggs from fecal samples using specific PCR assay by agarose gel electrophoresis. Lane 1 represents “positive control”, lanes 2 and 3 represent single egg of C. sinensis, lanes 4 and 5 represent three eggs of C. sinensis, lanes 6 and 7 represent five eggs of C. sinensis, lanes 8–14 represent representative positive fecal samples of cats, lanes 15–20 represent representative positive fecal samples of humans, lane 21 represents no-DNA control. M represents a DNA size marker (ordinate values in bp).
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