Parasitol Res (2011) 109:493–498 DOI 10.1007/s00436-011-2272-0
SHORT COMMUNICATION
A real-time multiplex-nested PCR system for coprological diagnosis of Echinococcus multilocularis and host species Anke Dinkel & Selina Kern & Anja Brinker & Rainer Oehme & Amélie Vaniscotte & Patrick Giraudoux & Ute Mackenstedt & Thomas Romig
Received: 14 October 2010 / Accepted: 26 January 2011 / Published online: 16 February 2011 # Springer-Verlag 2011
Abstract A hybridization probe-based real-time multiplexnested PCR system was developed for the simultaneous detection of Echinococcus multilocularis and host species directly from faecal samples. Species identification was determined by melting curve analysis. Specificity was assessed by using DNA extracted from various cestodes (E. multilocularis, Echinococcus granulosus (G1), Echinococcus ortleppi, Echinococcus canadensis (G6, G7), Taenia crassiceps, Taenia hydatigena, Taenia mustelae, Taenia pisiformis, Taenia serialis, Taenia taeniaeformis, Mesocestoides leptothylacus), carnivores (Vulpes vulpes, Vulpes corsac, Vulpes ferrilata, Canis familiaris, Felis catus, Martes foina), Microtus arvalis and Arvicola terrestris. The analytical sensitivity was 10 fg, evaluated with serially diluted DNA of E. multilocularis to 10 μl total DNA solution from E. multilocularis-negative canid faeces. Based on a comparison of 47 dog samples from China, the proportion of the E. multilocularis-positive-tested samples by the real-time multiplexnested PCR was moderately higher (38% vs. 30%) as when tested with a previously evaluated nested PCR with a sensitivity of 70–100%, depending on the number and A. Dinkel (*) : S. Kern : A. Brinker : U. Mackenstedt : T. Romig Department of Parasitology, University of Hohenheim, Emil-Wolff-Str.34, 70599 Stuttgart, Germany e-mail:
[email protected] R. Oehme State Health Office Baden-Wuerttemberg, Nordbahnhofstr. 135, 70191 Stuttgart, Germany A. Vaniscotte : P. Giraudoux Department of Chrono-environment, UMR UFC/CNRS 6249 aff. INRA, Universite de Franche-Comté, 25030 Besançon, France
gravidity status of worms present in the intestine (Dinkel et al., J Clin Microbiol 36:1871–1876, 1998). To assess the epidemiological applicability of this method, 227 canid faecal samples collected in the field were analysed. This newly developed real-time multiplex-nested PCR system is a specific, sensitive and reliable method for the detection of E. multilocularis and host species in faecal samples for epidemiological purposes.
Introduction A number of coprodiagnostic tests for Echinococcus multilocularis have been developed in the past, either based on the detection of coproantigens (Sakai et al. 1998; Deplazes et al. 1999) or the detection of parasite DNA by PCR (Bretagne et al. 1993; Monnier et al. 1996; Dinkel et al. 1998). These diagnostic approaches are particularly useful when living animals have to be examined (as is the case with companion animals) or where the necropsy of, e.g., fox carcasses is impractical. In the latter case, a sample of rectal content can be obtained through the anus. Important information for small-scale epidemiological studies can be derived from the examination of faecal samples collected in the environment (Giraudoux et al. 2003; Deplazes et al. 2004), in particular, when the spatial distribution and frequency of infective faeces need to be determined. One of the inherent disadvantages of this method is the difficulty to reliably determine the origin of the faeces. The biochemical analysis of bile acids by thin-layer chromatography has allowed the identification of fox faeces (Major et al. 1980), but this is a very laborious approach. Morphological analysis of hair in faeces should, in principle, determine the species of large carnivores as, e.g., fox hair is not present in dog faeces (Deplazes et al. 2003). However, such an approach has not yet been evaluated for
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this purpose. While the general characteristics of shape, smell, etc. are useful, some uncertainty often remain. In particular, the faeces of ‘urban’ foxes, due to their untypical diet, are difficult to distinguish from those of dogs. To identify carnivore species by molecular analysis of faeces, Farrell et al. (2000) used an approach consisting in amplifying a fragment of the mitochondrial (mt) cytochrome b gene followed by sequencing to differentiate between four carnivore species in Venezuela. Some authors have adopted an approach involving the amplification of a part of mt cytochrome b gene or mt D-loop region with conserved universal primers, followed by digestion with restriction enzymes to distinguish among alternative species (Foran et al. 1997a, b; Paxinos et al. 1997; Pilgrim et al. 1998; Hansen and Jacobsen 1999; Mills et al. 2000; Huettner et al. 2009). However, simpler approaches involving only DNA isolation, PCR amplification and detection of the amplification products are more appropriate for epidemiological purposes. For the identification of the Iberian lynx from faecal samples, Palomares et al. (2002) developed a method based on specific PCR of mt cytochrome b and D-loop region sequences. Nonaka et al. (2009) developed a multiplex PCR system for identifying the carnivore origin of faeces without the inclusion of parasite diagnostics with two separate primer mixtures against the D-loop region of mt DNA for fox, raccoon dog, dog, cat, raccoon and weasel, distinguishing the target animals by the size of their amplification products with a detection sensitivity of 1–10 pg of DNA. Such diagnostic amplification products can be designed to be short to facilitate the application of the method to fairly degraded material, such as faecal samples. Real-time PCR has the advantage compared to conventional PCR in that it is very fast and there is no need for postamplification handling, resulting in reducing the risk for amplicon contamination. In order to provide a simple method for the simultaneous identification of E. multilocularis and host species directly from faecal samples, we developed a real-time multiplex-nested PCR system.
Materials and methods Materials For specificity screening, the following samples of cestodes, canids and rodents were used : E. multilocularis (five metacestodes, Germany; two metacestodes, Spitzbergen), Echinococcus granulosus (G1) (metacestodes, Kenya), Echinococcus ortleppi (metacestode, Switzerland; metacestode, Vietnam), Echinococcus canadensis (G6) (metacestodes, Kenya), E. canadensis (G7) (metacestodes, Slovak Republic), Taenia crassiceps (metacestode, Germany), Taenia hydatigena (metacestode, Switzerland), Taenia
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mustelae (metacestode, Germany), Taenia pisiformis (adult, Australia), Taenia serialis (adult, Australia), Taenia taeniaeformis (metacestode, Germany), Mesocestoides leptothylacus (adult, Germany), Vulpes vulpes (red fox) (tissue, Germany and China), Vulpes corsac (sand fox) (faeces, zoo animal, Germany), Vulpes ferrilata (Tibetan fox) (tissue, China), Canis familiaris (dog) (blood, Germany), Felis catus (cat) (blood, Germany), Martes foina (stone marten) (tissue, Germany), Microtus arvalis (common vole) (tissue, Germany) and Arvicola terrestris (water vole) (tissue, Germany). Additionally, we used 180 non-inhibited field faecal samples collected of suspected dog and/or fox origin from China and 47 non-inhibited faecal samples from China supposed to be from dogs. DNA extraction DNA from faecal samples and tissue/blood samples was extracted as described previously (Dinkel et al. 1998, 2004). Real-time multiplex-nested PCR system For the specific detection of E. multilocularis and canid host species DNA of V. vulpes (red fox), V. ferrilata (Tibetan fox), V. corsac (sand fox) and C. lupus/familiaris (wolf/dog), two hybridization probe-based real-time LightCycler (Roche, Mannheim, Germany)-nested PCR assays were developed: 1. A real-time multiplex-nested PCR (rtm-nested PCR) with fluorescence resonance energy transfer (FRET) probes for the specific detection and discrimination of E. multilocularis and canid host species in one reaction, enabling the specific species identification by analysing the melting curves . Target sequence for E. multilocularis is part of the mitochondrial 12S rRNA gene, whereas a part of the mitochondrial cytochrome b gene from different carnivore species (Palomares et al. 2002; accession numbers AJ441328–AJ441338) was the basis from which primers and probes for the canid host species identification were designed. 2. A real-time-nested PCR (rt-nested PCR) with FRET probes only for the detection and discrimination of the host species described above by melting curve analysis. Both of the rtm- and rt-nested PCR were conducted in two steps: For the first preamplification duplex PCR, which allows the parallel amplification of DNA from various cestodes (355-bp fragment) and different carnivores (401-bp fragment), the primer mix contained a set of four primers:
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P60 short.for/P375 short.rev and CVF for/CVF rev (Dinkel et al. 2006). A total of 10 μl DNA (approximately 70 ng) from faecal samples was added to the 90-μl reaction mixture consisting of 15 mM Tris–HCl (pH 8.0), 50 mM KCl, 2.5 mM MgCl2, 200 μM of each dNTP, 30 pmol of each cestode primer (P60 short.for/P375 short. rev), 40 pmol of each carnivore primer (CVF for/CVF rev) and 2.5 units AmpliTaq Gold (Applied Biosystems). Hot start PCR was done for 40 cycles (denaturation for 30 s at 94°C, annealing for 1 min at 54°C and elongation for 40 s at 72°C) in a thermal block cycler (2720 Thermal Cycler, Applied Biosystems). In the second step, the LightCycler instrument (Version 1.5, Roche, Mannheim, Germany) was used for the amplification of the target DNA and to monitor the development of the PCR product after each cycle.
495 Table 1 Sequences of primers and probes Primer/probe
Primer/probe sequence (5′–3′)
P60.short.for P375.short.rev CVF.for CVF.rev
TGG TAC AGG ATT AGA TAC CC TGA CGG GCG GTG TGT ACC TTA ATG ACC AAC ATT CGA AA AGG/T ACA/G TAG/C CCC ATA/G AAA/T GC ACA ATA CCA TAT TAC AAC AAT ATT CCT ATC ATA TTT TGT AAG GTT GTT CTA TCA/T GCC/T TGA TGA/G AAC TTC GGA/G TCC AC/TA/G ATT CCA ATA/G TTT CAT GTC/T TCT CTA AAA CTA CAC AAA CTT ACA TTA CTA–FL LC705-ACA ATA ATA TCA AAC CAG ACA TAC ACC A–PH ATA CAC TAT ACA TCT GAC AC–FL LC640-GCT ACT GCT TTC TCA TCT G–PH
Pnest.for Pnest.rev CVF.light for CVF.light rev emulti-fl emulti-705
The rtm-nested PCR was performed with a final volume of 20 μl in sealed LightCycler glass capillaries, including 5 pmol of primer Pnest.for and Pnest.rev (Dinkel et al. 1998), 10 pmol of primer CVF light.for and CVF light.rev, 2 μM of each hybridization probe (emulti-fl, emulti-705, CaVuFe1-fl and CaVuFe2-640), 2 μl LightCyler DNA Master HybProbe (Roche, Mannheim, Germany), 2.5 mM MgCl2 and 1 μl PCR product of the first PCR. Cycling conditions included 15 s at 94°C, followed by 45 cycles of 4 s at 94°C, 15 s at 50°C and 25 s at 72°C. Differentiation was obtained by detection of mismatches with FRET probes using subsequent melting curve analysis performed with 4 s at 94°C, 15 s at 50° C and 25 s heating to 90°C with a ramping rate of 0.2°C/s for emulti-fl/emulti-705 and 10 s at 95°C, 10 s at 30°C and heating to 90°C (0.2°C/s) for CaVuFe1-fl and CaVuFe2-640. To increase the sensitivity in samples which gave no result for the identification of canid host species, we additionally used the rt-nested PCR. The PCR mixture contained 10 pmol of each primer (CVF light.for and CVF light.rev), 2 μM of each hybridization probe (CaVuFe1-fl and CaVuFe2-640), 2 μl LightCyler DNA Master HybProbe (Roche, Mannheim, Germany), 2.5 mM MgCl2 and 1 μl PCR product of the first PCR. Thermal cycling and melting curve analysis were done as described for rtm-nested PCR. The primer and hybridization probe sequences are given in Table 1. Additionally, we confirmed the detection of E. multilocularis by using a published nested PCR assay (Dinkel et al. 1998). All rtm-nested PCRs included a negative control to monitor possible contamination and the following PCR positive controls as references: E. multilocularis, V. vulpes, V. ferrilata, V. corsac and C. familiaris. Inhibition control To exclude false-negative results due to inhibition factors present in faecal samples, the isolated DNA from each faecal
CaVuFe1-fl CaVuFe2-640
sample underwent an inhibition control as follows: 100 ng of E. multilocularis DNA was added to each sample with negative result and the previously published cestode-specific PCR (cs PCR) (Dinkel et al. 1998, 2004) was performed. The sample was recorded as negative only if a signal was obtained; if not, the result was regarded as inconclusive. The overall inhibition rate for the panel of faecal samples used in this study was 11%. All figures for faecal samples in the following sections refer to non-inhibited samples only.
Results and discussion Specificity The carnivore primer pair CVF for/CVF rev was found to amplify the target sequence of all carnivore species, but not the other mammals tested (rodents). Primer pair P60.short. for/P375.short.rev amplified the target sequence of all cestode species which were tested. Species identification was obtained by detection of mismatches with FRET probes using a subsequent melting curve analysis. The canid DNAs differed from each other by base pair mismatches which allowed the distinct recognition and discrimination of their target DNAs based on the melting temperature (Tm) values: E. multilocularis 60.0°C, V. vulpes 56.0°C, V. corsac (three mismatches) 45.4°C (data not shown), V. ferrilata (four mismatches) 44.8°C and C. lupus/familiaris (six mismatches) 38.0°C
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(Fig. 1). Since the assay was designed to be homologous for V. vulpes DNA, the Tm value for the red fox was the highest of all canids tested. No cross-reactions were detected with DNA isolated from E. granulosus (G1), E. ortleppi, E. canadensis (G6, G7), T. crassiceps, T. hydatigena, T. mustelae, T. pisiformis, T. serialis, T. taeniaeformis, M. leptothylacus, M. arvalis, A. terrestris, F. catus and M. foina. Sensitivity The detection limit of the rtm-nested PCR was determined with serial tenfold dilutions (100 ng–1 fg) of genomic E. multilocularis DNA added to 10 μl total DNA solution from E. multilocularis-negative canid faeces. E. multilocularis DNA at 10 fg , when mixed together with total
faecal DNA, still could be identified by this rtm-nested PCR along with positive canid diagnostic. As an example for field application, we present the results of two series of non-inhibited environmental faecal samples from China (180 field samples collected from the provinces of Qinghai and Sichuan and 47 supposed dog samples from the province of Qinghai). Out of the 180 field samples collected, we detected 120 as dog/wolf, eight as red fox and 14 as Tibetan fox, while four samples showed different melting curves from those of the target species and 34 gave no result for host origin. Out of the 47 supposed dog samples, 24 were identified as dog/wolf, whereas 23 gave no result for host species. The E. multilocularis prevalence based on rtm-nested PCR was found to be 38% for the 47 supposed dog samples (18 of 47) and 14% for the 180 field samples collected (25 of 180).
a E. multilocularis (Tm 60.0oC)
b
b c
a b c
dog (Tm 38.0oC) tibetan fox (Tm 44.8oC) red fox (Tm 56.0oC)
a
Fig. 1 Melting curve analysis of E. multilocularis isolates (a) and different host species (a, dog; b, tibetan fox; c, red fox) with different melting peaks after real-time multiplex PCR (b)
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Based on a comparison of the 47 dog faecal samples from China, the proportion of the E. multilocularis-positive-tested samples by the real-time multiplex-nested PCR was moderately higher as that when tested by nested PCR (38% vs. 30%) which had been evaluated to give a sensitvity of 70–100%, depending on the number and gravidity status of worms present in the intestine (Dinkel et al. 1998). To increase the sensitivity of host species detection in 49 field samples collected from China which gave no result for the identification of the canid host, we additionally performed the rt-nested PCR. Fifteen additional faecal samples could thereby be identified (14C. lupus/familiaris and one V. vulpes), while 34 samples remained without result. Reproducibility A subset of 180 faecal samples from China were examined twice by different investigators using the rtm-nested PCR assay. The results, shown in Table 2, demonstrate that the rtm-nested PCR provided reproducible results. For the first time, we developed a real-time multiplexnested PCR assay for the simultaneous detection of E. multilocularis–DNA and host species DNA directly from faecal samples. This highly specific and sensitive approach involves only a few steps and can be used for epidemiological purposes. Fifty-seven of 227 faecal samples from the environment gave no result for host species DNA. This could be due to various factors. DNA from faecal samples collected in the environment could be degraded due to UV-light, high temperature, moisture and activity of DNases. Farrell et al. (2000) observed a reduced amplification success for faecal samples which were collected during the wet season and concluded that rain may cause more degradation than frozen precipitation and heavy rains might wash off the shed epithelial cells from the surface of the sample. DNA isolated from faeces collected in winter performed significantly better than those in summer and fresh faeces performed better than older samples (>2 days in summer, >7 days in winter) (Lucchini et al. 2002). Nonaka et al. (2009) reported that the total amount of DNA obtained from fox faeces was reduced by ageing under natural summer conditions for 8 weeks. Table 2 Comparison of the results of 180 faecal samples from China examined twice (A+B) using rtm-nested PCR E. multilocularis C. lupus/ V. ferrilata V. vulpes V. corsac familiaris A 27 Ba 24 a
104 106
15 14
6 7
0 0
All E. multilocularis and V. ferrilata samples positive in B were also positive in A. All C. lupus/familiaris and V. vulpes samples positive in A were also positive in B
Hybridization probe-based real-time PCR is known to detect extremely small quantities of DNA. The sensitivity of this rtm-nested PCR against E. multilocularis was found to be higher than that of a conventional nested PCR. The analytical sensitivity of our rtm-nested PCR was found to be 10 fg (one E. multilocularis egg had a DNA content of approximately 8 ng) (Rishi and McManus 1987). Still we cannot exclude false negative results because, even in hosts with mature infections of E. multilocularis, eggs and proglottids are not shed continuously and are not homogeneously distributed within the faeces. In our study, we detected four samples showing different melting curves from those of the target species and therefore produced no interpretable results due to the host origin of these faecal samples. Since our rtm-nested PCR showed a reliable specificity, we conclude that the origin of this DNA must be from other carnivores which were not included in our panel of isolates. The presence of DNA from prey species in the intestine of carnivores is consistently in discussion. In our study, we found no cross-reaction with tested rodent DNA. Theoretically, positive PCR results can also be obtained by amplifying DNA from immature E. multilocularis metacestodes which have been ingested by the fox together with the intermediate host (voles). However, as estimated before (Dinkel et al. 1998), calculations that consider the rate of mature and immature infections in voles and the prevalence and lifespan of the adult worm in foxes indicate that voles with immature metacestodes cannot be present in the intestines of more than 2% of foxes at a given time. We also cannot exclude the possibility of the detection of DNA from other ingested mammals including other canids. The multiplex PCR system, as presented here, was designed for field application on the Tibetan plateau (Vaniscotte et al., manuscript submitted for publication) and therefore includes the locally relevant species of canids. However, the system can easily be adapted on other geographic situations by including, e.g., raccoon dog, jackals, coyotes, etc. Due to the 100% sequence identity of the target sequences (Palomares et al. 2002), discrimination between domestic dogs and wolves is not possible. The multiplex feature of this assay is optional. If preferred, the components can be used as single-targeting assays without compromising the quality of the result. This makes this assay adaptable to circumstances that may not require the simultaneous detection of both host and parasite. Consequently, the presented rtm-nested PCR is a method with the possibility for extension to other Echinococcus species and genotypes and other carnivore hosts as well. Acknowledgements This work was supported by the NIH Fogarty International Center (grants number RFATW-00-002 and RO1 TW001565). The content is solely the responsibility of the authors
498 and does not necessarily represent the official views of the Fogarty International Center or the National Institutes of Health.
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