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On the risk of criminal manipulation in caviar trade by intended contamination of caviar with PCR products
Aquaculture 269 (2007) 130 – 134 www.elsevier.com/locate/aqua-online
On the risk of criminal manipulation in caviar trade by intended contamination of caviar with PCR products Sven Wuertz a,⁎, Mesfin Belay b , Frank Kirschbaum a a
Leibniz-Institute for Freshwater Ecology and Inland Fisheries, 12587 Berlin, Germany b DESIETRA GmbH, 36041 Fulda, Germany
Received 13 February 2007; received in revised form 21 May 2007; accepted 25 May 2007
Abstract The over-exploitation for caviar production has led to drastic decreases of sturgeon stocks worldwide and consequently, all sturgeon species have been listed on the CITES regulation. In order to fulfil the obligation to control caviar trade effectively, species identification can solely be performed by molecular techniques. Here, we show that these approaches are vulnerable towards manipulation and protocols should consider criminal intends to manipulate the caviar. Using artifically amplified DNA (cytochrome b gene) from Acipenser sturio we show, that unsalted eggs and caviar from A. baerii can be manipulated and artificial DNA is detected by DNA sequencing. The amount of PCR product required was determined by a dilution series, and clearly demonstrates that such manipulation is economically feasible as it comprises less than 5% of the actual price. Neither extensive washing nor additional treatment with a non-ionic tensid (0.1 and 0.01% Tween20) unmasked the cytochrome b sequence from A. baerii. Species identity could only be determined successfully by a DNase treatment of contaminated eggs. For a standard testing procedure currently asked for in context of the CITES convention, a DNase treatment should be integrated into the protocol. © 2007 Elsevier B.V. All rights reserved. Keywords: Acipenser; Sturgeon; Caviar; Species identification; Mislabelling; Manipulation; Masking DNA
1. Introduction From the 19th century on, sturgeon populations decreased rapidly and nowadays sturgeons and paddlefishes (Acipenseriformes) are among the most endangered freshwater fishes (IUCN, 2006). Their caviar is an extremely valuable product, achieving species-specific net import prizes/kg in 2006 ranging from US$1000– 1100 for farmed Siberian sturgeon and US$1350–1500 for farmed Osietra to US$2000–2200 for wild Sevruga, US$2200–2300 for Osietra and US$2600–2700 for ⁎ Corresponding author. Tel.: +49 3064181622; fax: +49 3064181750. E-mail address: [email protected] (S. Wuertz). 0044-8486/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2007.05.027
Beluga caviar (pers. comm. 2007, Zuther-Grauerholz, Managing Director Dieckmann-Hansen, Hamburg), thereby threatening the commercially harvested species with over-fishing (Pikitch et al., 2005). Consequently, all 27 sturgeon and paddlefish species worldwide were listed on the Annex II or I of the CITES convention (IUCN, 2006), providing tools for a more effective control of the international trade (CITES Conf. 12.7 Rev. CoP13). Still, despite the controls on caviar trade, illegal fishing continue to threaten many populations and illicit trade of mislabelled caviar is rarely uncovered (Ludwig, 2006; Raymakers, 2006; Pikitch et al., 2005). In order to fulfil the obligation to control international caviar trade, effective and precise species identification system is essential. In several studies species
S. Wuertz et al. / Aquaculture 269 (2007) 130–134
identification by molecular genetic techniques was performed, e.g. by PCR–RFLP (Ludwig et al., 2002), species-specific PCR (Birstein et al., 1998; DeSalle and Birstein, 1996) or direct sequencing (for review Ludwig, 2007) and there is a common agreement that a molecular approach is the method of choice for species identification. Recently, the IUCN/SSC Sturgeon Specialist Group advocated direct sequencing of the cytochrome b for species identification in the control of caviar trade, aiming at a universal protocol for species identification (Ludwig, 2007). Theoretically, the detection of such a selected target can be biased, if a similar sequence matching the priming sites is artificially introduced. For example a caviar sample might be masked by a PCR amplicon of the selected target derived from another species. This is easy to perform with regard to caviar since the target is known to a broad public as a consequence of the international demand for an uniform protocol in the framework of the CITES regulation. Subsequently, any caviar sample could be manipulated and “genetically mislabelled”, circumventing the CITES control measures. In this study, we investigated the potential threat of such a manipulation. Therefore, caviar samples from Siberian sturgeon A. baerii were contaminated with a cytochrome b PCR amplicon derived from A. sturio. Different washing protocols and DNase digestion were performed to reverse the contamination. In order to assess the minimum
131
concentration and the economic costs of the mislabelling, supplementation with a dilution series of the PCR amplicon was carried out separately. 2. Materials and methods For the manipulation, a tissue sample was taken from the dorsal fin of a European sturgeon A. sturio reared at the facilities of the Institute for Freshwater Ecology and Inland Fisheries. Samples were stored in Queen's tissue buffer [0.25 M EDTA, NaCl staturated, 20% DMSO, pH 8] until DNA was extracted with the DNA extraction kit according to the manufacturer's protocol (Qiagen, Germany). The concentration of purified DNA was determined spectrophotometrically and quality was confirmed by agarose gel electrophoresis. PCR amplification of a 1300 bp fragment including the cytochrome b gene was carried out in 50 μl volume with the primers CytbF4 5'-AATgACTTgAAAAACCACCgTT-3' and CytbR1292 5'-TTAgCTTTgggAgTTAAggg-3' [95 °C for 5 min, 35 cycles: 95 °C for 30 s, 60 °C for 30 s, 72 °C for 1 min, 72 °C for 10 min; 1.5 mM MgCl2, 1× PCR buffer, 0.4 μM each primer, 0.2 mM dNTP, 1.3 U Promega GoTaq, 80 ng DNA]. Subsequently, reaction vials were pooled, and the PCR product was confirmed by sequencing and diluted to a working solution of 20 ng/μl. Fig. 1 provides a schematic outline of the experiments. In brief, caviar and unsalted eggs were obtained from a
Fig. 1. Scheme of the experiments performed on caviar and unsalted eggs, illustrating the succession of 1) manipulation with a PCR product (20 ng/ μl) or a dilution series of this product (10− 2, 10− 4, 10− 6, 10− 8), 2) washing steps and 3) post-treatments with a) 0.1% and b) 0.01% of a non-ionic tensid (Tween) and c) a DNase digestion and subsequent denaturation of the enzyme in ethanol. Veggs — volume of eggs, −control — negative control, +control — positive control, RDD — digestion buffer (Qiagen).
132
S. Wuertz et al. / Aquaculture 269 (2007) 130–134
Siberian sturgeon A. baerii at the commercial farm DESIETRA GmbH during the process of caviar production. Unsalted eggs (eggs) or caviar were incubated for 20 min in two volumes (Veggs — volume of the eggs/ caviar) manipulation solution, a 1:10 (10− 1), a 1:100 (10− 2), a 1:1000 (10− 3), a 1:10,000 (10− 4), a 1:105 (10− 5), and a 1:106 (10− 6) dilution of this manipulation solution. Successive washings (3×) were performed with 50 Veggs phosphate buffered saline PBS [3 mM KCl, 1.5 mM KH2PO4, 0.14 M NaCl, 8 mM, pH 7.5]. In two treatments, an additional washing step in Tween (0.1% or
0.01%, 10 Veggs) was carried out, followed by three washings in PBS (20 Veggs). Furthermore, a DNase treatment was performed to digest the DNA attached to the surface before actual DNA extraction: Per egg, 4 μl (20 K units) DNA solution, 10 μl RDD and 87.5 μl H2O were incubated for 20 min at RT. In order to avoid further DNA digestion, the eggs were subsequently washed in 100% EtOH p.a. (20 Veggs) to denature the enzyme. All samples were stored in Queen's tissue buffer until processed. Again, DNA extraction was carried out with the DNeasy kit (Qiagen), followed by PCR amplification
Fig. 2. Alignment of the cytochrome b gene from A. baerii sequenced from caviar and unsalted eggs (− controls) and from PCR product amplified from A. sturio and subsequently used for the manipulation (+controls and individual fish). Sequence identities were confirmed by alignment with deposited sequences (A. baerii: AF006137,AF40479,AF217207,AF006172,AF006123,X95054,AF404779,AJ245825,AJ245828; A. sturio: AJ245825,AJ549117,AJ245839,AJ428497,AF217209,AF006134,AF006145,AF006176), diagnostic substitutions (97) are not shaded.
S. Wuertz et al. / Aquaculture 269 (2007) 130–134
as described above. Sequencing was performed with a capillary sequencer CEQ 8800 (Beckman Coulter, Germany) according to the manufacturer's protocol. Sequences were aligned using BioEdit 7.0.0. Diagnostic substitutions are given in Fig. 2.
133
3. Results There was no difference between the results of the experiments conducted with unsalted eggs and those performed with caviar. After DNA extraction, gel electrophoresis of all samples did not reveal a distinct 1300 kb band indicating the introduced PCR product. After manipulation, the artificial PCR product was identified by sequencing in the +control (20 ng/μl DNA) and in the 10− 1 dilution. For the manipulation with 10− 2, 10− 3, 10− 4, 10− 5, 10− 6 dilutions of the manipulation solution, sequencing identified A. baerii. There were no double signals observed in the chromatogram of the +control and 10− 1 that indicated an underlying A. baerii sequence. In the 10− 2 treatment, some double signals indicated the PCR product (Fig. 3), but the A. baerii like sequence was clearly analysed on the grounds of the dominant signals. The post-treatment with Tween20 (0.01%, 0.1%) did not uncover the original A. baerii sequence. Again, the A. sturio PCR product was identified and no double signals indicative of A. baerii were observed. In this study, only post-treatment with DNase revealed the original A. baerii sequence on the genomic level. 4. Discussion
Fig. 3. Double signals (arrows) observed after manipulation of caviar from A. baerii with a PCR product of A. sturio (1:100 dilution, corresponding to 0.2 ng/μl), analysed as a) A. sturio, b) A. baerii, and c) no prevailance (Y).
The increasing sequence information acquired in DNA databases such as NCBI, EMBL and DDBJ over the past years comprises mitochondrial cytochrome b sequences of all sturgeon species (Ludwig, 2007). This culminated in an advance to include molecular species identification into the existing CITES regulations, prepared and discussed on the 2nd Status Workshop on the Identification of Acipenseriformes Species in Trade (29th Sep.–1st Oct. 2006, Berlin, Germany). In general, species identification involving most of the commercially harvested species is feasible applying PCR-based techniques (Ludwig, 2007; Birstein et al., 1998). Nevertheless, some species cannot be distinguished due to a lack of diagnostic substitutions or unsolved phylogenetic position (Ludwig, 2007) and increasing aquaculture of hybrids may complicate the control measures. In addition, criminal intention to manipulate such measures should be considered as demonstrated here: Our results clearly show that the most promising method – namely sequencing of PCR-amplified cytochrome b – is vulnerable towards intentional contamination with an artificial PCR product and may be used to mislabel any illicit caviar. Considering the actual value of caviar (e.g. $2000/kg), such manipulation is economically feasible: The manipulation with a dilution series clearly showed that 2 ng/μl PCR product (double of the eggs volume) is sufficient to mislabel caviar and unsalted eggs under the
134
S. Wuertz et al. / Aquaculture 269 (2007) 130–134
experimental conditions applied. Here, we estimate costs of approximately $40–100 for the manipulation of 1 kg of caviar (40–50 cents per reaction). The multiple use of a manipulation solution or economic optimisation of the PCR might extremely reduce these costs. Therefore, criminal manipulation represents a threat that has to be accounted for. A variety of methods has been used for caviar/ sturgeon species identification including species-specific PCR (Birstein et al., 1998; DeSalle and Birstein, 1996), DNA sequencing (data stored in NCBI), SSCP (Rehbein et al., 1999), PCR–RFLP (Ludwig et al., 2002), AFLP (Congiu et al., 2002) and microsatellites (Jenneckens et al., 2001). Currently, only speciesspecific PCR, PCR–RFLP and DNA sequencing could be used for identification of the traded species, targeting mitochondrial cytochrome b (Ludwig, 2007). Still, it should be mentioned that some closely species cannot be discriminated, for example the A. gueldenstaedti – A. baerii – A. naccarii – A. naccarii – A. persicus complex. Among these three techniques, a RFLP approach could be established that is not vulnerable towards manipulation with PCR products. Still, the integration of the DNase digestion offers the possibility to counteract manipulation effectively. Consequently, DNA sequencing can be regarded as the method of choice for caviar identification in our point of view (for a detailed discussion on caviar identification methods see Ludwig, 2007). Although Tween20 is a solvent in molecular applications that can be used to solve nucleic acids from surfaces (Helenius, 1979), neither washing with 0.1% nor 0.01% removed the contaminant. Higher concentrations were not recommendable since concentrations between 0.05% and 0.5% are used for cell lysis for DNA extraction (Hall et al., 2006; Nam et al., 2003; Vince et al., 1998). In this study, only the DNase treatment removed the contamination successfully. It is therefore the recommended procedure and which can easily be integrated in any DNA extraction protocol. Acknowledgements This study was supported in part by the Ministry of Education and Research (BMBF). We would like to thank Peter Löschau for his technical assistance.
References Birstein, V.J., Doukakis, P., Sorkin, B., DeSalle, R., 1998. Population aggregation analysis of three caviar-producing species of sturgeons and implications for the species identification of black caviar. Cons. Biol. 12, 766–775. Congiu, L., Fontana, F., Patarnello, T., Rossi, R., Zane, L., 2002. The use of AFLP in sturgeon identification. J. Appl. Ichthyol. 18, 286–289. DeSalle, R., Birstein, V.J., 16-5-1996. PCR identification of black caviar. Nature 381, 197–198. Hall, J.A., Felnagle, E., Fries, M., Spearing, S., Monaco, L., Steele, A., 2006. Evaluation of cell lysis procedures and use of a micro fluidic system for an automated DNA-based cell identification in interplanetary missions. Planet. Space Sci. 54, 1600–1611. Helenius, A., 1979. Properties of detergents. Methods Enzymol. 56, 734–749. IUCN, 2006. Red List of Endangered Species. www.iucnredlist.Org status 19. January. Jenneckens, I., Meyer, J.N., Horstgen-Schwark, G., May, B., Debus, L., Wedekind, H., Ludwig, A., 2001. A fixed allele at microsatellite locus LS-39 exhibiting species-specificity for the black caviar producer Acipenser stellatus. J. Appl. Ichthyol. 17, 39–42. Ludwig, A., 2006. A sturgeon view on conservation genetics. Europ. J. Wildlife Res. 52, 3–8. Ludwig, A., 2007. Identification of Acipenseriformes species in trade. IUCN/SSC Sturgeon Specialist Group and the IUCN Species Programme. IUCN, Gland, Switzerland. Ludwig, A., Debus, L., Jenneckens, I., 2002. A molecular approach to control the international trade in black caviar. Int. Rev. Hydrobiol. 87, 661–674. Nam, Y.K., Park, J.E., Kim, K.K., Kim, D.S., 2003. A rapid and simple PCR-based method for analysis of transgenic fish using a restricted amount of fin tissue. Transg. Res. 12, 523–525. Pikitch, E.K., Doukakis, P., Lauck, L., Chakrabarty, P., Erickson, D.L., 2005. Status, trends and management of sturgeon and paddlefish fisheries. Fish Fish. 6, 233–265. Raymakers, C., 2006. CITES, the convention on international trade in endangered species of wild fauna and flora: its role in the conservation of Acipenseriformes. J. Appl. Ichthyol. 22, 53–65. Rehbein, H., Gonzales-Sotelo, C., Perez-Martin, R., Quinteiro, J., Rey-Mendez, M., Pryde, S., Mackie, I.M., Santos, A.T., 1999. Differentiation of sturgeon caviar by single strand conformation polymorphism (PCR–SSCP) analysis. Arch. Lebensm. Hyg. 50, 13–17. Vince, A., Poljak, M., Seme, K., 1998. DNA extraction from archival Giemsa-stained bone-marrow slides: comparison of six rapid methods. Br. J. Haematol. 101, 349–351.
On the risk of criminal manipulation in caviar trade by intended contamination of caviar with PCR products Sven Wuertz a,⁎, Mesfin Belay b , Frank Kirschbaum a a
Leibniz-Institute for Freshwater Ecology and Inland Fisheries, 12587 Berlin, Germany b DESIETRA GmbH, 36041 Fulda, Germany
Received 13 February 2007; received in revised form 21 May 2007; accepted 25 May 2007
Abstract The over-exploitation for caviar production has led to drastic decreases of sturgeon stocks worldwide and consequently, all sturgeon species have been listed on the CITES regulation. In order to fulfil the obligation to control caviar trade effectively, species identification can solely be performed by molecular techniques. Here, we show that these approaches are vulnerable towards manipulation and protocols should consider criminal intends to manipulate the caviar. Using artifically amplified DNA (cytochrome b gene) from Acipenser sturio we show, that unsalted eggs and caviar from A. baerii can be manipulated and artificial DNA is detected by DNA sequencing. The amount of PCR product required was determined by a dilution series, and clearly demonstrates that such manipulation is economically feasible as it comprises less than 5% of the actual price. Neither extensive washing nor additional treatment with a non-ionic tensid (0.1 and 0.01% Tween20) unmasked the cytochrome b sequence from A. baerii. Species identity could only be determined successfully by a DNase treatment of contaminated eggs. For a standard testing procedure currently asked for in context of the CITES convention, a DNase treatment should be integrated into the protocol. © 2007 Elsevier B.V. All rights reserved. Keywords: Acipenser; Sturgeon; Caviar; Species identification; Mislabelling; Manipulation; Masking DNA
1. Introduction From the 19th century on, sturgeon populations decreased rapidly and nowadays sturgeons and paddlefishes (Acipenseriformes) are among the most endangered freshwater fishes (IUCN, 2006). Their caviar is an extremely valuable product, achieving species-specific net import prizes/kg in 2006 ranging from US$1000– 1100 for farmed Siberian sturgeon and US$1350–1500 for farmed Osietra to US$2000–2200 for wild Sevruga, US$2200–2300 for Osietra and US$2600–2700 for ⁎ Corresponding author. Tel.: +49 3064181622; fax: +49 3064181750. E-mail address: [email protected] (S. Wuertz). 0044-8486/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2007.05.027
Beluga caviar (pers. comm. 2007, Zuther-Grauerholz, Managing Director Dieckmann-Hansen, Hamburg), thereby threatening the commercially harvested species with over-fishing (Pikitch et al., 2005). Consequently, all 27 sturgeon and paddlefish species worldwide were listed on the Annex II or I of the CITES convention (IUCN, 2006), providing tools for a more effective control of the international trade (CITES Conf. 12.7 Rev. CoP13). Still, despite the controls on caviar trade, illegal fishing continue to threaten many populations and illicit trade of mislabelled caviar is rarely uncovered (Ludwig, 2006; Raymakers, 2006; Pikitch et al., 2005). In order to fulfil the obligation to control international caviar trade, effective and precise species identification system is essential. In several studies species
S. Wuertz et al. / Aquaculture 269 (2007) 130–134
identification by molecular genetic techniques was performed, e.g. by PCR–RFLP (Ludwig et al., 2002), species-specific PCR (Birstein et al., 1998; DeSalle and Birstein, 1996) or direct sequencing (for review Ludwig, 2007) and there is a common agreement that a molecular approach is the method of choice for species identification. Recently, the IUCN/SSC Sturgeon Specialist Group advocated direct sequencing of the cytochrome b for species identification in the control of caviar trade, aiming at a universal protocol for species identification (Ludwig, 2007). Theoretically, the detection of such a selected target can be biased, if a similar sequence matching the priming sites is artificially introduced. For example a caviar sample might be masked by a PCR amplicon of the selected target derived from another species. This is easy to perform with regard to caviar since the target is known to a broad public as a consequence of the international demand for an uniform protocol in the framework of the CITES regulation. Subsequently, any caviar sample could be manipulated and “genetically mislabelled”, circumventing the CITES control measures. In this study, we investigated the potential threat of such a manipulation. Therefore, caviar samples from Siberian sturgeon A. baerii were contaminated with a cytochrome b PCR amplicon derived from A. sturio. Different washing protocols and DNase digestion were performed to reverse the contamination. In order to assess the minimum
131
concentration and the economic costs of the mislabelling, supplementation with a dilution series of the PCR amplicon was carried out separately. 2. Materials and methods For the manipulation, a tissue sample was taken from the dorsal fin of a European sturgeon A. sturio reared at the facilities of the Institute for Freshwater Ecology and Inland Fisheries. Samples were stored in Queen's tissue buffer [0.25 M EDTA, NaCl staturated, 20% DMSO, pH 8] until DNA was extracted with the DNA extraction kit according to the manufacturer's protocol (Qiagen, Germany). The concentration of purified DNA was determined spectrophotometrically and quality was confirmed by agarose gel electrophoresis. PCR amplification of a 1300 bp fragment including the cytochrome b gene was carried out in 50 μl volume with the primers CytbF4 5'-AATgACTTgAAAAACCACCgTT-3' and CytbR1292 5'-TTAgCTTTgggAgTTAAggg-3' [95 °C for 5 min, 35 cycles: 95 °C for 30 s, 60 °C for 30 s, 72 °C for 1 min, 72 °C for 10 min; 1.5 mM MgCl2, 1× PCR buffer, 0.4 μM each primer, 0.2 mM dNTP, 1.3 U Promega GoTaq, 80 ng DNA]. Subsequently, reaction vials were pooled, and the PCR product was confirmed by sequencing and diluted to a working solution of 20 ng/μl. Fig. 1 provides a schematic outline of the experiments. In brief, caviar and unsalted eggs were obtained from a
Fig. 1. Scheme of the experiments performed on caviar and unsalted eggs, illustrating the succession of 1) manipulation with a PCR product (20 ng/ μl) or a dilution series of this product (10− 2, 10− 4, 10− 6, 10− 8), 2) washing steps and 3) post-treatments with a) 0.1% and b) 0.01% of a non-ionic tensid (Tween) and c) a DNase digestion and subsequent denaturation of the enzyme in ethanol. Veggs — volume of eggs, −control — negative control, +control — positive control, RDD — digestion buffer (Qiagen).
132
S. Wuertz et al. / Aquaculture 269 (2007) 130–134
Siberian sturgeon A. baerii at the commercial farm DESIETRA GmbH during the process of caviar production. Unsalted eggs (eggs) or caviar were incubated for 20 min in two volumes (Veggs — volume of the eggs/ caviar) manipulation solution, a 1:10 (10− 1), a 1:100 (10− 2), a 1:1000 (10− 3), a 1:10,000 (10− 4), a 1:105 (10− 5), and a 1:106 (10− 6) dilution of this manipulation solution. Successive washings (3×) were performed with 50 Veggs phosphate buffered saline PBS [3 mM KCl, 1.5 mM KH2PO4, 0.14 M NaCl, 8 mM, pH 7.5]. In two treatments, an additional washing step in Tween (0.1% or
0.01%, 10 Veggs) was carried out, followed by three washings in PBS (20 Veggs). Furthermore, a DNase treatment was performed to digest the DNA attached to the surface before actual DNA extraction: Per egg, 4 μl (20 K units) DNA solution, 10 μl RDD and 87.5 μl H2O were incubated for 20 min at RT. In order to avoid further DNA digestion, the eggs were subsequently washed in 100% EtOH p.a. (20 Veggs) to denature the enzyme. All samples were stored in Queen's tissue buffer until processed. Again, DNA extraction was carried out with the DNeasy kit (Qiagen), followed by PCR amplification
Fig. 2. Alignment of the cytochrome b gene from A. baerii sequenced from caviar and unsalted eggs (− controls) and from PCR product amplified from A. sturio and subsequently used for the manipulation (+controls and individual fish). Sequence identities were confirmed by alignment with deposited sequences (A. baerii: AF006137,AF40479,AF217207,AF006172,AF006123,X95054,AF404779,AJ245825,AJ245828; A. sturio: AJ245825,AJ549117,AJ245839,AJ428497,AF217209,AF006134,AF006145,AF006176), diagnostic substitutions (97) are not shaded.
S. Wuertz et al. / Aquaculture 269 (2007) 130–134
as described above. Sequencing was performed with a capillary sequencer CEQ 8800 (Beckman Coulter, Germany) according to the manufacturer's protocol. Sequences were aligned using BioEdit 7.0.0. Diagnostic substitutions are given in Fig. 2.
133
3. Results There was no difference between the results of the experiments conducted with unsalted eggs and those performed with caviar. After DNA extraction, gel electrophoresis of all samples did not reveal a distinct 1300 kb band indicating the introduced PCR product. After manipulation, the artificial PCR product was identified by sequencing in the +control (20 ng/μl DNA) and in the 10− 1 dilution. For the manipulation with 10− 2, 10− 3, 10− 4, 10− 5, 10− 6 dilutions of the manipulation solution, sequencing identified A. baerii. There were no double signals observed in the chromatogram of the +control and 10− 1 that indicated an underlying A. baerii sequence. In the 10− 2 treatment, some double signals indicated the PCR product (Fig. 3), but the A. baerii like sequence was clearly analysed on the grounds of the dominant signals. The post-treatment with Tween20 (0.01%, 0.1%) did not uncover the original A. baerii sequence. Again, the A. sturio PCR product was identified and no double signals indicative of A. baerii were observed. In this study, only post-treatment with DNase revealed the original A. baerii sequence on the genomic level. 4. Discussion
Fig. 3. Double signals (arrows) observed after manipulation of caviar from A. baerii with a PCR product of A. sturio (1:100 dilution, corresponding to 0.2 ng/μl), analysed as a) A. sturio, b) A. baerii, and c) no prevailance (Y).
The increasing sequence information acquired in DNA databases such as NCBI, EMBL and DDBJ over the past years comprises mitochondrial cytochrome b sequences of all sturgeon species (Ludwig, 2007). This culminated in an advance to include molecular species identification into the existing CITES regulations, prepared and discussed on the 2nd Status Workshop on the Identification of Acipenseriformes Species in Trade (29th Sep.–1st Oct. 2006, Berlin, Germany). In general, species identification involving most of the commercially harvested species is feasible applying PCR-based techniques (Ludwig, 2007; Birstein et al., 1998). Nevertheless, some species cannot be distinguished due to a lack of diagnostic substitutions or unsolved phylogenetic position (Ludwig, 2007) and increasing aquaculture of hybrids may complicate the control measures. In addition, criminal intention to manipulate such measures should be considered as demonstrated here: Our results clearly show that the most promising method – namely sequencing of PCR-amplified cytochrome b – is vulnerable towards intentional contamination with an artificial PCR product and may be used to mislabel any illicit caviar. Considering the actual value of caviar (e.g. $2000/kg), such manipulation is economically feasible: The manipulation with a dilution series clearly showed that 2 ng/μl PCR product (double of the eggs volume) is sufficient to mislabel caviar and unsalted eggs under the
134
S. Wuertz et al. / Aquaculture 269 (2007) 130–134
experimental conditions applied. Here, we estimate costs of approximately $40–100 for the manipulation of 1 kg of caviar (40–50 cents per reaction). The multiple use of a manipulation solution or economic optimisation of the PCR might extremely reduce these costs. Therefore, criminal manipulation represents a threat that has to be accounted for. A variety of methods has been used for caviar/ sturgeon species identification including species-specific PCR (Birstein et al., 1998; DeSalle and Birstein, 1996), DNA sequencing (data stored in NCBI), SSCP (Rehbein et al., 1999), PCR–RFLP (Ludwig et al., 2002), AFLP (Congiu et al., 2002) and microsatellites (Jenneckens et al., 2001). Currently, only speciesspecific PCR, PCR–RFLP and DNA sequencing could be used for identification of the traded species, targeting mitochondrial cytochrome b (Ludwig, 2007). Still, it should be mentioned that some closely species cannot be discriminated, for example the A. gueldenstaedti – A. baerii – A. naccarii – A. naccarii – A. persicus complex. Among these three techniques, a RFLP approach could be established that is not vulnerable towards manipulation with PCR products. Still, the integration of the DNase digestion offers the possibility to counteract manipulation effectively. Consequently, DNA sequencing can be regarded as the method of choice for caviar identification in our point of view (for a detailed discussion on caviar identification methods see Ludwig, 2007). Although Tween20 is a solvent in molecular applications that can be used to solve nucleic acids from surfaces (Helenius, 1979), neither washing with 0.1% nor 0.01% removed the contaminant. Higher concentrations were not recommendable since concentrations between 0.05% and 0.5% are used for cell lysis for DNA extraction (Hall et al., 2006; Nam et al., 2003; Vince et al., 1998). In this study, only the DNase treatment removed the contamination successfully. It is therefore the recommended procedure and which can easily be integrated in any DNA extraction protocol. Acknowledgements This study was supported in part by the Ministry of Education and Research (BMBF). We would like to thank Peter Löschau for his technical assistance.
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