http://informahealthcare.com/mdn ISSN: 1940-1736 (print), 1940-1744 (electronic) Mitochondrial DNA, Early Online: 1–4 ! 2014 Informa UK Ltd. DOI: 10.3109/19401736.2014.963823
SHORT COMMUNICATION
A duplex polymerase chain reaction assay for the identification of goat cashmere and sheep wool Qing-Rong Geng
Mitochondrial DNA Downloaded from informahealthcare.com by Selcuk Universitesi on 01/30/15 For personal use only.
College of Life Science and Technology, Yancheng Teachers University, Yancheng, Jiangsu Province, P.R China
Abstract
Keywords
In this article attempts were made to establish one-step duplex PCR assay for the identification of goat cashmere and sheep wool. Primers were selected from published papers or designed in the well-conserved region of mitochondrial D-loop genes after alignment of the available sequences in the GenBank database. A fragment of 294 bp from cashmere goat was amplified and three PCR fragments including a bright main band of approximately 404 bp in length were obtained from sheep. The duplex PCR was found to be effective in detecting mixed samples precisely when sheep wool was mixed to goat cashmere with the relative proportion of over 9.09%. The duplex PCR could be considered as a simple and promising method in identification of goat cashmere and sheep wool.
Duplex PCR, goat cashmere, identification, sheep wool
Introduction Cashmere goats (Capra hircus) produce one of the finest animal fibres. The soft and light fibres from the downy undercoat of cashmere goats are always described as cashmere. The total annual world production of cashmere fleece is about 15,000– 20,000 tones (Dubeuf et al., 2004; Zhang & Jia, 2009), of which only 17–50% are fine cashmere fibres (Holst & Clarke, 1982). Goat cashmere is often termed as specialty or luxury fibre because of its high economic value, softness, luster, warmness and scarcity (McCarthy, 1998; McGregor et al., 2011). Because goat cashmere is expensive and scarce, merchants usually substitute them with cheap sheep (Ovis aries) wool to obtain lots of economic profits. The assessment of authenticity of the cashmere and its related products is very important and useful for avoiding unfair competition among producers, allowing consumers to have accurate information about their acquired products. Several analytical methods have been applied for the identification of goat cashmere and sheep wool. At present, the identification of cashmere and wool mostly depends on traditional methods which mainly include macroscopical identification, microscopical examination, physical and chemical experiment (Robson, 2000; She et al., 2002; Shi & Yu, 2008; Vineis et al., 2014; Zoccola et al., 2013). However, the application of these methods is restricted in evaluating cashmere and wool due to different technological processes. More recently, DNA molecules have been the target compounds for species identification due to their high stability compared with other technologies. Moreover, the DNA based methods like polymerase chain reaction (PCR) have been successfully applied to authentication of animal species (Fajardo et al., 2010). These techniques take advantage of the high Correspondence: Qing-Rong Geng, College of Life Science and Technology, Yancheng Teachers University, Yancheng, Jiangsu 224051, P.R. China. Tel: +86-0515-88239395. Fax: +86-0515-88239395. E-mail: [email protected]
History Received 10 July 2014 Revised 31 August 2014 Accepted 7 September 2014 Published online 26 September 2014
specificity and sensitivity of PCR-based methods to detect very low amounts of DNA. A primary aspect for successfully detecting a species by PCR is to choose adequate genetic markers to develop the assay. Both nuclear and mitochondrial genes have been broadly targeted for the identification of species. The use of mitochondrial DNA (mtDNA) sequences offers a series of advantages over other genetic markers with the characteristics of simple in complexity, more conservative, rapid evolution, and high number of copies per cell (Girish et al., 2004; Gray, 1989; Rokas et al., 2003). However, attempts to use PCR as a quantitative tool for cashmere and wool authentication are still very scarce (Geng et al., 2012; Wan et al., 2011). This study will describe a simple a fast, reliable and straightforward approach using a duplex PCR technique to amplify species-specific fragments of the mitochondrial D-Loop gene to identify the mixed cashmere and wool.
Materials and methods Samples Goat cashmere samples (n ¼ 78) from different cashmere goat individuals were obtained from a farm at Shaanxi Province of China. Sheep wool samples (n ¼ 72) from different sheep individuals were obtained from a farm at Jiangsu Province of China. Reference samples were prepared with blood samples acquired from cashmere goat and sheep. Blood samples were used as controls for evaluating the specificity of primers used in the study. A total of five blood samples were collected from each species, respectively. Mitochondrial DNA extraction Mitochondrial DNA was extracted using the method as described by Geng et al. (2012) with some minor modifications. Briefly, all goat cashmere and sheep wool samples were cleaned prior to mitochondrial DNA extraction by successively shaking for 10 s in 95% ethanol to wipe off sundries. The air dried samples of about
2
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20 mg were then cut into 1 mm fragments and were put into the 2 ml microcentrifuge tube. The mitochondrial DNA was extracted using Tissue and Hair Extraction Kit (for use with DNA IQÔ, Promega, Madison, WI) following the manufacturer’s protocol. The extracted mitochondrial DNA was quantified and diluted to 50 ng/ml.
Mitochondrial DNA, Early Online: 1–4
were prepared with the following weight/weight ratios: 12:1, 11:1, 10:1, 9:1, 8:1, 6:1, 3:1, 1:1, 1:3, 1:6, 1:8, 1:9, 1:10, 1:11 and 1:12. The mitochondrial DNA extraction from mixture, duplex PCR amplification and detection were operated following the same procedure described above.
Results
Mitochondrial DNA Downloaded from informahealthcare.com by Selcuk Universitesi on 01/30/15 For personal use only.
Primer design The principle of screening primers was that each pair of primers had intraspecies-universality and interspecies-specificity. Mitochondrial D-loop sequences of goat (Capra hircus) and sheep (Ovis aries) were downloaded from GenBank. Speciesspecific primers were designed and confirmed for specificity by using the Primer-BLAST program (http://www.ncbi.nlm.nih.gov/ tools/primer-blast/). Sheep-specific primers were published by Karabasanavar et al. (2011). The goat-specific primers were used for the amplification of a 294 bp DNA fragment (forward 50 TTCTTCAGGGCCATCTCATC-30 ; reverse 50 -GCGGATGCATG GTGAAAT-30 ). The sheep-specific primers were used for the amplification of a 404 bp DNA fragment (forward 50 -CCACCC ACGGACACGAG-30 ; reverse 50 -AGTTCAATGCCCTATATGC TTCAG-30 ). The primers were synthesized by Nanjing GenScript Biotechnology Co., Ltd., Nanjing, China. Simplex PCR In a preliminary phase of this research, primers specificity was assessed with DNA extracted from whole blood samples of cashmere goat and sheep. All goat cashmere and sheep wool samples were tested by the same method in the next experiment. PCR amplification was performed in a final volume of 25 ml containing 2.5 ml of 10 PCR buffer (100 mM Tris-HCl, pH 9.0, 15 mM MgCl2, 500 mM KCl and 0.1% gelatin), 1 ml of 10 mM dNTPs mix, 1ml of each primer (10 mM), 1 U of Taq DNA polymerase, 100 ng of DNA template. Amplification was performed in a Thermal Cycler C1000 (Bio-Rad, Hercules, CA). PCR cycling parameters were as follows: 32 cycles each consisted of 94 C for 30 s, 57 C for 30 s, and 72 C for 30 s, with a initial hot start at 94 C for 5 min and a final extension at 72 C for 10 min. The amplified fragments were analyzed by electrophoresis in a 1% agarose gel carried out in TBE buffer for 20 min at 120 V, stained with ethidium bromide. The agarose gel was visualised under UV light and a digital image was obtained using a ChemiDoc XRS + Gel Documentation System (Bio-Rad, Hercules, CA).
Simplex PCR specificity The direct simplex amplifications of two pair of species-specific primers used in this study were first performed using the whole blood samples. This was done to test the primers’ efficiency, specificity and to evaluate the possibility of applying direct PCR for cashmere and wool authentication. A fragment of 294 bp from cashmere goat mitochondrial D-loop gene was amplified and three PCR fragments including a bright main band of approximately 404 bp in length were obtained from mitochondrial D-loop gene in sheep. To detect possible cross-reactions, each pair of primers was challenged in simplex PCR with non-target species. As expected, a cross-reaction was not observed and no PCR product was observed from the negative controls. The results showed that direct PCR could be used to identify species from cashmere goat and sheep blood samples. Amplifications were subsequently carried out with mitochondrial DNA extracted from goat cashmere and sheep wool, respectively. In simplex PCR, amplification with goat specific primer set showed a consistent PCR signal corresponding to product size of 294 bp from goat cashmere samples (Figure 1A). Similarly, only desired amplifications were obtained from sheep wool samples when sheep-specific primer set was used (Figure 1B). In these experiments, each species-specific
Duplex PCR and sensitivity validation For the simultaneous detection of two species, a one-step duplex PCR was developed using two primer sets previously designed for the simplex PCR. The amplification by duplex PCR was carried out in a final volume of 25 ml containing 2.5 ml of 10 PCR buffer (100 mM Tris-HCl, pH 9.0, 15 mM MgCl2, 500 mM KCl and 0.1% gelatin), 1 ml of 10 mM dNTPs mix, 1.2ml and 1.5ml each of goat and sheep primers (10 mM), 1.5 U of Taq DNA polymerase, 150 ng of DNA template. Amplification was performed in a Thermal Cycler C1000 (Bio-Rad, Hercules, CA). The PCR cycling parameters were set as follows: 32 cycles each consisted of 94 C for 45 s, 55 C for 45 s, and 72 C for 45 s, with an initial hot start at 94 C for 5 min and a final extension at 72 C for 10 min. The amplified fragments were visualized by electrophoresis in a 10% polyacrylamide gel and stained with silver nitrate. Electrophoresis was carried out in TBE buffer for 150 min at 150 V at room temperature. The gel was visualized under white light and a digital image was recorded. To determine the sensitivity of the duplex PCR in detecting mixed samples, a serial mixture of goat cashmere and sheep wool
Figure 1. Specificity of simplex PCR products using goat primers (A) and sheep primers (B) from goat cashmere and sheep wool samples. M: DL2000 DNA marker, fragments range from 2000 bp, 1000 bp, 750 bp, 500 bp, 250 bp to 100 bp vertically. (A) lanes 1–4: goat cashmere samples; lanes 5–8: sheep wool samples; C: negative control. (B) lanes 1–6: sheep wool samples; lanes 7–9: goat cashmere samples; C: negative control.
DOI: 10.3109/19401736.2014.963823
primer pair produced PCR products only from its target species and generated the expected PCR products. The results showed the applicability of the species-specific primers for the identification of goat cashmere and sheep wool.
Mitochondrial DNA Downloaded from informahealthcare.com by Selcuk Universitesi on 01/30/15 For personal use only.
Testing samples by duplex PCR and PCR sensitivity The same primers used in simplex PCR were applied in duplex PCR amplification. The duplex PCR was carried out for coamplification of both goat and sheep target DNA segment in a single PCR reaction. Briefly, duplex PCR was established to produce expected bands in a single tube and the conditions were optimized with temperature, primer concentrations and extension time to prevent non-specific reaction. When duplex PCR was carried out on samples, the set of primers retained the same specificity and no cross-reaction from non-target species was found (data not shown). The sensitivity of duplex PCR was quantified by amplification of known amount of binary mixtures from goat cashmere and sheep wool. It was found that the goat specific assay was sensitive to detect goat cashmere and sheep wool mixtures when their ratio was 1:10 (Figure 2A). The sheep-specific primers successfully produced amplification from goat cashmere and sheep wool mixtures when their ratio was 10:1 (Figure 2B). A similar degree of sensitivity in detection of cashmere and wool mixtures was observed with duplex PCR assay.
Discussion Species authenticity and traceability of fibres are important topics, not only for the consumers but also for the textile
Figure 2. Sensitivity of duplex PCR in identification of goat cashmere and sheep wool mixtures. M: DL2000 DNA marker, fragments range from 2000 bp, 1000 bp, 750 bp, 500 bp, 250 bp to 100 bp vertically. (A) C: negative control (wool sample); lanes 1–8: mixed cashmere and wool samples with different ratio of 1:1, 1:3, 1:6, 1:8, 1:9, 1:10, 1:11, 1:12. (B) C: negative control (cashmere sample); lanes 1–8: mixed cashmere and wool samples with different ratio of 1:1, 3:1, 6:1, 8:1, 9:1, 10:1, 11:1, 12:1.
Identification of goat cashmere and sheep wool
3
industry, traders, retailers and regulators. Over the last decades, full attention has been turning towards implementation of molecular genetic approaches for species identification because of their high specificity and sensitivity, as well as rapid processing time and low cost. Among DNA-based methods, PCR is the most well-developed molecular technique till now and provides a simple, rapid, highly sensitive and specific tool for detecting constituents of species origin (Bottero & Dalmasso, 2011; Doosti et al., 2014; Eaqub et al., 2012). PCR amplification is based on the hybridization of specific oligonucleotides to a target DNA and synthesis of millions of DNA copies flanked by these primers. The amplification of DNA fragments, followed by gel electrophoresis for fragment size verification, is the simplest PCR strategy applied to evaluate the presence of a species. Additional confirmation methods or examination of PCR products can be accomplished by sequencing of DNA amplicons (PCR-sequencing), analysis of PCR-single strand conformation polymorphism (PCR-SSCP), simultaneous amplification of two or more fragments with different primer pairs (multiplex PCR), analysis of PCR-restriction fragment length polymorphism (PCR-RFLP), and real-time fluorescence PCR assays (Fajardo et al., 2010). Multiplex PCR allows the simultaneous amplification of more than one target sequence in a single PCR, which saves considerable time and effort and thereby decreases the number of reactions to be performed. Compared to other PCR systems, multiplex PCR, in which many primers are used together for the amplification of more than one target region, is a hopeful technique to save costs and enhance the speed, efficiency and reliability of analysis for the simultaneous identification of various species. Multiplex PCR has been proven successful in identification of species by numerous reports (Ghovvati et al., 2008; Nakyinsige et al., 2012; Rentsch et al., 2013; Ryu et al., 2013). The application of the multiplex PCR was also demonstrated useful in quantitative detection of mixtures (Mafra et al., 2004, 2007; Soares et al., 2010). The obtained results showed that the well-developed methodology of multiplex PCR can be effectively used for quantitative detection of mixtures, representing a lower cost, reliable, specific and sensitive alternative to realtime PCR. In this study, we have developed a duplex PCR assay to identify cashmere and wool with high accuracy. To our knowledge, this is the first study to discriminate cashmere and wool using duplex PCR in a single tube. Combining the two pairs of primers allowed simultaneous detection of cashmere and wool, each of the two pairs of primers exclusively amplified the targeted gene of the specific species and thus. This single assay provides critical information for different species. In addition, duplex PCR was applied to monitor the presence of wool in cashmere. It was able to successfully detect a serial mixture of goat cashmere and sheep wool at weight ratios from 10:1 to 1:10 in a reaction. The duplex PCR was found to be effective in detecting mixed samples precisely when sheep wool was mixed to goat cashmere with the relative proportion over 9.09%. It could be easily identified whether sheep wool was added to goat cashmere when the wool was more than the percentage of 9.09%. It was revealed that PCR-RFLP technique could identify sheep wool from goat cashmere when the ration of wool was more than the percentage of 11.1% (Geng et al., 2012). Therefore, the duplex PCR method was more sensitive than PCR-RFLP analysis in identification of mixed cashmere and wool. The duplex PCR described in this paper proved to be very sensitive, with a very low detection limit. The possibility of detecting lower levels of sheep wool in goat cashmere would be interesting from a theoretical point of view and helpful in practice.
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Conclusions The duplex PCR described in this study, was proved to be fast, low-cost, sensitive and reliable which could be considered as a simple and promising method in identification of goat cashmere and sheep wool.
Declaration of interest This research was supported by the National Natural Science Foundation of China (31101684 and 31101747), the Natural Science Foundation of Jiangsu Province (BK20141259) and sponsored by Qing Lan Project of Jiangsu Province. The author reports no conflicts of interest. The author alone is responsible for the content and writing of this article.
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References Bottero MT, Dalmasso A. (2011). Animal species identification in food products: Evolution of biomolecular methods. Vet J 190:34–8. Doosti A, Ghasemi DP, Rahimi E. (2014). Molecular assay to fraud identification of meat products. J Food Sci Technol 51:148–52. Dubeuf JP, Morand-Fehr P, Rubino R. (2004). Situation, changes and future of goat industry around the world. Small Ruminant Res 51: 165–73. Eaqub AM, Kashif M, Uddin K, Hashim U, Mustafa S, Man YBC. (2012). Species authentication methods in foods and feeds: The present, past, and future of Halal forensics. Food Anal Methods 5: 935–55. Fajardo V, Gonza´lez I, Rojas M, Garcı´a T, Martı´n R. (2010). A review of current PCR-based methodologies for the authentication of meats from game animal species. Trends Food Sci Technol 21:408–21. Geng RQ, Yuan C, Chen YL. (2012). Identification of goat cashmere and sheep wool by PCR-RFLP analysis of mitochondrial 12S rRNA gene. Mitochondrial DNA 23:466–70. Ghovvati S, Nassiri MR, Mirhoseini SZ, Heravi Moussavi A, Javadmanesh A. (2008). Fraud identification in industrial meat products by multiplex PCR assay. Food Control 20:696–9. Girish PS, Anjaneyulu ASR, Viswas KN, Anand M, Rajkumar N, Shivakumar BM, Bhaskar S. (2004). Sequence analysis of mitochondrial 12S rRNA gene can identify meat species. Meat Sci 66:551–6. Gray WM. (1989). Origin and evolution of mitochondrial DNA. Annu Rev Cell Biol 5:25–50. Holst P, Clarke W. (1982). Skin and fleece characteristics of two groups of feral goats. Aus J Exp Agriculture Animal Husbandry 22:173–6. Karabasanavar NS, Singh SP, Umapathi V, Kumar D, Patil G, Shebannavar SN. (2011). A highly specific PCR assay for
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identification of raw and heat treated mutton (Ovis aries). Small Ruminant Res 100:153–8. Mafra I, Ferreira IM, Faria MA, Oliveira BP. (2004). A novel approach to the quantification of bovine milk in ovine cheeses using a duplex polymerase chain reaction method. J Agric Food Chem 52:4943–7. Mafra I, Roxo A, Ferreira IMPLVO, Oliveira MBPP. (2007). A duplex polymerase chain reaction for the quantitative detection of cows’ milk in goats’ milk cheese. Int Dairy J 17:1132–8. McCarthy BJ. (1998). Specialty animal fibres. Textiles 1:6–8. McGregor BA, Kerven C, Toigonbaev S. (2011). Sources of variation affecting cashmere grown in the Pamir mountain districts of Tajikistan and implications for industry development. Small Ruminant Res 99: 7–15. Nakyinsige K, Man YBC, Sazili AQ. (2012). Halal authenticity issues in meat and meat products. Meat Sci 91:207–14. Rentsch J, Weibel S, Ruf J, Eugster A, Beck K, Ko¨ppel R. (2013). Interlaboratory validation of two multiplex quantitative real-time PCR methods to determine species DNA of cow, sheep and goat as a measure of milk proportions in cheese. Eur Food Res Technol 236: 217–27. Robson D. (2000). Animal fiber analysis using imaging techniques. Textile Res J 70:116–20. Rokas A, Ladoukakis E, Zouros E. (2003). Animal mitochondrial DNA recombination revisited. Trends Ecol Evol 18:411–17. Ryu J, Park SH, Yeom YS, Shrivastav A, Lee SH, Kim UR, Kim HY. (2013). Simultaneous detection of Listeria species isolated from meat processed foods using multiplex PCR. Food Control 32:659–64. She FH, Kong LX, Nahavandi S, Kouzani AZ. (2002). Intelligent animal fiber classification with artificial neural networks. Textile Res J 72: 594–600. Shi XJ, Yu WD. (2008). Identification of animal fiber based on scale shape. Image Signal Process 3:573–7. Soares S, Amaral JS, Mafra I, Oliveira MB. (2010). Quantitative detection of poultry meat adulteration with pork by a duplex PCR assay. Meat Sci 85:531–6. Vineis C, Tonetti C, Paolella S, Pozzo P, Sforza S. (2014). A UPLC/ESIMS method for identifying wool, cashmere and yak fibres. Textile Res J 84:953–8. Wan J, Li B, Ming J, Xue Y. (2011). A method for quantifying mixed goat cashmere and sheep wool. Forensic Sci Int 208:139–42. Zhang LZ, Jia YS. (2009). Analysis of cashmere market and developmental strategies for China’s cashmere industry. J Beijing Technol Business Univ (Soc Sci) 6:117–21. Zoccola M, Lu N, Mossotti R, Innocenti R, Montarsolo A. (2013). Identification of wool, cashmere, yak, and angora rabbit fibers and quantitative determination of wool and cashmere in blend – A near infrared spectroscopy study. Fibers Polymers 14:1283–9.
SHORT COMMUNICATION
A duplex polymerase chain reaction assay for the identification of goat cashmere and sheep wool Qing-Rong Geng
Mitochondrial DNA Downloaded from informahealthcare.com by Selcuk Universitesi on 01/30/15 For personal use only.
College of Life Science and Technology, Yancheng Teachers University, Yancheng, Jiangsu Province, P.R China
Abstract
Keywords
In this article attempts were made to establish one-step duplex PCR assay for the identification of goat cashmere and sheep wool. Primers were selected from published papers or designed in the well-conserved region of mitochondrial D-loop genes after alignment of the available sequences in the GenBank database. A fragment of 294 bp from cashmere goat was amplified and three PCR fragments including a bright main band of approximately 404 bp in length were obtained from sheep. The duplex PCR was found to be effective in detecting mixed samples precisely when sheep wool was mixed to goat cashmere with the relative proportion of over 9.09%. The duplex PCR could be considered as a simple and promising method in identification of goat cashmere and sheep wool.
Duplex PCR, goat cashmere, identification, sheep wool
Introduction Cashmere goats (Capra hircus) produce one of the finest animal fibres. The soft and light fibres from the downy undercoat of cashmere goats are always described as cashmere. The total annual world production of cashmere fleece is about 15,000– 20,000 tones (Dubeuf et al., 2004; Zhang & Jia, 2009), of which only 17–50% are fine cashmere fibres (Holst & Clarke, 1982). Goat cashmere is often termed as specialty or luxury fibre because of its high economic value, softness, luster, warmness and scarcity (McCarthy, 1998; McGregor et al., 2011). Because goat cashmere is expensive and scarce, merchants usually substitute them with cheap sheep (Ovis aries) wool to obtain lots of economic profits. The assessment of authenticity of the cashmere and its related products is very important and useful for avoiding unfair competition among producers, allowing consumers to have accurate information about their acquired products. Several analytical methods have been applied for the identification of goat cashmere and sheep wool. At present, the identification of cashmere and wool mostly depends on traditional methods which mainly include macroscopical identification, microscopical examination, physical and chemical experiment (Robson, 2000; She et al., 2002; Shi & Yu, 2008; Vineis et al., 2014; Zoccola et al., 2013). However, the application of these methods is restricted in evaluating cashmere and wool due to different technological processes. More recently, DNA molecules have been the target compounds for species identification due to their high stability compared with other technologies. Moreover, the DNA based methods like polymerase chain reaction (PCR) have been successfully applied to authentication of animal species (Fajardo et al., 2010). These techniques take advantage of the high Correspondence: Qing-Rong Geng, College of Life Science and Technology, Yancheng Teachers University, Yancheng, Jiangsu 224051, P.R. China. Tel: +86-0515-88239395. Fax: +86-0515-88239395. E-mail: [email protected]
History Received 10 July 2014 Revised 31 August 2014 Accepted 7 September 2014 Published online 26 September 2014
specificity and sensitivity of PCR-based methods to detect very low amounts of DNA. A primary aspect for successfully detecting a species by PCR is to choose adequate genetic markers to develop the assay. Both nuclear and mitochondrial genes have been broadly targeted for the identification of species. The use of mitochondrial DNA (mtDNA) sequences offers a series of advantages over other genetic markers with the characteristics of simple in complexity, more conservative, rapid evolution, and high number of copies per cell (Girish et al., 2004; Gray, 1989; Rokas et al., 2003). However, attempts to use PCR as a quantitative tool for cashmere and wool authentication are still very scarce (Geng et al., 2012; Wan et al., 2011). This study will describe a simple a fast, reliable and straightforward approach using a duplex PCR technique to amplify species-specific fragments of the mitochondrial D-Loop gene to identify the mixed cashmere and wool.
Materials and methods Samples Goat cashmere samples (n ¼ 78) from different cashmere goat individuals were obtained from a farm at Shaanxi Province of China. Sheep wool samples (n ¼ 72) from different sheep individuals were obtained from a farm at Jiangsu Province of China. Reference samples were prepared with blood samples acquired from cashmere goat and sheep. Blood samples were used as controls for evaluating the specificity of primers used in the study. A total of five blood samples were collected from each species, respectively. Mitochondrial DNA extraction Mitochondrial DNA was extracted using the method as described by Geng et al. (2012) with some minor modifications. Briefly, all goat cashmere and sheep wool samples were cleaned prior to mitochondrial DNA extraction by successively shaking for 10 s in 95% ethanol to wipe off sundries. The air dried samples of about
2
Q.-R. Geng
20 mg were then cut into 1 mm fragments and were put into the 2 ml microcentrifuge tube. The mitochondrial DNA was extracted using Tissue and Hair Extraction Kit (for use with DNA IQÔ, Promega, Madison, WI) following the manufacturer’s protocol. The extracted mitochondrial DNA was quantified and diluted to 50 ng/ml.
Mitochondrial DNA, Early Online: 1–4
were prepared with the following weight/weight ratios: 12:1, 11:1, 10:1, 9:1, 8:1, 6:1, 3:1, 1:1, 1:3, 1:6, 1:8, 1:9, 1:10, 1:11 and 1:12. The mitochondrial DNA extraction from mixture, duplex PCR amplification and detection were operated following the same procedure described above.
Results
Mitochondrial DNA Downloaded from informahealthcare.com by Selcuk Universitesi on 01/30/15 For personal use only.
Primer design The principle of screening primers was that each pair of primers had intraspecies-universality and interspecies-specificity. Mitochondrial D-loop sequences of goat (Capra hircus) and sheep (Ovis aries) were downloaded from GenBank. Speciesspecific primers were designed and confirmed for specificity by using the Primer-BLAST program (http://www.ncbi.nlm.nih.gov/ tools/primer-blast/). Sheep-specific primers were published by Karabasanavar et al. (2011). The goat-specific primers were used for the amplification of a 294 bp DNA fragment (forward 50 TTCTTCAGGGCCATCTCATC-30 ; reverse 50 -GCGGATGCATG GTGAAAT-30 ). The sheep-specific primers were used for the amplification of a 404 bp DNA fragment (forward 50 -CCACCC ACGGACACGAG-30 ; reverse 50 -AGTTCAATGCCCTATATGC TTCAG-30 ). The primers were synthesized by Nanjing GenScript Biotechnology Co., Ltd., Nanjing, China. Simplex PCR In a preliminary phase of this research, primers specificity was assessed with DNA extracted from whole blood samples of cashmere goat and sheep. All goat cashmere and sheep wool samples were tested by the same method in the next experiment. PCR amplification was performed in a final volume of 25 ml containing 2.5 ml of 10 PCR buffer (100 mM Tris-HCl, pH 9.0, 15 mM MgCl2, 500 mM KCl and 0.1% gelatin), 1 ml of 10 mM dNTPs mix, 1ml of each primer (10 mM), 1 U of Taq DNA polymerase, 100 ng of DNA template. Amplification was performed in a Thermal Cycler C1000 (Bio-Rad, Hercules, CA). PCR cycling parameters were as follows: 32 cycles each consisted of 94 C for 30 s, 57 C for 30 s, and 72 C for 30 s, with a initial hot start at 94 C for 5 min and a final extension at 72 C for 10 min. The amplified fragments were analyzed by electrophoresis in a 1% agarose gel carried out in TBE buffer for 20 min at 120 V, stained with ethidium bromide. The agarose gel was visualised under UV light and a digital image was obtained using a ChemiDoc XRS + Gel Documentation System (Bio-Rad, Hercules, CA).
Simplex PCR specificity The direct simplex amplifications of two pair of species-specific primers used in this study were first performed using the whole blood samples. This was done to test the primers’ efficiency, specificity and to evaluate the possibility of applying direct PCR for cashmere and wool authentication. A fragment of 294 bp from cashmere goat mitochondrial D-loop gene was amplified and three PCR fragments including a bright main band of approximately 404 bp in length were obtained from mitochondrial D-loop gene in sheep. To detect possible cross-reactions, each pair of primers was challenged in simplex PCR with non-target species. As expected, a cross-reaction was not observed and no PCR product was observed from the negative controls. The results showed that direct PCR could be used to identify species from cashmere goat and sheep blood samples. Amplifications were subsequently carried out with mitochondrial DNA extracted from goat cashmere and sheep wool, respectively. In simplex PCR, amplification with goat specific primer set showed a consistent PCR signal corresponding to product size of 294 bp from goat cashmere samples (Figure 1A). Similarly, only desired amplifications were obtained from sheep wool samples when sheep-specific primer set was used (Figure 1B). In these experiments, each species-specific
Duplex PCR and sensitivity validation For the simultaneous detection of two species, a one-step duplex PCR was developed using two primer sets previously designed for the simplex PCR. The amplification by duplex PCR was carried out in a final volume of 25 ml containing 2.5 ml of 10 PCR buffer (100 mM Tris-HCl, pH 9.0, 15 mM MgCl2, 500 mM KCl and 0.1% gelatin), 1 ml of 10 mM dNTPs mix, 1.2ml and 1.5ml each of goat and sheep primers (10 mM), 1.5 U of Taq DNA polymerase, 150 ng of DNA template. Amplification was performed in a Thermal Cycler C1000 (Bio-Rad, Hercules, CA). The PCR cycling parameters were set as follows: 32 cycles each consisted of 94 C for 45 s, 55 C for 45 s, and 72 C for 45 s, with an initial hot start at 94 C for 5 min and a final extension at 72 C for 10 min. The amplified fragments were visualized by electrophoresis in a 10% polyacrylamide gel and stained with silver nitrate. Electrophoresis was carried out in TBE buffer for 150 min at 150 V at room temperature. The gel was visualized under white light and a digital image was recorded. To determine the sensitivity of the duplex PCR in detecting mixed samples, a serial mixture of goat cashmere and sheep wool
Figure 1. Specificity of simplex PCR products using goat primers (A) and sheep primers (B) from goat cashmere and sheep wool samples. M: DL2000 DNA marker, fragments range from 2000 bp, 1000 bp, 750 bp, 500 bp, 250 bp to 100 bp vertically. (A) lanes 1–4: goat cashmere samples; lanes 5–8: sheep wool samples; C: negative control. (B) lanes 1–6: sheep wool samples; lanes 7–9: goat cashmere samples; C: negative control.
DOI: 10.3109/19401736.2014.963823
primer pair produced PCR products only from its target species and generated the expected PCR products. The results showed the applicability of the species-specific primers for the identification of goat cashmere and sheep wool.
Mitochondrial DNA Downloaded from informahealthcare.com by Selcuk Universitesi on 01/30/15 For personal use only.
Testing samples by duplex PCR and PCR sensitivity The same primers used in simplex PCR were applied in duplex PCR amplification. The duplex PCR was carried out for coamplification of both goat and sheep target DNA segment in a single PCR reaction. Briefly, duplex PCR was established to produce expected bands in a single tube and the conditions were optimized with temperature, primer concentrations and extension time to prevent non-specific reaction. When duplex PCR was carried out on samples, the set of primers retained the same specificity and no cross-reaction from non-target species was found (data not shown). The sensitivity of duplex PCR was quantified by amplification of known amount of binary mixtures from goat cashmere and sheep wool. It was found that the goat specific assay was sensitive to detect goat cashmere and sheep wool mixtures when their ratio was 1:10 (Figure 2A). The sheep-specific primers successfully produced amplification from goat cashmere and sheep wool mixtures when their ratio was 10:1 (Figure 2B). A similar degree of sensitivity in detection of cashmere and wool mixtures was observed with duplex PCR assay.
Discussion Species authenticity and traceability of fibres are important topics, not only for the consumers but also for the textile
Figure 2. Sensitivity of duplex PCR in identification of goat cashmere and sheep wool mixtures. M: DL2000 DNA marker, fragments range from 2000 bp, 1000 bp, 750 bp, 500 bp, 250 bp to 100 bp vertically. (A) C: negative control (wool sample); lanes 1–8: mixed cashmere and wool samples with different ratio of 1:1, 1:3, 1:6, 1:8, 1:9, 1:10, 1:11, 1:12. (B) C: negative control (cashmere sample); lanes 1–8: mixed cashmere and wool samples with different ratio of 1:1, 3:1, 6:1, 8:1, 9:1, 10:1, 11:1, 12:1.
Identification of goat cashmere and sheep wool
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industry, traders, retailers and regulators. Over the last decades, full attention has been turning towards implementation of molecular genetic approaches for species identification because of their high specificity and sensitivity, as well as rapid processing time and low cost. Among DNA-based methods, PCR is the most well-developed molecular technique till now and provides a simple, rapid, highly sensitive and specific tool for detecting constituents of species origin (Bottero & Dalmasso, 2011; Doosti et al., 2014; Eaqub et al., 2012). PCR amplification is based on the hybridization of specific oligonucleotides to a target DNA and synthesis of millions of DNA copies flanked by these primers. The amplification of DNA fragments, followed by gel electrophoresis for fragment size verification, is the simplest PCR strategy applied to evaluate the presence of a species. Additional confirmation methods or examination of PCR products can be accomplished by sequencing of DNA amplicons (PCR-sequencing), analysis of PCR-single strand conformation polymorphism (PCR-SSCP), simultaneous amplification of two or more fragments with different primer pairs (multiplex PCR), analysis of PCR-restriction fragment length polymorphism (PCR-RFLP), and real-time fluorescence PCR assays (Fajardo et al., 2010). Multiplex PCR allows the simultaneous amplification of more than one target sequence in a single PCR, which saves considerable time and effort and thereby decreases the number of reactions to be performed. Compared to other PCR systems, multiplex PCR, in which many primers are used together for the amplification of more than one target region, is a hopeful technique to save costs and enhance the speed, efficiency and reliability of analysis for the simultaneous identification of various species. Multiplex PCR has been proven successful in identification of species by numerous reports (Ghovvati et al., 2008; Nakyinsige et al., 2012; Rentsch et al., 2013; Ryu et al., 2013). The application of the multiplex PCR was also demonstrated useful in quantitative detection of mixtures (Mafra et al., 2004, 2007; Soares et al., 2010). The obtained results showed that the well-developed methodology of multiplex PCR can be effectively used for quantitative detection of mixtures, representing a lower cost, reliable, specific and sensitive alternative to realtime PCR. In this study, we have developed a duplex PCR assay to identify cashmere and wool with high accuracy. To our knowledge, this is the first study to discriminate cashmere and wool using duplex PCR in a single tube. Combining the two pairs of primers allowed simultaneous detection of cashmere and wool, each of the two pairs of primers exclusively amplified the targeted gene of the specific species and thus. This single assay provides critical information for different species. In addition, duplex PCR was applied to monitor the presence of wool in cashmere. It was able to successfully detect a serial mixture of goat cashmere and sheep wool at weight ratios from 10:1 to 1:10 in a reaction. The duplex PCR was found to be effective in detecting mixed samples precisely when sheep wool was mixed to goat cashmere with the relative proportion over 9.09%. It could be easily identified whether sheep wool was added to goat cashmere when the wool was more than the percentage of 9.09%. It was revealed that PCR-RFLP technique could identify sheep wool from goat cashmere when the ration of wool was more than the percentage of 11.1% (Geng et al., 2012). Therefore, the duplex PCR method was more sensitive than PCR-RFLP analysis in identification of mixed cashmere and wool. The duplex PCR described in this paper proved to be very sensitive, with a very low detection limit. The possibility of detecting lower levels of sheep wool in goat cashmere would be interesting from a theoretical point of view and helpful in practice.
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Q.-R. Geng
Conclusions The duplex PCR described in this study, was proved to be fast, low-cost, sensitive and reliable which could be considered as a simple and promising method in identification of goat cashmere and sheep wool.
Declaration of interest This research was supported by the National Natural Science Foundation of China (31101684 and 31101747), the Natural Science Foundation of Jiangsu Province (BK20141259) and sponsored by Qing Lan Project of Jiangsu Province. The author reports no conflicts of interest. The author alone is responsible for the content and writing of this article.
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