1 ŻYWNOŚĆ. Nauka. Technologia. Jakość, 2012, 5 (84), PAVOL BAJZIK, JOSEF GOLIAN, RADOSLAV ŽIDEK, MARTIN KRALL, MARIA WALCZYCKA, JOANNA TKACZEWSKA IDENTIFICATION OF THE COMMON CARP (CYPRINUS CARPIO) SPECIES USING REAL-TIME PCR METHODS S u m m a r y Before being put out onto the market many fish species sold around the world need to be processed, which may result in the subsequent removal of characteristics used for their classification (head, fins, internal organs). The biochemical characterization of fish species could be achieved using proteins or DNA sequences as species-specific markers. However, since different fish products undergo different processes, the method of analysis has to be chosen according to the modifications undergone by fish constituents during processing. As DNA molecules are more resistant than proteins to various processes (including thermal treatment), DNA analysis appears to be a promising method for fish species identification. For the species identification of the Common Carp (Cyprinus carpio) among 15 different freshwater fish species a specific predesigned molecular - genetic marker of Common Carp (Cyprinus carpio) was used, which comes from the mtdna control D - loop area. Next we analyzed the presence of mtdna in DNA isolates of the 15 kinds of freshwater fish and compared them with the Common Carp markers by using the following two PCR identification methods. The isolates were diluted to 10 % concentration, using the TaqMan Real-Time PCR method and the SYBR Green Real-Time PCR method. The results of using the optimized the SYBR Green Real-Time PCR method for species identification of the Common Carp (C. carpio) pointed to its suitability. We were able to create an analysis of the monitored standard curve which represented the PCR positive control (C. carpio), containing the characteristic melting peak (up to the melting point C). A single peak indicated a single product (C. carpio) which can be verified upon characterization of the PCR product by agarose gel electrophoresis. The TaqMan Real-Time PCR method with a TaqMan probe is a very sensitive and reliable method of authentication used on food of animal origin. The suitability of this method, which we used for species identification of the Common Carp (C. carpio), was confirmed. Thanks to using this method, already in the 17th cycle of the PCR amplification procedure, the presence of the Common Carp gene (C. carpio) was detected in the positive control and not detected in the rest of the fish samples. Key words: species identification, Common Carp (Cyprinus carpio), Real-Time PCR method P. Bajzik, Ing., PhD Student, J. Golian, PhD Student, R. Židek, PhD Student, M. Krall, PhD Student, Department of Food Hygiene and Safety, Faculty of Biotechnology and Food Sciences, Slovak University of Agriculture, M. Walczycka, dr, J. Tkaczewska doktorantka, Katedra Przetwórstwa Produktów Zwierzęcych, Wydz. Technologii Żywności, Uniwersytet Rolniczy w Krakowie, ul. Balicka 122, Kraków
2 IDENTIFICATION OF THE COMMON CARP (CYPRINUS CARPIO) SPECIES USING 167 Introduction Over the last years there has been a tremendous growth in fish consumption due to changes in consumer attitudes towards health and nutrition . Depending on their source (i.e., natural or processed), the quality and price of these products, even from the same fish species, vary with the production location (i.e., domestic or imported). Labeling regulations of commercial fishery products also differ among countries. Consequently, techniques are needed for authentication of commercial fishery products in order to guarantee accurate labeling and to prevent unfair competition . The increase in international trade and global seafood consumption, along with fluctuations in the supply and demand of different fish and seafood species, have resulted in intentional product mislabeling. The effects of species substitution are far-reaching and include economic fraud, health hazards, and illegal trade of protected species . The international fish market, estimated by the FAO at around 60 billion tons/year, may imply more than 20,000 species of fish . For instance, about 420 species of fish are sold on the German market alone . Market globalization, large numbers of both exploited species and processed fish products explained why the substitution of a less valuable species for a valuable one (representing a commercial fraud), may be a common phenomenon difficult to detect. For instance, a study on food fish in the United States revealed that three-quarters of fish sold as the threatened red snapper Lutjanus campechanus were mislabeled and belonged to other species . Likewise, Pepe et al.  found that 84.2 % (16/19) of surimi-based fish products sold as Theragra chalcogramma were actually prepared from species different from the one declared . Fish species identification is traditionally based on external morphological features. Yet, in many cases fish, and especially at their different developmental stages are difficult to identify by morphological features characteristic. DNA-based identification methods offer an analytically powerful addition or even an alternative to immunological techniques such as immunodiffusion and ELISA . Allozyme analysis has traditionally been used to authenticate the species of commercial fish sharing a few morphological features. However, recently DNA analysis has been developing rapidly as an alternative, since it is common and highly sensitive. In particular, analysis of mitochondrial DNA (mtdna), which has species-specific features in the base sequences and a high genetic stability due to maternal inheritance, has been used widely in the study of phylogeny and population structure of fish and in the authentication of fish products. Complete mtdna sequences of many fish species are already available in DNA databases, i.e., GenBank, EMBL, DDBJ and MitBASE [7, 14, 17, 31]. Thus, mtdna analysis is useful for authentication of ingredients of commercial fishery products. The mtdna is of maternal inheritance and has no re-
3 168 Pavol Bajzik, Josef Golian, Radoslav Židek, Martin Krall, Maria Walczycka, Joanna Tkaczewska combination in all vertebrates, so that the sequence of mtdna is more conservative [3, 23]. The mtdna lies in the inner membrane of mitochondria and is easily affected by oxidative phosphorylation. The rate of base substitution on mtdna is higher than that on genomic DNA [16, 27]. Genetic identification of species is based on the principle of DNA polymorphism, or genetic variations that take place as a result of naturally occurring mutations in the genetic code . To detect species-specific genetic polymorphism, DNA is first extracted from the target organism and then the DNA fragment(s) of interest is amplified using PCR. The resulting PCR amplicons are then analyzed to reveal the characteristic polymorphism under study . The present study was undertaken to develop a simple, rapid and accurate method based on the Real-Time PCR analysis of the specific pre-designed molecular - genetic marker which comes from the mtdna control D - loop area, for identification of the Common Carp (Cyprinus carpio) among different fish species. Material and Methods Fish Samples Frozen samples of 15 fish species (table 1) were obtained from and authenticated by the Department of Poultry Husbandry and Small Livestock at the Slovak University of Agriculture in Nitra. DNA extraction Fish samples of 300 mg were minced using a sterile surgical blade and transferred into a 2 ml Eppendorf tube. DNA was extracted from 300 mg of homogenized portions using the protocol of NucleoSpin Food Isolation Kit (Macherey-Nagel) for isolation of genomic DNA from food. DNA isolates were then diluted to 10 % concentration. Primers SYBR Green Real-Time PCR method The specific marker for species identification of the Common Carp (Cyprinus Carpio) was amplified using the following primers: Carp1-F (5 -TGGCATCTGGTTCCTATTTCA-3 ), Carp1-R (5 -CCAAAGGGGGCACTATGTAA-3 ) designed by Židek and Golian, 2008 .
4 IDENTIFICATION OF THE COMMON CARP (CYPRINUS CARPIO) SPECIES USING 169 Names and categories of fish samples studied. Nazwy gatunkowe i kategorie badanych próbek ryb. T a b l e 1 Number of samples Numer próbki Common name Nazwy zwyczajowa Scientific Name Genus / Species Nazwy gatunkowe Rodzaj/Gatunek 1. Brown trout / Pstrąg potokowy Salmo trutta fario 2. Alpine bullhead / Głowacz pręgopłetwy Cottus poecilopus 3. Grayling / Lipień pospolity Thymallus thymallus 4. Rainbow trout / Pstrąg tęczowy Oncorhynchus mykiss 5. Black bullhead / Sumik czarny Ameiurus melas 6. European eel / Węgorz europejski Anguilla anguilla 7. Northern pike / Szczupak pospolity Esox lucius 8. Wels catfish / Sum pospolity Silurus glanis 9. Sterlet / Czeczuga Acipenser ruthenus 10. European perch / Okoń europejski Perca fluviatilis 11. Freshwater bream / Leszcz Abramis brama 12. European chub / Kleń europejski Leuciscus cephalus 13. Common barbel / Brzanka pospolita Barbus barbus 14. Common nase / Świnka zwyczajna Chondrostoma nasus 15. Rainbow trout / Pstrąg tęczowy Oncorhynchus mykiss 16. Common Carp / Karp zwyczajny Cyprinus carpio TaqMan Real-Time PCR method The designed primers and TaqMan probe are specific for all lines of the specific pre-designed molecular - genetic marker which comes from the mtdna control D - loop area, for identification of the Common Carp (Cyprinus carpio). Primer pairs and TaqMan probe were prepared in final form by Roche, Slovakia. Sequences of primers and TaqMan probes were as follows: D Loop F (5 -CATCTGGTTCCTATTTCAGGGA-3 ), D Loop R (5 -GGCACTATGTAAGGATAAGTTGAACT-3 ), TM LNA (LC640-TGCACTTGAGATAAAAGTATGTAA+T+T+CT-BBQ). PCR amplification SYBR Green Real-Time PCR method The thermal cycling profile was as follows: pre-incubation of the samples at 95 C for 2 min. It was subsequently repeated by 50 cycles with a temperature profile: denaturation at 95 C for 45 s, annealing at 60 C for 45 s and extension at 72 C for
5 170 Pavol Bajzik, Josef Golian, Radoslav Židek, Martin Krall, Maria Walczycka, Joanna Tkaczewska 1 min 20 s with a final extension for 10 min at 72 C. The melting curve of PCR products was launched by heating samples to 95 C and immediate cooling to 65 C for 15 seconds. The sample was heated at a rate of 0.1 C per second and the fluorescence was measured after each temperature change of one tenth degree. PCR amplification was carried out in the capillary cycler LightCycler 1.5 using LightCycler software (version 4.5). TaqMan Real-Time PCR method The LightCycler TaqMan Master kit (Roche) was used. The thermal cycling profile was as follows: pre-incubation of the sample at 95 C for 10 min. It was subsequently repeated by 45 cycles with a temperature profile: denaturation at 95 C for 10 s, annealing at 63 C for 30 s and extension for 20 s at 72 C, followed by the measurement of fluorescence. The final cooling was at 30 s at 40 C. PCR amplification was carried out in the capillary cycler LightCycler 1.5 using LightCycler software (version 4.5). Results and Discussion SYBR Green Real-Time PCR method As shown in Fig. 1 (fish samples 3, 6, 7, 12, 16; 17- H 2 O), the earliest possible increase in the fluorescence and the creation of a specific curve with its exponential and linear phases was possible in the positive control (Sample 16 - C. carpio) only. In other fish samples a minimal increase in fluorescence was detected (Table 1- see samples). Based on the results we can create the standard curve (Fig. 2). The standard curve representing the PCR positive control (Sample 16 - C. carpio) contains the characteristic peak (formed at the melting point C), which shows the right use of the specific primer pair. For other curves of the PCR products of other fish samples (samples 1 to 15, and sample 17 the negative control - H 2 O), non-specific peaks were observed (Table 1 - see samples).
6 IDENTIFICATION OF THE COMMON CARP (CYPRINUS CARPIO) SPECIES USING 171 Positive control Cyprinus carpio / Pozytywna kontrola Other fish samples/ Próbki innych ryb Fig. 1. The curves of the fluorescence during the PCR amplification for individual fish samples by using SYBR Green Real-Time PCR method. Rys. 1. Krzywe fluorescencyjne uzyskane w trakcie amplifikacji PCR dla poszczególnych próbek ryb przy użyciu metody SYBR Green Real-Time PCR. T a b l e 2 Crossing thresholds of fish samples studied after PCR amplification by using SYBR Green Real-Time PCR method. Piki temperatury charakterystyczne dla badanych gatunków ryb metodą amplifikacji PCR przy użyciu techniki SYBR Green Real-Time PCR. Number of samples Numer próbki Scientific Name Genus / Species Nazwy gatunkowe Rodzaj/Gatunek C t - (Crossing threshold) Pik maksymalny topnienia 15 Cyprinus carpio (positive control) 11,33 3 Thymallus thymallus 27,66 12 Leuciscus cephalus 31,36 7 Esox lucius 32,93 6 Anguilla anguilla 33,87
7 172 Pavol Bajzik, Josef Golian, Radoslav Židek, Martin Krall, Maria Walczycka, Joanna Tkaczewska Other fish samples / Próbki innych ryb Positive control Cyprinus carpio / Pozytywna kontrola Melting of PCR products / Topnienie produktów z PCR Fig. 2. The curves of the melting PCR products in individual fish samples generated by the SYBR Green Real-Time PCR method. Rys. 2. Krzywe topnienia produktów PCR poszczególnych próbek ryb otrzymane za pomocą metody SYBR Green Real-Time PCR. TaqMan Real-Time PCR method As shown in Fig. 3, the earliest possible increase in fluorescence intensity and above the measurable nonspecific background in the positive control (Sample 16 - C. carpio) was in the 17 th cycle of the PCR amplification. The fluorescence intensity corresponds to the shape of the curve, which passes from exponential phase to the linear growth phase. For Samples: 3 (T. thymallus), 4 (O. mykiss), 6 (A. anguilla), 7 (E. lucius) and 11 (A. brama) the nonspecific background levels were visible to about the 40 th cycle of the PCR amplification. This is already the late phase of the PCR amplification in which we can not clearly demonstrate the specificity and affinity of the studied fish species with the positive control (C. carpio). For Samples 1 (S. trutta fario), 2 (C. poecilopus) 5 (A. melas), 8 (S. glanis), 9 (A. ruthenus), 10 (P. fluviatilis), 12 (L. cephalus), 13 (B. barbus), 14 (C. nasus), 15 (O. mykiss), and 17 (H 2 O - negative control) no changes in fluorescence intensity were observed, so those samples did not create the characteristic curve whether in their exponential or linear phase, i. e. there were excess levels of nonspecific background (Table 1- see samples). The specificity and sensitivity of this technique, combined with its high speed, robustness, reliability, and the possibility of automation, contribute to the adequacy of the method for quantifying fish species in fishery products. Thus, for instance, Sotelo et al.  used the TaqMan assay for the identification and quantification of Cod (Gadus morhua). Trotta et al.  used the Real-Time PCR for the identification of fish fillets from Grouper (Genera Epinephelus and Mycteroperca) and common substitute species. Hird et al.  used it for the detection and quantification of Haddock (Mela-
8 IDENTIFICATION OF THE COMMON CARP (CYPRINUS CARPIO) SPECIES USING 173 nogrammus aeglefinus). The presence of this fish in concentrations of up to 7 % in raw or slightly heat treated products could be detected. In another work, López and Pardo  applied the TaqMan Real-Time PCR technology for the identification and quantification of albacore and Yellow fin tuna (Thunnus albacares). Positive control Cyprinus carpio / Pozytywna kontrola Other fish samples / Próbki innych ryb Fig. 3. Rys. 3. The curves of the fluorescence during the PCR cycles in individual fish samples by using TaqMan Real-Time PCR method. Krzywe fluorescencyjne otrzymane z analizy PCR, za pomocą TaqMan Real-Time PCR, indywidualnych próbek ryb. The development of analytical methods for fish species identification may help to detect and avoid willful, as well as unintentional substitution of different fish species and thus enforce labeling regulations [2, 12, 15, 29]. Numerous methods have been developed to authenticate various fish species in a wide range of food products, including soup and dried fins , surimi , fish roe , spicy roe , fish tails , canned sardine and sardine-type products , and canned tuna . The development of these molecular methods helps not only to protect both consumers and producers from frauds, but may also help to protect fish species from over-exploitation or illegal trafficking . Conclusions 1. The results of using the optimized SYBR Green Real-Time PCR method for species identification of the Common Carp (Cyprinus carpio) prove its suitability. We were able to create an analysis of the monitored melting curves i.e. the curve representing the PCR positive control (Cyprinus carpio) contains the characteristic peak (up to the melting point C), which shows us the right use of the specific primer pair. Our results confirmed the suitability of this method for the identification of the species of the Common Carp (Cyprinus carpio).
9 174 Pavol Bajzik, Josef Golian, Radoslav Židek, Martin Krall, Maria Walczycka, Joanna Tkaczewska 2. The TaqMan Real-Time PCR method, using a TaqMan probe, is a very sensitive and reliable way of authentication of food of animal origin. Our results confirmed the suitability of this method for species identification of the Common Carp (Cyprinus carpio). Acknowledgments This work was a part of the project VEGA 1/1074/11 Authentication and traceability of food in relation to allergenic and no allergenic components, using molecular genetic method. Literatura  Aranishi F., Okimoto T., Ohkubo M.: Molecular identification of commercial spicy pollack roe products by PCRRFLP analysis. J. Food Sci., 2005, 70,  Asensio L.: PCR-based methods for fish and fishery products authentication. Trends Food Sci. Technol., 2007, 18, doi: /j.tifs  Bromham L., Eyre-Walker A., Smith N.H., Smith J.M.: Mitochondrial Steve: Paternal inheritance of mitochondrial in humans. Trend. Ecol. Evol., 2003, 18, 2-4.  Gil L.A.: PCR-based methods for fish and fishery products authentication. Food Sci. Technol., 2007, 18,  Hird H.J., Hold G.L., Chrishol, J., Reece P., Russell V.J., Brown J., et al.: Development of a method for the quantification of haddock (Melanogrammus aeglefinus) in commercial products using realtime PCR. Eur. Food Res. Technol., 2005, 220,  Hoelzel A.R.: Shark fishing in fin soup. Conserv. Genet., 2001, 2, doi: /a:  Ishiguro N., Miya M., Nishida M.: Complete mitochondrial DNA sequence of ayu Plecoglossus altivelis. Fish. Sci., 2001, 67,  Jérôme M., Lemaire C., Verrez-Bagnis V.: Directsequencing method for species identification of canned sardine and sardine-type products. J. Agric. Food Chem., 2003, 51, doi: /jf034652t.  Klossa-Kilia E., Papasotiropoulos V., Kilias G.: Authentication of Messolongi (Greece) fish roe using PCRRFLP analysis of 16s RNA mtdna segment. Food Control, 2002, 13, doi: /s (01)  Kochzius M., Nolte M., Weber H.: DNA microarrays for identifying fishes. Mar. Biotechnol., 2008, 10, doi: /s  Liu Z.J., Cordes J.F.: DNA marker technologies and their applications in aquaculture genetics. Aquaculture, 2004, 238,  Lockley A.K., Bardsley R.G.: DNA-based methods for food authentication. Trends Food Sci. Technol., 2000, 11,  López I., Pardo M.A.: Application of relative quantification Taqman real-time polymerase chain reaction technology for the identification and quantification of Thunnus alalunga and Thunnus albacares. J. Agricul. Food Chem., 2005, 53,  Mabuchi K., Miya M., Azuma Y. Nishida M.: Independent evolution of the specialized pharyngeal jaw apparatus in cichlid and labrid fishes. BMC Evol. Biol., 2007, 7, 10.  Mafra I., Ferreira I., Beatriz M.: Food authentication by PCR-based methods. Eur. Food Res. Technol., 2008, 227,
10 IDENTIFICATION OF THE COMMON CARP (CYPRINUS CARPIO) SPECIES USING 175  Mandavilli B.S., Santos J. H., Houten, B.V.: Mitochondrial DNA repair and agong. Mutation Res., 2002, 509,  Manchado M., Catanese G., Infante C.: Complete mitochondrial DNA sequence of the Atlantic bluefin tuna Thunnus thynnus. Fish. Sci., 2004, 70,  Marko P.B., Lee S.C., Rice A.M.: Fisheries: mislabelling of a depleted reef fish. Nature, 2004, 430,  Mitsutoshi N., Kazuhiko M., Tadanori A., Katsuaki S., Tsutomu M.: Analytical methods for quantification of relative flying fish paste content in processed sea food (ago-noyaki) Food Sci. Technol. Res., 2010, 16 (5),  Pepe T., Trotta M., Di Marco I., Fish species identification in surimi-based products. J. Agric. Food Chem., 2007, 55,  Rasmussen R.S., Morrissey M.T.: DNA-based methods for the identification of commercial fish and seafood species. Compr. Rev. Food Sci. Food Saf., 2008, 7,  Rehbein H., Mackie I., Pryde S.: Fish species identification in canned tuna by PCR-SSCP: validation by a collaborative study and investigation of intra-species variability of the DNA-patterns. Food Chem., 1999, 64,  Rokas A., Ladoukakis E., Zouros E.: Animal mitochondrial DNA recombination revisited. Trend Ecol. Evol., 2003, 18,  Rosalee S., Rasmussen and Michael T. Morrissey.: DNA-Based Methods for the Identification of Commercial Fish and Seafood Species. Comprehensive Reviews in Food Science and Food Safety, 2008, 7,  Sanjuan A., Raposo-Guillan J., Comesana A.: Geneticidentification of Lophius budegassa and L. piscatorius by PCR-RFLP analysis of a mitochondrial trnaglu/cytochrome b segment. J. Food Sci., 2002, 67,  Sotelo C.G., Chapela M.J., Rey M., Pe rez-martı n R.I.: Development of an identification and quantitation system for cod (Gadus morhua) using taqman assay. In: First Joint Trans-Atlantic Fisheries Technology Conference, Reykjavik, Iceland, 2003, pp  Stoneking M., Soodyall H.: Human evolution and the mitochondrial genome. C urr. Opin. Genetic Dev., 1996, 6,  Teletchea T.: Molecular identification methods of fish species: reassessment and possible applications. Reviews in Fish Biology and Fisheries, 2009, 19,  Teletchea T., Maudet C., Hȁnni C.: Food and forensic molecular identification: update and challenges. Trends Biotech., 2005, 23,  Trotta M., Schönhuth S., Pepe T., Cortesi M. L., Puyet, A., Bautista J.M.: A multiplex-pcr method for use in real-time PCR for identification of fish fillets from grouper (Epinephelus spp. and Mycteroperca spp.) and common substitute species. J. Agric. Food Chem., 2005, 53,  Yanagimoto T., Kitamura T., Kobayashi T.: Complete nucleotide sequence and variation of mitochondrial DNA from 10 individuals of walleye pollock, Theragra chalcogramma. Fish. Sci., 2004, 70,  Židek R., Golian J.: Detection of carp (Cyprinius carpio) proteins using genetic markers. Proteiny - Sborník příspěvků V. ročníku medzinárodní konference, 2008, pp
11 176 Pavol Bajzik, Josef Golian, Radoslav Židek, Martin Krall, Maria Walczycka, Joanna Tkaczewska IDENTIFIKACJA GATUNKU KARP ZWYCZAJNY (CYPRINUS CARPIO) PRZY UŻYCIU REAL-TIME PCR S t r e s z c z e n i e Przed wprowadzeniem do obrotu ryby są wstępnie przetwarzane, co może spowodować usunięcie tych części anatomicznych (tj. głowy, płetwy, organy wewnętrzne), na podstawie których identyfikuje się gatunek. Do identyfikacji ryb można wtedy zastosować charakterystykę biochemiczną, którą dla danego gatunku mogą być specyficzne białka lub sekwencja DNA specyficzne markery danego gatunku ryb. Jednak ryby podlegają różnym procesom przetwórczym, w związku z czym musi być opracowana taka metoda ich biochemicznej identyfikacji, która byłaby zgodna ze zmianami składników tkanki ryb, zachodzącymi podczas tego przetwarzania. Cząsteczki DNA są stosunkowo odporne na czynniki przetwórcze (włącznie z obróbką termiczną), dlatego analiza sekwencji DNA może być przydatną metodą do identyfikacji gatunkowej ryb. W celu gatunkowej identyfikacji karpia zwyczajnego, spośród 15 innych gatunków ryb słodkowodnych, opracowano specyficzny molekularno-genetyczny marker karpia zwyczajnego, który pochodził z mdna (z obszaru pętli kontrolnej D). Następnie analizowano izobaty mdna z 15 różnych gatunków ryb słodkowodnych, porównując je do markera karpia za pomocą dwóch metod identyfikacji PCR. Izolaty były rozcieńczane do 10 % stężenia w obu stosowanych metodach oznaczeń tj. TaqMan Real-Time PCR i SYBR Green Real-Time PCR. Na podstawie wyników badań karpia zwyczajnego (Cyprinus carpio), uzyskanych zoptymalizowaną metodą SYBR Green Real-Time PCR, wykazano jej przydatność do identyfikacji gatunkowej. Monitorowano krzywą standardową topnienia PCR (z maksimum w temp. 80,72 C) świadczącą o pozytywnej weryfikację Cyprinus carpio oraz krzywe topnienia pozostałych próbek ryb. Maksima topnienia poszczególnych (15) próbek ryb były następnie weryfikowane metodą elektroforezy żelowej na agarozie. Druga z zastosowanych metod TaqMan Real-Time PCR, z wykorzystaniem próbnika TaqMan, jest bardzo dokładną i wrażliwą metodą identyfikacji (potwierdzania autentyczności) karpia zwyczajnego (Cyprinus carpio). Dzięki zastosowanie metody amplifikacji już w 17. cyklu potwierdzono obecność, poprzez PCR, genu Cyprinus carpio w próbce kontrolnej i brak obecności tego genu w pozostałych próbkach ryb. Słowa kluczowe: identyfikacja gatunku, karp zwyczajny, metoda PCR
Introduction To Real Time Quantitative PCR (qpcr) SABiosciences, A QIAGEN Company www.sabiosciences.com The Seminar Topics The advantages of qpcr versus conventional PCR Work flow & applications Factors
Essentials of Real Time PCR About Real-Time PCR Assays Real-time Polymerase Chain Reaction (PCR) is the ability to monitor the progress of the PCR as it occurs (i.e., in real time). Data is therefore collected
Protocols Internal transcribed spacer region (ITS) region Niklaus J. Grünwald, Frank N. Martin, and Meg M. Larsen (2013) The nuclear ribosomal RNA (rrna) genes (small subunit, large subunit and 5.8S) are
SR3_3E S h i m a d z u R e v i e w DNA Analytical Technology to Identify the Actual Composition of Foods - Application of the MultiNA Microchip Electrophoresis System - by Yuji SOGABE Abstract Recently,
Roche Applied Science Technical Note No. LC 18/2004 Purpose of this Note Assay Formats for Use in Real-Time PCR The LightCycler Instrument uses several detection channels to monitor the amplification of
Forensic DNA Testing Terminology ABI 310 Genetic Analyzer a capillary electrophoresis instrument used by forensic DNA laboratories to separate short tandem repeat (STR) loci on the basis of their size.
User manual imegen Anchovies II ID kit Anchovies species (E. encrasicolus and E. japonicus) DNA detection by sequencing Reference: Made in Spain The information in this guide is subject to change without
Real-Time PCR Vs. Traditional PCR Description This tutorial will discuss the evolution of traditional PCR methods towards the use of Real-Time chemistry and instrumentation for accurate quantitation. Objectives
Scientific Working Group on DNA Analysis Methods Validation Guidelines for DNA Analysis Methods Table of Contents Introduction.2 1. Definitions. 2 2. General Considerations 3 3. Developmental Validation..5
APPLICATION NOTE Real-Time PCR Real-time PCR: Understanding C t Real-time PCR, also called quantitative PCR or qpcr, can provide a simple and elegant method for determining the amount of a target sequence
Protocol for GenomePlex Whole Genome Amplification from Formalin-Fixed Parrafin-Embedded (FFPE) tissue Application Guide... 2 I. Description... 2 II. Product Components... 2 III. Materials to be Supplied
GM and non GM supply chains: Their CO EXistence and TRAceability Outcomes of Co Extra Comparison of different real time PCR chemistries and their suitability for detection and quantification of genetically
DNA and Forensic Science Micah A. Luftig * Stephen Richey ** I. INTRODUCTION This paper represents a discussion of the fundamental principles of DNA technology as it applies to forensic testing. A brief
Deutsche Akkreditierungsstelle GmbH German Accreditation Body Annex to the Accreditation Certificate D-PL-13372-01-00 according to DIN EN ISO/IEC 17025:2005 Period of validity: 26.03.2012 to 25.03.2017
Quantitative Telomerase Detection Kit (QTD Kit) Catalog No. MT3010, MT3011, MT3012 For Research Use Only. Not for use in diagnostic procedures 1 Table of Contents 1. Introduction Background Product Overview
DNA Sequence Analysis Two general kinds of analysis Screen for one of a set of known sequences Determine the sequence even if it is novel Screening for a known sequence usually involves an oligonucleotide
Mitochondrial DNA Analysis Lineage Markers Lineage markers are passed down from generation to generation without changing Except for rare mutation events They can help determine the lineage (family tree)
BioScience quantitative real-time PCR, grain, simplex DNA extraction: PGS0426 RT-PCR: PGS0494 & PGS0476 This method describes a Real-time semi-quantitative TaqMan PCR procedure for the determination of
Definition of Minimum Performance Requirements for Analytical Methods of GMO Testing European Network of GMO Laboratories (ENGL) 13 October 2008 Date of application: 13 April 2009 INTRODUCTION The scope
Biotechnology Chapter 20: Biotechnology: DNA Technology & Genomics The BIG Questions How can we use our knowledge of DNA to: o Diagnose disease or defect? o Cure disease or defect? o Change/improve organisms?
Topic J10+11: Molecular-biological methods + Clinical virology I (hepatitis A, B & C, HIV) To study: PCR, ELISA, your own notes from serology reactions Task J10/1: DNA isolation of the etiological agent
Cat. # R010A For Research Use PrimeSTAR HS DNA Polymerase Product Manual Table of Contents I. Description...3 II. III. IV. Components...3 Storage...3 Features...3 V. General Composition of PCR Reaction
Validating Microarray Data Using RT 2 Real-Time PCR Products Introduction: Real-time PCR monitors the amount of amplicon as the reaction occurs. Usually, the amount of product is directly related to the
Real-time quantitative RT -PCR (Taqman) Author: SC, Patti Lab, 3/03 This is performed as a 2-step reaction: 1. cdna synthesis from DNase 1-treated total RNA 2. PCR 1. cdna synthesis (Advantage RT-for-PCR
An introduction to PCR and real-time PCR Dr. F. Waldherr CONGEN Biotechnologie GmbH DNA basic principles DNA carries the information of life on Earth DNA is present in all organisms (some viruses use RNA)
Lecture 13: DNA Technology DNA Sequencing Genetic Markers - RFLPs polymerase chain reaction (PCR) products of biotechnology DNA Sequencing determine order of nucleotides in a strand of DNA > bases = A,
Forensic Science International 167 (2007) 1 7 www.elsevier.com/locate/forsciint Species-specific oligonucleotides and multiplex PCR for forensic discrimination of two species of scallops, Placopecten magellanicus
HiPer RT-PCR Teaching Kit Product Code: HTBM024 Number of experiments that can be performed: 5 Duration of Experiment: Protocol: 4 hours Agarose Gel Electrophoresis: 45 minutes Storage Instructions: The
Revised Fall 2011 The Techniques of Molecular Biology: Forensic DNA Fingerprinting The techniques of molecular biology are used to manipulate the structure and function of molecules such as DNA and proteins
DNA Fingerprinting Unless they are identical twins, individuals have unique DNA DNA fingerprinting The name used for the unambiguous identifying technique that takes advantage of differences in DNA sequence
Cat. # RR420A For Research Use SYBR Premix Ex Taq (Tli RNaseH Plus) Product Manual Table of Contents I. Description... 3 II. Principle... 3 III. Components... 4 IV. Materials Required but not Provided...
IIID 14. Biotechnology in Fish Disease Diagnostics: Application of the Polymerase Chain Reaction (PCR) Background Infectious diseases caused by pathogenic organisms such as bacteria, viruses, protozoa,
Table of Contents General PCR Methods Page PCR Protocol Selection Guide...65.1 Basic PCR... 66.1.1 Hot Start PCR - The new Standard... 184.108.40.206 Reagents and Equipment Required... 220.127.116.11 General Considerations
Biological Sciences Initiative HHMI PCR Polymerase Chain Reaction PCR is an extremely powerful technique used to amplify any specific piece of DNA of interest. The DNA of interest is selectively amplified
Revision No.: ZJ0002 Issue Date: Aug 7 th, 2008 HBV Quantitative Real Time PCR Kit Cat. No.: HD-0002-01 For Use with LightCycler 1.0/LightCycler2.0/LightCycler480 (Roche) Real Time PCR Systems (Pls ignore
Genetics 10201232 Faculty of Agriculture and Veterinary Medicine Instructor: Dr. Jihad Abdallah Topic 15:Recombinant DNA Technology 1 Recombinant DNA Technology Recombinant DNA Technology is the use of
Product Bulletin Human Identification Quantifiler Human DNA Quantification Kit Quantifiler Y Human Male DNA Quantification Kit The Quantifiler kits produce reliable and reproducible results, helping to
Lectures 26 and 27 recombinant DNA technology I. oal of genetics A. historically - easy to isolate total DNA - difficult to isolate individual gene B. recombinant DNA technology C. why get the gene? 1.
Real-time PCR Kit for detection of mutation G1691A in Factor V gene using the Rotor-Gene Analyzer 3000/6000/Q (Corbett Research) Cat.# G0102 Institute of Applied Biotechnologies a.s. User manual Version
PRODUCT ULLETIN SYR Select Master Mix SYR Select Master Mix Highly specific and sensitive quantitation SYR Select Master Mix offers advanced performance at an affordable price. SYR Select Master Mix is
BIOTECHNOLOGY What can we do with DNA? Biotechnology Manipulation of biological organisms or their components for research and industrial purpose Usually manipulate DNA itself How to study individual gene?
PowerPoint Lecture Presentations prepared by Mindy Miller-Kittrell, North Carolina State University C H A P T E R 8 Recombinant DNA Technology The Role of Recombinant DNA Technology in Biotechnology Biotechnology
PrecisionPLUS TM PrecisionFAST TM Precision TM Mastermix Instructions for use of PrimerDesign Precision TM Mastermix/Precision Plus TM Precision TM FAST Mastermix Mastermix and MM MasterMix Contents Introduction
Super One-step RT-PCR Kit for Virus Detection For fast and sensitive one-step RT-PCR with highly specificity www.tiangen.com/en SD121221 Super One-step RT-PCR Kit Kit Contents Contents Cat. no. SD103 SD103
qstar mirna qpcr Detection System Table of Contents Table of Contents...1 Package Contents and Storage Conditions...2 For mirna cdna synthesis kit...2 For qstar mirna primer pairs...2 For qstar mirna qpcr
GENOTYPING ASSAYS AT ZIRC A. READ THIS FIRST - DISCLAIMER Dear ZIRC user, We now provide detailed genotyping protocols for a number of zebrafish lines distributed by ZIRC. These protocols were developed
A Modified Wizard SV Genomic DNA Purification System Protocol to Purify Genomic DNA... A Modified Wizard SV Genomic DNA Purification System Protocol to Purify Genomic DNA from Shed Reptile Skin ABSTRACT
Detection of Multiple Reporter Dyes in Real-time, On-line PCR Analysis with the LightCycler System Gregor Sagner, Cornelia Goldstein, and Rob van Miltenburg Roche Molecular Biochemicals, Penzberg, Germany
Use of the Agilent 2100 Bioanalyzer and the DNA LabChip in the Analysis of PCR Amplified Mitochondrial DNA Application Homeland Security/Forensics Author Mark Jensen Agilent Technologies, Inc. 2850 Centerville
Real time and Quantitative (RTAQ) PCR or.. for this audience so I have an outlier and I want to see if it really is changed Nigel Walker, Ph.D. Laboratory of Computational Biology and Risk Analysis, Environmental
Gene Mapping Techniques OBJECTIVES By the end of this session the student should be able to: Define genetic linkage and recombinant frequency State how genetic distance may be estimated State how restriction
PicoMaxx High Fidelity PCR System Instruction Manual Catalog #600420 (100 U), #600422 (500 U), and #600424 (1000 U) Revision C Research Use Only. Not for Use in Diagnostic Procedures. 600420-12 LIMITED
Chapter 20. Biotechnology: DNA Technology & Genomics 2003-2004 The BIG Questions How can we use our knowledge of DNA to: diagnose disease or defect? cure disease or defect? change/improve organisms? What
Action for Proposal for Implementation of Biotechnology Instruction at Fort Lauderdale High School Valerie Ruwe Fort Lauderdale High School Abstract As part of the Next Generation Strategic Plan, the Florida
Protocol Introduction to TaqMan and SYBR Green Chemistries for Real-Time PCR Copyright 2008, 2010 Applied Biosystems. All rights reserved. Ambion and Applied Biosystems products are for Research Use Only.
BacReady TM Multiplex PCR System Technical Manual No. 0191 Version 10112010 I Description.. 1 II Applications 2 III Key Features.. 2 IV Shipping and Storage. 2 V Simplified Procedures. 2 VI Detailed Experimental
Beginner s Guide to Real-Time PCR 02 Real-time PCR basic principles PCR or the Polymerase Chain Reaction has become the cornerstone of modern molecular biology the world over. Real-time PCR is an advanced
CHAPTER ONE: INTRODUCTION... 1 STUDY GOALS AND OBJECTIVES... 1 REASONS FOR DOING THIS STUDY... 1 SCOPE AND FORMAT... 1 METHODOLOGY AND INFORMATION SOURCES... 2 INTENDED AUDIENCE... 2 ANALYST CREDENTIALS...
Log In Sign Up Biotechnology Test Test 15 Matching Questions Regenerate Test 1. Plasmid 2. PCR Process 3. humulin 4. pluripotent 5. polymerase chain reaction (PCR) a b Is much smaller than the human genome,
POLYMERASE CHAIN REACTION (PCR) TECHNOLOGIES AND GLOBAL MARKETS BIO087C August 2016 Shalini Shahani Dewan Project Analyst ISBN: 1-62296-333-4 BCC Research 49 Walnut Park, Building 2 Wellesley, MA 02481
Techne qpcr test Human Herpes Virus 4 (Epstein Barr) nonglycosylated membrane protein (BNRF1) gene 150 tests For general laboratory and research use only 1 Introduction to Human Herpes Virus 4 (Epstein
Technical Note Dissociation Curve Analysis T08-003A General Introduction to Data Analysis The aim of this Technical Note is to explain the principle of dissociation curve analysis and to guide you through
Lecture 38: DNA Fingerprinting (DNA technology) The most awesome and powerful tool acquired by man since the splitting of atoms - The Time Magazine (USA) Conventional fingerprint of an individual comes
DNA analysis of the Metro Chinese Cemetery samples Ripan S. Malhi University of Illinois Urbana-Champaign Report on the Extraction of DNA from the Metro Chinese cemetery samples Summary The eight samples
Recombinant DNA technology (genetic engineering) involves combining genes from different sources into new cells that can express the genes. Recombinant DNA technology has had-and will havemany important
Development of two Novel DNA Analysis methods to Improve Workflow Efficiency for Challenging Forensic Samples Sudhir K. Sinha, Ph.D.*, Anne H. Montgomery, M.S., Gina Pineda, M.S., and Hiromi Brown, Ph.D.
ab185916 Hi-Fi cdna Synthesis Kit Instructions for Use For cdna synthesis from various RNA samples This product is for research use only and is not intended for diagnostic use. Version 1 Last Updated 1
Lit. # L0126 Rev. 8/99 Competitive PCR Guide Table of Contents I. What is Competitive PCR? A. Difficulties of Quantitative Analysis in Normal PCR... 2 B. Principle of Competitive PCR... 3 C. Competitive
TM Primerdesign Ltd TM Primerdesign Ltd Human Herpes Virus 1 (Herpes simplex type 1) Capsid assembly and DNA maturation gene genesig Standard Kit 150 tests DNA testing Everything... Everyone... Everywhere...
DNA CLONING DNA CLONING: amplification of unique DNA molecules In vitro-pcr In vivo-in different host cells POLYMERASE CHAIN REACTION (PCR) PCR THE POLYMERASE CHAIN REACTION (PCR) PROVIDES AN EXTREMELY
Stratagene QPCR Mouse Reference Total RNA Instruction Manual Catalog #750600 Revision C.0 For Research Use Only. Not for use in diagnostic procedures. 750600-12 LIMITED PRODUCT WARRANTY This warranty limits
Bio 100 DNA Technology 1 Chapter 12 - DNA Technology Among bacteria, there are 3 mechanisms for transferring genes from one cell to another cell: transformation, transduction, and conjugation 1. Transformation
Next Generation Polymerase Chain Reaction Developed by Nobel laureate Kary Mullis in the 1980s, Polymerase Chain Reaction (PCR) is a molecular technology that allows fast and in vitro. It has since become
qpcr Symposium 2007 - Weihenstephan Speed Matters - Fast ways from template to result March 28, 2007 Dr. Thorsten Traeger Senior Scientist, Research and Development - 1 - Overview Ạgenda Fast PCR The Challenges
GenScript BloodReady TM Multiplex PCR System Technical Manual No. 0174 Version 20040915 I Description.. 1 II Applications 2 III Key Features.. 2 IV Shipping and Storage. 2 V Simplified Procedures. 2 VI
RealStar HBV PCR Kit 1.0 11/2012 RealStar HBV PCR Kit 1.0 For research use only! (RUO) Product No.: 201003 96 rxns INS-201000-GB-02 Store at -25 C... -15 C November 2012 altona Diagnostics GmbH Mörkenstraße
Your consent to our cookies if you continue to use this website.