This is surely a contributions of Science to the analysis of our marine food at the micro-level.
I can recalled that in early 90's when I was saying about DNA chips inplanted into the Human brain who have suffer from brain defects, friends & audients were thinking that, this guy is crazy. Now, the Nano-Tech have been advance further to do more to save life.
Howver, based from the feedback in China, many people are suspect of having cancer due to consumming the DNA altered soy. Hence, I hope
scientist would not do any DNA alterations to the fish & shell fish.
Identification of fish and shellfish by protein and DNA analysis
Hartmut Rehbein
Research Department for Fish Quality, Federal Research Centre for Nutrition and Food, Palmaille 9, D-22767 Hamburg, Germany. Tel. 0049-40-38905-167. Fax 0049-40-38905-262. E-mail hartmut.rehbein@ibt.bfa-fisch.de
1. Introduction
The number of aquatic organisms consumed by humans is considerably underestimated by most people. At a rough estimate, world-wide, about 5000 species of fish, crustaceans and molluscs are finding their way to the dining table. Consumers are often not aware of the great variety of seafood available, especially in countries where fish consumption is low. Confusion also arises because it is not uncommon to use one name for several different fish species, some of which may differ in sensory qualities.
Mislabelling of fishery products occurs, especially when valued and popular species like sturgeon (Huso and Acipenser spp., delivering black caviar), blue-fin tuna (Thunnus thynnus) or even some stocks of cod (Gadus morhua) are heavily over-exploited and substitutes are sold. Consumers need to be protected against fraud, and fair trade has to be safeguarded. Equally, endangered species or populations must be protected from extermination.
Against this background, food control authorities need reliable, fast and cheap methods for authentication of all kinds of seafood. At the present time two different analytical techniques are mainly in use: protein electrophoresis and polymerase chain reaction (PCR)-based DNA-analysis.
2. Protein electrophoresis
The edible parts of most kinds of seafood consists of muscle tissue. This is mainly light muscle in the majority of cases.
If the origin of a raw fish fillet has to be determined, isoelectric focusing (IEF) of water-soluble muscle proteins is the method of choice. Firstly, proteins are extracted by homogenizing the light muscle of the fillet with water, this step is followed by centrifugation to precipitate non-dissolved material. Extracted proteins are applied to a polyacrylamide gel containing ampholytes. During electrophoresis a pH-gradient is established within the gel from the anode to the cathode, and the proteins take positions in the gel according to their pI-value. The pI is defined as the pH at which the net charge of the protein is zero. After being visualized by staining with Coomassie dye or silver, the bands of water-soluble muscle proteins are arranged in patterns, which are specific for a given fish species (Figure 1). Unknown samples are identified by running references side by side in the same gel. Recently, protein patterns of about one hundred fish species have been compiled in an electronic database available on the World Wide Web (http://vm.cfsan.fda.gov/~frf/rfe0.html) Known as the Regulatory Fish Encyclopedia (RFE), it contains gel images and tabulated pIs which can be used as references.
Before the application of PCR to fish species identification, several other methods of protein electrophoresis were in use for analysing various fish products containing denatured protein. Examples are sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), separating proteins according to their mass, or urea IEF. Both methods are applicable for the identification of cooked, fried or smoked products.
3. DNA analysis
In the course of fish processing their DNA is degraded from high molecular weight strands to short sections. However, even under severe heating conditions, for example, canned tuna, sequences of 100�200 base pairs in length are maintained allowing amplification by PCR.
Fish species identification by PCR comprises the following steps:
* solubilization of tissue by means of detergents and protease K
* extraction of DNA using organic chemicals like phenol/chloroform, or one of the numerous commercially available kits using specific binding of DNA to resins or membranes
* amplification of target DNA by PCR using species-specific, universal or randomly-binding primers
* detection and characterization of the PCR product.
Species-specific primers have the advantage that further characterization of the amplicon is not necessary, as the appearance of a band in a gel or the increase in fluorescence intensity in real-time PCR indicates the presence of fish DNA possessing specific primer binding sites. Specific PCR systems have been developed, for example, to identify black caviar produced from sturgeon roe, to differentiate between sole (Solea solea) and Greenland halibut (Reinhardtius hippoglossoides) and for discrimination of blue-fin tuna (Thunnus thynnus) and bonito (Sarda sarda).
In contrast to specific primers, so-called �universal� primers are selected from conservative regions of the nuclear or mitochondrial genome to react with a large number of different species. The cytochrome b gene is one of most popular targets for PCR with universal primers, as it has been sequenced for a large number of animals for subsequent phylogenetic studies.
PCR products are either sequenced, or analysed by less expensive techniques like RFLP- (restriction fragment length polymorphism) or SSCP- (single strand conformation polymorphism) analysis, to obtain the information needed to identify the sample.
The RFLP-method consists of cutting the generated amplicon by sequence-selective restriction endonucleases, and separation of the resulting DNA fragments according to their length using electrophoresis. Differences in sequences, i.e. in cutting sites for enzymes, between amplicons result in different patterns of DNA fragments. By selecting suitable targets and enzymes, species-specific patterns are obtained (Figure 2). At the present time RFLP is the method most commonly used for fish species identification.
In contrast to RFLP, SSCP, which is a very popular technique in medical mutation research, has not yet been applied very often for fish species identification. SSCP analysis comprises the following steps:
* after PCR is complete, the assay mix is diluted with weak alkaline solution and formamide
* the mixture is heated to 95�C, and then rapidly cooled in iced-water (by this treatment double stranded DNA dissociates into single strands). The single-stranded DNA folds into conformations according to sequence.
* Finally, the single-stranded DNA is analysed by native PAGE�under conditions of temperature, gel matrix and buffer composition suitable for separation of different conformers. In this way, differences in amplicon sequences are translated into specific patterns of single-stranded DNA bands.
SSCP gives very good results for short DNA strands (100-500 bases in length), which are prevalent in fishery products. For identification purposes unknown samples and references have to be run on the same gel (Figure 3).
PCR with randomly-binding primers has been used rarely for fish species identification. Random amplified polymorphic DNA (RAPD) analysis uses short primers (10-20 bases), which anneal to DNA at low temperatures at sites which have not been pre-selected. Under these PCR conditions, fingerprints of DNA bands are obtained after electrophoretic separation of PCR products. In several studies these fingerprints were found to be species specific, but the technique is difficult to standardize. Results are strongly dependent on the quality of DNA, as well as conditions of PCR and gel electrophoresis.
4. Future prospects
A number of PCR-based methods for authentication of all kinds of fish and other seafood have been developed, but only RFLP analysis has been validated for use as an official method. As international trade of fishery products is increasing continuously, a universal database for identification of all commercially important fish and shellfish species should be developed. The database should comprise DNA sequences, recommendations for PCR primers, restriction endonucleases, and information about the potential of SSCP analysis and other methods.
In the future DNA micro assays may be developed for fish species identification. However, the costs of development of DNA chips are rather high, and the market for food control analysis is relatively small. Read More....
Food Science Central from IFIS Publishing
I can recalled that in early 90's when I was saying about DNA chips inplanted into the Human brain who have suffer from brain defects, friends & audients were thinking that, this guy is crazy. Now, the Nano-Tech have been advance further to do more to save life.
Howver, based from the feedback in China, many people are suspect of having cancer due to consumming the DNA altered soy. Hence, I hope
scientist would not do any DNA alterations to the fish & shell fish.
Identification of fish and shellfish by protein and DNA analysis
Hartmut Rehbein
Research Department for Fish Quality, Federal Research Centre for Nutrition and Food, Palmaille 9, D-22767 Hamburg, Germany. Tel. 0049-40-38905-167. Fax 0049-40-38905-262. E-mail hartmut.rehbein@ibt.bfa-fisch.de
1. Introduction
The number of aquatic organisms consumed by humans is considerably underestimated by most people. At a rough estimate, world-wide, about 5000 species of fish, crustaceans and molluscs are finding their way to the dining table. Consumers are often not aware of the great variety of seafood available, especially in countries where fish consumption is low. Confusion also arises because it is not uncommon to use one name for several different fish species, some of which may differ in sensory qualities.
Mislabelling of fishery products occurs, especially when valued and popular species like sturgeon (Huso and Acipenser spp., delivering black caviar), blue-fin tuna (Thunnus thynnus) or even some stocks of cod (Gadus morhua) are heavily over-exploited and substitutes are sold. Consumers need to be protected against fraud, and fair trade has to be safeguarded. Equally, endangered species or populations must be protected from extermination.
Against this background, food control authorities need reliable, fast and cheap methods for authentication of all kinds of seafood. At the present time two different analytical techniques are mainly in use: protein electrophoresis and polymerase chain reaction (PCR)-based DNA-analysis.
2. Protein electrophoresis
The edible parts of most kinds of seafood consists of muscle tissue. This is mainly light muscle in the majority of cases.
If the origin of a raw fish fillet has to be determined, isoelectric focusing (IEF) of water-soluble muscle proteins is the method of choice. Firstly, proteins are extracted by homogenizing the light muscle of the fillet with water, this step is followed by centrifugation to precipitate non-dissolved material. Extracted proteins are applied to a polyacrylamide gel containing ampholytes. During electrophoresis a pH-gradient is established within the gel from the anode to the cathode, and the proteins take positions in the gel according to their pI-value. The pI is defined as the pH at which the net charge of the protein is zero. After being visualized by staining with Coomassie dye or silver, the bands of water-soluble muscle proteins are arranged in patterns, which are specific for a given fish species (Figure 1). Unknown samples are identified by running references side by side in the same gel. Recently, protein patterns of about one hundred fish species have been compiled in an electronic database available on the World Wide Web (http://vm.cfsan.fda.gov/~frf/rfe0.html) Known as the Regulatory Fish Encyclopedia (RFE), it contains gel images and tabulated pIs which can be used as references.
Before the application of PCR to fish species identification, several other methods of protein electrophoresis were in use for analysing various fish products containing denatured protein. Examples are sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), separating proteins according to their mass, or urea IEF. Both methods are applicable for the identification of cooked, fried or smoked products.
3. DNA analysis
In the course of fish processing their DNA is degraded from high molecular weight strands to short sections. However, even under severe heating conditions, for example, canned tuna, sequences of 100�200 base pairs in length are maintained allowing amplification by PCR.
Fish species identification by PCR comprises the following steps:
* solubilization of tissue by means of detergents and protease K
* extraction of DNA using organic chemicals like phenol/chloroform, or one of the numerous commercially available kits using specific binding of DNA to resins or membranes
* amplification of target DNA by PCR using species-specific, universal or randomly-binding primers
* detection and characterization of the PCR product.
Species-specific primers have the advantage that further characterization of the amplicon is not necessary, as the appearance of a band in a gel or the increase in fluorescence intensity in real-time PCR indicates the presence of fish DNA possessing specific primer binding sites. Specific PCR systems have been developed, for example, to identify black caviar produced from sturgeon roe, to differentiate between sole (Solea solea) and Greenland halibut (Reinhardtius hippoglossoides) and for discrimination of blue-fin tuna (Thunnus thynnus) and bonito (Sarda sarda).
In contrast to specific primers, so-called �universal� primers are selected from conservative regions of the nuclear or mitochondrial genome to react with a large number of different species. The cytochrome b gene is one of most popular targets for PCR with universal primers, as it has been sequenced for a large number of animals for subsequent phylogenetic studies.
PCR products are either sequenced, or analysed by less expensive techniques like RFLP- (restriction fragment length polymorphism) or SSCP- (single strand conformation polymorphism) analysis, to obtain the information needed to identify the sample.
The RFLP-method consists of cutting the generated amplicon by sequence-selective restriction endonucleases, and separation of the resulting DNA fragments according to their length using electrophoresis. Differences in sequences, i.e. in cutting sites for enzymes, between amplicons result in different patterns of DNA fragments. By selecting suitable targets and enzymes, species-specific patterns are obtained (Figure 2). At the present time RFLP is the method most commonly used for fish species identification.
In contrast to RFLP, SSCP, which is a very popular technique in medical mutation research, has not yet been applied very often for fish species identification. SSCP analysis comprises the following steps:
* after PCR is complete, the assay mix is diluted with weak alkaline solution and formamide
* the mixture is heated to 95�C, and then rapidly cooled in iced-water (by this treatment double stranded DNA dissociates into single strands). The single-stranded DNA folds into conformations according to sequence.
* Finally, the single-stranded DNA is analysed by native PAGE�under conditions of temperature, gel matrix and buffer composition suitable for separation of different conformers. In this way, differences in amplicon sequences are translated into specific patterns of single-stranded DNA bands.
SSCP gives very good results for short DNA strands (100-500 bases in length), which are prevalent in fishery products. For identification purposes unknown samples and references have to be run on the same gel (Figure 3).
PCR with randomly-binding primers has been used rarely for fish species identification. Random amplified polymorphic DNA (RAPD) analysis uses short primers (10-20 bases), which anneal to DNA at low temperatures at sites which have not been pre-selected. Under these PCR conditions, fingerprints of DNA bands are obtained after electrophoretic separation of PCR products. In several studies these fingerprints were found to be species specific, but the technique is difficult to standardize. Results are strongly dependent on the quality of DNA, as well as conditions of PCR and gel electrophoresis.
4. Future prospects
A number of PCR-based methods for authentication of all kinds of fish and other seafood have been developed, but only RFLP analysis has been validated for use as an official method. As international trade of fishery products is increasing continuously, a universal database for identification of all commercially important fish and shellfish species should be developed. The database should comprise DNA sequences, recommendations for PCR primers, restriction endonucleases, and information about the potential of SSCP analysis and other methods.
In the future DNA micro assays may be developed for fish species identification. However, the costs of development of DNA chips are rather high, and the market for food control analysis is relatively small. Read More....
Food Science Central from IFIS Publishing
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