SR13_003E
S h i m a d z u R e v i e w
by Yuji SOGABE 1
Recently, methods to identify the actual composition of various foods have been developed because of progress in gene-analysis technology.
An electrophoretic pattern required for this type of analysis can be obtained by using MCE-202 MultiNA Microchip Electrophoresis System.
Examples of analysis data obtained using MultiNA to identify species of meat, tuna, and rice are shown.
Keywords : DNA, Identifying the species, Qualitative PCR, Microchip electrophoresis
In recent years, there have been many news reports in the mass media about imported foods, functional foods, foods containing recombinant products, increased incidence of food allergies, and frequent deceptive representation of food products. The consumers are increasingly demanding added value in food products, such as nutritional benefits, safety, branding, high quality, etc. At the same time, because of worsening of the economy, there is also an increased demand for cheaper food materials and products. Proper labeling of food products has been specified in the JAS Law (Act on
Standardization and Proper Quality Labeling of Agricultural and
Forestry Products), Food Sanitation Act, Act against Unjustifiable
Premiums and Misleading Representations, Measurement Act, and the
Health Promotion Act1), 2). Among these, the details of the JAS Law have been revised to specify a quality labeling standards system. The
Food Sanitation Act, on the other hand, mandates that the animal species of origin has to be specified for meat, as also the kinds of meat used as raw material for processed meat products. The act also mandates accurate labeling of the name of the food product, the place of its production, etc. Thus, the manufacturers and distributors have to accurately convey to the consumers the information that the latter can use for choosing a commercial product.
Among cases of deceptive labeling, those related to the source organism can have wide-ranging negative impact not just on the consumers but also on distributors, manufacturers, and growers, and even on the rights of breeders who developed the varieties. In the case of processed or cooked foods and their raw materials, it is difficult to ascertain the species of origin from the external features alone. Moreover, deceptive labeling of food items only rarely leads to immediate and obvious health problems like food poisoning, allergic reactions, etc. Therefore, even if there is a mix-up, mislabeling or deceptive representation in the course of distribution of a food product, considerable time often elapses before it is detected. A rapid, handy and accurate analytical method based on sound scientific principles is required for setting up a monitoring system for prevention and control of deceptive representation of food products. Various analytical methods that utilize scientific advances of recent years have been developed. I shall introduce here examples of using the microchip electrophoresis system MCE-202 MultiNA3) for gene analysis undertaken to identify the source organisms of food products.
Immunological methods (ELISA, etc.) that use proteins specific to certain species as markers, and physicochemical methods (high performance liquid chromatography, etc.) that exploit the differences in the protein and other components present in different meats have been in use for identification of the source organisms of food products4). These methods can identify the animal species with a certain level of certainty, but their drawbacks include the cumbersome procedures required and difficulties in use with heat-processed foods and foods containing materials from more than one animal species.
Thus, there is a need to develop rapid, handy and highly accurate analytical methods.
Various analytical methods have been developed along with the recent advances in gene analysis technology. One such technique, which has now become a mainstay, is qualitative analysis based on polymerase chain reaction (PCR). In the field of qualitative PCR, many methods have been developed utilizing the polymorphism of the cytochrome gene region of mitochondrial DNA (mtDNA). mtDNA is widely used to identify the type of organism as it is more abundant than nuclear DNA and the cytochrome gene in mtDNA has genetic characteristics specific to the organism. The analytical procedure for identifying the organism through qualitative PCR is shown in Fig. 1. In general it follows the steps of extraction of the DNA from samples, PCR, and electrophoresis.
In some of the methods, a more detailed analysis is made possible by inserting a step of treatment with restriction enzymes, etc. after the
PCR and then analyzing the separation patterns. DNA extraction from the sample is the most labor-intensive among these steps. However,
DNA extraction kits optimized for certain species (of animals, plants, etc.) are now available, which makes this step relatively easy. Primer sets used in the PCR for certain methods of identification are also available commercially as kits. Thus, the methods can be relatively easily employed if the equipment is available.
1-1/Received June 13, 2012
1 Global Application Development Center, Analytical & Measuring Instruments
Division, Shimadzu Corporation, Kyoto, Japan

Analysis of the
PCR product
(MultiNA)
Treatment with restriction enzymes
Analysis of fragments produced
(MultiNA)
Analysis of electrophoresis patterns
Fig. 1 Experiment procedure of identification of organisms
using qualitative PCR method
With meat products packaged as fresh single meats, we can identify the source species in many cases from the color and texture of the meat. However, if it is minced meat, and further if meats of more than one species are mixed, it becomes very difficult to determine the identity from external features. Immunological methods like ELISA and methods of soluble protein analysis have been conventionally used for testing meat. As the external clues are very much suppressed in processed or cooked meats, it becomes almost impossible to identify the type of meat. The aforesaid conventional methods, however, have the drawbacks of requiring cumbersome pretreatment of samples and not being applicable to heat-treated processed foods and processed foods containing more than one type of meat. Matsunaga et al.
1) had developed a method of identifying animal species of meats through qualitative PCR that targeted the cytochrome b gene of mtDNA and analysis of the amplified product by electrophoresis.
I shall introduce here an example where 5 kinds of single meats, namely, chicken, beef, lamb, pork and horsemeat, were analyzed by the above method. DNA was extracted from each sample and used as template for PCR. The primers used by Matsunaga et al. were partly modified and used for the PCR. The PCR products were analyzed by the MCE-202 MultiNA system. The results are shown in Fig. 2. PCR products of size 218, 268, 331, 359 and 430 bp are amplified specifically in chicken, beef, lamb, pork and horsemeat respectively.
The analysis results shown here demonstrate the presence of the amplification products of the respective sizes. I then prepared mixtures of 3 types of meat, i.e., chicken, beef and pork, at different ratios for analysis. 1 to 20% each of two other meats was mixed with the main component of each mixture. These samples were processed and analyzed using MCE-202 MultiNA in the same way as the single meat samples. The results (Fig. 3) showed that even the presence of as little as 1% meat of another species could be clearly detected.
Fig. 2 Analytical results of PCR products from five kinds of meat respectively with MultiNA

Sample ID
Pork (%)
Beef (%)
Chicken (%)
C1
0
100
0
C2
1
98
1
C3
5
90
5
C4
10
80
10
C5
20
60
20
C6
100
0
0
C7
98
1
1
C8
90
5
5
C9
80
10
10
C10
60
20
20
C11
0
0
100
C12
1
1
98
D1
5
5
90
D2
10
10
80
D3
20
20
60
Pork
Beef
Chicken
Fig. 3 Analytical results of PCR products from three kinds of meat mixture with MultiNA
Tuna fish, which Japanese are very fond of, is mostly sold as fresh cut pieces or in a cooked form, and it is very rare that a whole fish would be bought by an individual consumer. Under such conditions it is almost impossible to identify the tuna species from the external features of the product. With bluefin tuna, for example, some of it is branded according to the site of capture and sold at very high prices.
Because of this, mix-up, mislabeling, deceptive representation, etc. actually occurs during the distribution of the product, which has become a social problem. Tuna is mostly traded in the uncooked form and the distribution is very rapid, it requires a rapid, handy and accurate technique of species identification. The Food and Agricultural
Materials Inspection Center and the National Research Institute of
Fisheries Science of the Fisheries Research Agency have jointly brought out a manual on a method of identifying tuna species using
Polymerase Chain Reaction-Restriction Fragment Length Polymorphism
(PCR-RFLP) 5) . In this method, DNA is first extracted from the tuna sample. PCR is then carried out using the extracted DNA as the template, targeting gene sequences specific to each tuna species, present in their mtDNA. The PCR-amplified DNA is then digested using
3 types of restriction enzymes, namely, Alu I, Mse I and Tsp 509 I. The
DNA fragments generated by these enzymes yield cleavage patterns when separated by electrophoresis. The tuna species is then identified by analyzing these patterns.
I obtained cleavage patterns of the meat of the 6 tuna species,
Atlantic bluefin tuna ( Thunnus thynnus ), southern bluefin tuna ( T. maccoyii ),
α
and
β
genotypes of bigeye tuna ( T. obesus ), yellowfin tuna ( T. albacares ) and albacore tuna ( T. alalunga ) using the above method and MultiNA. The results are given in Fig. 4. Atlantic bluefin tuna, bigeye tuna β type and albacore tuna were identified here as they showed distinctive cleavage patterns after treatment with the restriction enzyme Alu I (marked with stars in “ Alu I treated” of Fig.
4). Southern bluefin tuna, bigeye tuna
α
type and yellowfin tuna could not be differentiated as they showed identical cleavage patterns.
Therefore, I next used the restriction enzyme Mse I to obtain the cleavage patterns. Southern bluefin tuna could be identified as it showed a distinctive pattern with Mse I (marked with a star in “ Mse I treated” of Fig. 4). The use of the restriction enzyme Tsp 509 I enabled identification of the remaining bigeye tuna
α
type and yellowfin tuna from the cleavage patterns (marked with stars in “ Tsp 509 I treated” of
Fig. 4).

Alu I treated Mse I treated
Fig. 4 Analytical results of PCR-RFLP products from Thunnus
Tsp 509 I treated
Fig. 5 Analytical results of PCR products of rice DNA

Estimates of the number of rice varieties available in Japan vary from approximately 300 to 500. The JAS Law mandates labeling with variety name and region of production of the rice. Even the same rice variety can have different brand names depending on the region where it has been grown. Popular rice brands are evaluated as being very tasty and are therefore highly priced. In recent years, with increased branding of rice, there has been widespread disguising of the area of production and willful mixing of different types of rice. As the rice goes through the processes of threshing, husking and polishing, it becomes progressively more difficult to identify the variety from its appearance. There are methods of identifying rice varieties from their taste, but it is difficult for ordinary consumers to use such methods. Various analytical techniques have been therefore developed for scientific verification of the information declared on labels. These include the qualitative PCR method that analyzes the loci of genes specific to different varieties, the Random Amplified Polymorphic DNA
(RAPD) method that uses relatively short random primers, the
Sequence Tagged Sites (STS) method wherein primers for STS selected by RAPD analysis and sequenced are used, the Simple Sequence
Repeat (SSR) or microsatellite method which exploits the difference in amplified fragment lengths of microsatellites, and the Single
Nucleotide Polymorphisms (SNPs) method which detects a change of one base pair that defines the variety. Kits that can perform the steps from DNA extraction to PCR for these methods are now commercially available.
I shall introduce an example of identifying a rice variety using the STS method6). DNA was extracted from 20 grains of each of 10 different rice samples. PCR was performed using a kit that included STS primers. The results of electrophoresis of the PCR amplified product using the MultiNA system are shown in Fig. 5. This analytical kit can be used for multiplex PCR with 4 different sets of STS primers. After that, the amplified product is analyzed by electrophoresis to obtain the separation pattern. In Fig. 5, the right most lane represents the DNA marker solution provided with the PCR kit. It contains 4 types of DNA fragments, of size about 650, 770, 870 and 1600 bp. The rice samples were identified by analyzing the patterns for bands of the same fragment size as present in the DNA marker solution. Variety
Koshihikari should show 3 of the PCR amplified products
(approximately 650, 770 and 870 bp) but not the approximately 1600 bp band. The samples other than of Koshihikari should show amplification product separation patterns different from that of
Koshihikari. Among the 10 samples analyzed here, 5 showed the
3-band pattern characteristic of Koshihikari, and thus were identified as Koshihikari. The remaining 5 showed patterns different from the
Koshihikari pattern and were identified as non-Koshihikari varieties.
I have introduced here the use of gene analysis technology for species/variety identification of food products, with examples of analysis of meat, tuna and rice, and shown that the microchip electrophoresis system MCE-202 MultiNA can be used for such analyses. More systematic analytical methods should be developed in the future for rapid and accurate processing of large amounts of samples through handy procedures. Automation of the analytical procedures is one aspect of such labor-saving efforts, and it is expected that MultiNA would be an analytical system that could play a role in this endeavor.
References
1) Appended "Guideline of labeling under Food Sanitation Act" (Re:
Notification by the Director of the Environmental Health Bureau of the Ministry of Welfare on Nov. 8, 1979 "About Labeling under
Food Sanitation Act" )
2) JAS analytical test handbook: genetically modified food quality, labeling analysis manual for individual products, the revised edition,
The Food and Agricultural Materials Inspection Center, Japan (2002)
3) K. Suzuki, A. Arai, S. Utsunomiya et al., Shimadzu Review, 64, 117
(2008)
4) T. Matsunaga, K. Shibata, J. Yamada, Y. Shinmura, "Effects of
Processing Conditions on Species Identification of Meat Products" ,
NIPPON SHOKUHIN KAGAKU KOGAKU KAISHI, 46, 187(1999)
5) "Tuna Species Identification Manual" , Food and Agricultural
Materials Inspection Center / Fisheries Research Agency, Apr. 27,
2005
6) K. Ohtsubo, S. Nakamura, T. Imamura, "Development of the primer sets for identification of a rice cultivar, Koshihikari, by PCR" ,
Nippon Nogeikagaku Kaishi, 76, 4, 388(2002)