Download 1. Introduction 2. Analytical methods of identifying source species of

SR13_003E
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DNA Analytical Technology to Identify the Actual Composition of Foods
- Application of the “MultiNA” Microchip Electrophoresis System by Yuji SOGABE1
Abstract
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
1. Introduction
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.
1-1/Received June 13, 2012
1
Global Application Development Center, Analytical & Measuring Instruments
Division, Shimadzu Corporation, Kyoto, Japan
2. Analytical methods of identifying source
species 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.
3. Examples of analysis for identifying the
source species of meats
PCR product
Treatment with
restriction enzymes
Analysis of the
PCR product
(MultiNA)
Analysis of
fragments produced
(MultiNA)
Analysis of electrophoresis patterns
Identification of organism
Fig. 1 Experiment procedure of identification of organisms
Horsemeat
Pork
Lamp
Beef
Chicken
using qualitative PCR method
Horsemeat
PCR
Lamp
Pork
Purified DNA
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.
Beef
DNA extraction
Chicken
Sample
Relative migration time (%)
Fig. 2 Analytical results of PCR products from five kinds of meat respectively with MultiNA
Sample ID
C1
C2
C3
C4
C5
C6
C7
C8
C9
C10
C11
C12
D1
D2
D3
Pork (%)
0
1
5
10
20
100
98
90
80
60
0
1
5
10
20
Beef (%)
100
98
90
80
60
0
1
5
10
20
0
1
5
10
20
0
1
5
10
20
0
1
5
10
20
100
98
90
80
60
Chicken (%)
Pork
Beef
Chicken
Fig. 3 Analytical results of PCR products from three kinds of meat mixture with MultiNA
4. Example of analysis for identifying tuna
species
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 Tsp509 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 Tsp509 I enabled
identification of the remaining bigeye tuna α type and yellowfin tuna
from the cleavage patterns (marked with stars in “Tsp509 I treated” of
Fig. 4).
Yellowfin tuna
Bigeye tuna α type
Ladder
Yellowfin tuna
Bigeye tuna α type
Southern bluefin tuna
Ladder
Albacore tuna
Yellowfin tuna
Bigeye tuna β type
Bigeye tuna α type
Southern bluefin tuna
Atlantic bluefin tuna
Ladder
Koshihikari
Another variety
Koshihikari
Another variety
Koshihikari
Koshihikari
Koshihikari
Another variety
Another variety
DNA Marker
Solution
Another variety
Fig. 5 Analytical results of PCR products of rice DNA
PC
NC
Ladder
Tsp509 I treated
Mse I treated
Alu I treated
Fig. 4 Analytical results of PCR-RFLP products from Thunnus
Sample
Koshihikari
(3 bands)
5. Example of analysis for identifying rice
varieties
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.
6. Conclusion
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)