JP5388049B2 - Specific detection method for flying fish - Google Patents

Description

This invention is specifically and rapidly detect and primer set for identifying DN A comprising a gene encoding the mitochondrial 16S ribosomal RNA of flying fish such method for detecting the flying fish include, and kit for detecting flying fish such.

  Foods provide information to consumers by displaying the names of raw materials, the origin of raw materials, and the expiration date. The displayed content is reflected in the price, but the authenticity of the displayed content was basically based on the trust relationship between the producer or manufacturer and the consumer. However, since the number of fraud cases related to food labeling has increased since the mid-1990s, establishment of a method for verifying display contents from various angles is desired.

In the past, fish contained in fish or processed foods were identified using morphological techniques when there was an original form, or electrophoresis of proteins and isozymes when there was no original form. In many cases, identification is difficult if the protein is similar or if the protein is significantly denatured by heating or the like. On the other hand, due to recent developments in genetic engineering, highly sensitive DNA analysis techniques have attracted attention and are beginning to be used for the identification of various samples. According to the DNA analysis technique, it can be identified regardless of the presence or absence of the form, even if the state of the sample is bad. Tuna (Non-patent Document 1), Suzuki (Non-patent Document 2), Eel (Patent Document 1) Application examples are found in mackerel (Non-patent Document 3). However, until now, there are no literatures that have been tested for DNA fish analysis. There was also no technology that could detect multiple species of flying fish as a group.
J. et al. L. Ram, M .; L. Ram, F .; F. Baidon. J. et al. Agric. Food. Chem, 44, 1996, 2460-2467. Tomoyuki Tsuji et al., Shokuhin Magazine, 51, 2004, 471-476 Japanese Patent No. 3950546 K. Sezaki, Y. et al. Kuboshima, I .; Mitani, A.M. Fukui, S .; Watebe. Nippon Suisan Gakashi, 67, 2001, 17-22 Nagase M, Aimi T, Suginaka K, Kitamoto Y, Morinaga T .; Fishers Science, 71, 2005, 914-923

The present invention provides a primer set, a method for detecting flying squirrel, and a kit for detecting flying squirrel for specific and rapid detection and identification of DNA containing a gene encoding mitochondrial 16S ribosomal RNA of flying squirrel This is the issue.

As a result of intensive studies to solve the above-mentioned problems, the present inventors have isolated DNA containing a gene encoding the mitochondrial 16S ribosomal RNA of flying squirrel, and further, quickly and rapidly improving the flying squirrel contained in the subject. The present inventors have succeeded in producing a primer that can be detected with sensitivity, and have completed the present invention .

The present invention includes a 5'-side primer capable of at least a portion hybridizes's of any one of the nucleotide sequences represented by SEQ ID NO: 6 is the nucleotide sequence of the gene encoding the mitochondrial 16S ribosomal RNA of flying fish such , Primer sets A, B, and C consisting of a combination with a 3 ′ primer that can hybridize with at least a part of any one of the nucleotide sequences represented by SEQ ID NOs: 1 to 6. Primer set A includes the 5′-side primer represented by SEQ ID NO: 7 and the 3′-side primer represented by SEQ ID NO: 8. Primer set B includes the 5′-side primer represented by SEQ ID NO: 9, and the 3′-side primer represented by SEQ ID NO: 10. Primer set C includes the 5′-side primer represented by SEQ ID NO: 11 and the 3′-side primer represented by SEQ ID NO: 12.

Furthermore, the present invention is characterized by amplifying a DNA containing a gene encoding a mitochondrial 16S ribosomal RNA of a flying squirrel, using any one of the primer sets described above, for a subject suspected to contain flying squirrels. This is a method for detecting flying fish. Furthermore, the present invention is a kit for detecting flying fish, comprising any of the above primer sets.

The present invention, a method for detecting the flying fish such for the subject suspected of containing specific and rapid detection and identification for primers for flying fish acids, and flying fish such is provided.

Hereinafter, the present invention will be described in detail.
The gene of the present invention contains DNA encoding mitochondrial 16S ribosomal RNA of flying fish, and can be obtained as follows.

1. 1. Isolation of mitochondrial 16S ribosomal RNA gene and its adjacent region (1) Preparation of genomic DNA As sources of genomic DNA, there are Cyperusurus hetarius, Cyspelurus heterurus, Cypselurus ausu, and Cyprus urp. ), Cypselurus poeciloterus, Cypselurus cyanopterus, Cypselurus antoncichi , and other flying fish.

  For example, genomic DNA can be extracted and purified from the fish muscle by a generally known method such as a method using a surfactant or a method of performing a heat treatment. It is also possible to use a commercially available DNA extraction / purification kit (such as DNeasy Tissue Kit from QIAGEN). The target of collection of genomic DNA is not particularly limited, and it is extracted and prepared from any growth stage, low-order processed products such as dried fish, and high-order processed products mixed with other ingredients such as kamaboko. can do.

(2) Synthesis of universal primer In general, eukaryotic mitochondrial DNA containing flying fish is known to have a structure as shown in FIG. The inventor has clarified the entire nucleotide sequence of mitochondrial DNA for the flying fish (Cypselurus hiraii, AB182653) ( Non-patent Document 4) . The mitochondrial 16S ribosomal RNA is encoded by comparing this with the nucleotide sequences of the walleye pollock (Theragra charcograma, AB182300), maji (Trachurus japonicus, AP003091), and water flies (Exocoetus volitans, AP002933), which have been similarly revealed. A part of the region was universally amplified, and primer set B having specific restriction enzyme reaction sites, namely SEQ ID NO: 9 and SEQ ID NO: 10, was invented. Using this, the genomic DNA obtained in the above (1) as a template, intends row polymerase chain reaction (also referred to as PCR).

(3) Determination of base sequence After subcloning the obtained PCR fragment into an appropriate vector, the base sequence is determined. Alternatively, direct sequencing is performed using PCR fragments. The base sequence is determined using an automatic base sequencer (for example, 3100 DNA sequencer manufactured by ABI). Once the base sequence of the gene of the present invention is determined, then by chemical synthesis, by PCR using the genomic DNA of this gene as a template, or by hybridizing with a DNA fragment having the base sequence as a probe, DNA containing the gene for mitochondrial 16S ribosomal RNA of flying fish can be obtained.

(4) Synthesis of flying fish detection primer SEQ ID NO: 1 to SEQ ID NO: 6 exemplify the nucleotide sequence of flying fish mitochondrial 16S ribosomal RNA gene. Then, these nucleotide sequences can be compared with the already-identified flying fish, a sequence characteristic of flying fish can be found, and a synthetic oligonucleotide having the sequence can be used as a primer for detecting flying fish. In addition, these base sequences are compared with walleye pollock (Theragra charcograma, AB182300), maji (Trachurus japonicus, AP003091), damselfish (Exocoetus volitans, AP002933), etc. Synthetic oligonucleotides can be used as detection reaction confirmation primers.

For example, the 5′-side primer and 3′-side primer that can be used for flying fish detection can be selected from the region of the base sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2. However, it does not include the area a lot of GC content, the region that does not hybridize with the primer What happened is preferable.

  For example, as the 5′-side primer, in SEQ ID NO: 1 or SEQ ID NO: 2, no difference is seen between species, such as the 171st to 197th and the 198th to 235th in which differences from other fish species are seen, A region consisting of at least 10-50 bases, more preferably 15-25 bases is preferred. Further, as the 3 ′ primer, at least 10 to 50 bases, such as the 278th to 319th and the 308th to 358th positions, in which the difference between the species is similarly not seen and the difference from other fish species is seen, more preferably Is preferably a region consisting of 15 to 25 bases. These primers can be used in any arbitrary combination.

For example, a 5′-side primer and a 3′-side primer that can be used as a positive control for confirming a detection reaction can be selected from the region of the base sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2. However, it does not include the area a lot of GC content, the region that does not hybridize with the primer What happened is preferable.

  For example, as the 5′-side primer, the ninth to 25th, the 137th to 156th, the 374th to the 402th, etc. in which no difference from other fish species is seen in SEQ ID NO: 1 or SEQ ID NO: 2, etc. A region consisting of at least 10 to 50 bases, more preferably 15 to 25 bases is preferred. The 3 ′ primer is preferably a region consisting of at least 10 to 50 bases, more preferably 15 to 25 bases, from the 427th to 480th positions where no difference from other fish species is observed. These primers can be used in any arbitrary combination.

Examples of the base sequence of the primer for detecting flying fish of the present invention are SEQ ID NO: 7 and SEQ ID NO: 8 (primer set A) , and SEQ ID NO: 9 and SEQ ID NO: 10 (primer set B). Moreover, the base sequence of the primer for detection reaction confirmation of this invention is illustrated to sequence number 11 and sequence number 12 (primer set C) . However , the oligonucleotide of the present invention also includes an oligonucleotide that can hybridize with at least a part of a gene containing DNA comprising any one of the nucleotide sequences represented by SEQ ID NOs: 1 to 6. Moreover, the primer used in this invention is not limited to these.

2. Detection of flying fish using the primer of the present invention (1) Preparation of analyte sample solution Preparation of analyte sample solution that seems to contain flying fish is fish, blood, viscera, scales, salmon, sashimi, dried fish, smoked Then, genomic DNA is extracted and purified from a boiled, dashi, dashi, fish egg, soy sauce with dashi, kamaboko, etc. by a generally known method such as a method using a surfactant or a heat treatment method to obtain a sample solution.

(2) Detection of flying fish by PCR The mitochondrial 16S ribosomal RNA gene is amplified using the flying fish detection primer having a sequence specific to flying fish as described in 1. and the amplified fragment is fractionated by agarose gel electrophoresis, polyacrimido gel electrophoresis, etc. The gel can be stained with ethidium bromide or the like, and the presence or absence of the amplified fragment can be confirmed under a UV lamp.

  Amplification can be performed by PCR according to a standard protocol, and a target DNA fragment can be specifically amplified from a small amount of sample. That is, the DNA sample is denatured into a single strand. Next, a 5 ′ primer complementary to the 5 ′ side of one strand of the target DNA sequence, a 3 ′ primer complementary to the 3 ′ side of the other strand, a DNA sequence used as a template, and four types of deoxy Using a reaction system containing nucleoside triphosphates (dATP, dCTP, dGTP, and dTTP or dUTP) and a DNA polymerase, the template DNA and the above two primers are annealed. A complementary strand is then synthesized on each template from DNA polymerase and four deoxynucleoside triphosphates. After the resulting double strand is made into a single strand by thermal denaturation, the same annealing and complementary strand synthesis procedures are repeated using each strand as a template, so that only the portion sandwiched between the primers is exponential. Can be increased. As the DNA polymerase used in the PCR method or the like, it is preferable to use a polymerase that is heat resistant at the DNA cleavage temperature, such as Taq polymerase.

  In the present invention, the DNA fragment to be amplified is a DNA fragment containing a mitochondrial 16S ribosomal RNA gene possessed by flying fish. In the PCR method or the like, the DNA sequence of the portion sandwiched between the primers is amplified, so the size of the DNA fragment depends on the type of primer used. In one example of the present invention, when the above primer sets A and C are used, DNA fragments of about 100 bp are each amplified. Next, the amplified DNA fragment is subjected to electrophoresis using an agarose gel or acrylamide gel, and then stained with ethidium bromide or the like, and the presence or absence of the amplified fragment can be confirmed under a UV lamp.

In addition, by using the method of the present invention, flying fish such as fillets and sashimi that have no prototype and cannot be identified morphologically, processed fish such as dried fish and smoked fish, and other flying fish such as kamaboko High-order processed products containing other fish species and multiple food additives can also be accurately identified. Furthermore, the primer of the present invention can be used as a kit for detecting flying fish in a subject that seems to contain flying fish , in whole or in combination.

  Examples of the present invention will be specifically described below, but the present invention is not limited thereto.

Identification of flying fish mitochondrial DNA Preparation of DNA from various fishes Total DNA was extracted using DNA Isolation Kit (GE Healthcare Bioscience). Total DNA was determined for DNA concentration by measuring absorbance at 260 nm with a spectrophotometer.

PCR was performed using Thermal Cycler Personal (Takara Bio) using 10 ng of the prepared total DNA as a template. PCR uses SEQ ID NO: 7 and SEQ ID NO: 8 (primer set A) and SEQ ID NO: 11 and SEQ ID NO: 12 (primer set C), respectively. The total amount of the reaction solution is 100 μL, and after preheating at 94 ° C. for 2 minutes, A denaturation step at 94 ° C. for 30 seconds, an annealing step at 55 ° C. for 30 seconds, and an extension step at 72 ° C. for 30 seconds were performed under conditions of 30 cycles. After completion of PCR, 10 μL of the PCR product was taken and subjected to 3% agarose gel electrophoresis.

  The results are shown in FIGS. With the primer set A, which is a primer for detecting flying fish, PCR products of about 100 bp were confirmed for all seven kinds of flying fish, but no other four fish species such as walleye pollack, minamidara, maji, and kurakaketragis were confirmed. On the other hand, about 100 bp of PCR products were confirmed for all 11 types of samples by primer set C, which is a detection reaction confirmation primer. This revealed that it is possible to distinguish flying fish and other fish.

Identification of flying mitochondrial mitochondrial DNA by RFLP analysis In order to examine whether or not flyingfish can be applied to RFLP, the nucleotide sequence information (SEQ ID NOs: 1 to 6) of various flying fish and the already known nucleotide sequence information ( RFLP was examined using Cypselurus hirai, AB182653), Walleye chalcogramma, AB182300, and horse mackerel (Tracurus jamonicus, AP003091).

  In each of the nine types of base sequences, a restriction enzyme recognition site was searched using GENETYX (Genetics), which is a commercially available analysis software, and an AfaI recognition site and an MfeI recognition site common to flying fishes were confirmed.

  Preparation of DNA from a variety of fishes. Total DNA was extracted using Care Bioscience. Total DNA was determined for DNA concentration by measuring absorbance at 260 nm with a spectrophotometer.

  PCR was performed using Thermal Cycler Personal (Takara Bio) using 10 ng of the prepared total DNA as a template. PCR uses SEQ ID NO: 9 and SEQ ID NO: 10 (primer set B), the total volume of the reaction solution is 100 μL, after preheating at 94 ° C. for 2 minutes, denaturation step at 94 ° C. for 30 seconds, and annealing at 60 ° C. for 30 seconds The process and the extension process at 72 ° C. for 30 seconds were performed under the condition of 30 cycles. After the PCR was completed, the PCR product was purified using QIAquick PCR Purification Kit (Qiagen), and the absorbance at 260 nm was measured with a spectrophotometer to determine the DNA concentration.

Digestion was performed on 50 ng of the PCR product by incubating with restriction enzymes AfaI and MfeI at 37 ° C. for 1 hour, respectively . Each digest was then subjected to 4% agarose gel electrophoresis.

  The results are shown in FIGS. In both types of restriction enzymes, the seven kinds of flying fish were confirmed to have bands of about 300 bp and about 200 bp, and the PCR product was digested. On the other hand, the 500 bp band was confirmed in the other four fish species, Alaska pollack, Minamidara, Maaji, and Kuraketoragis, and the PCR product was not digested. This revealed that it is possible to distinguish flying fish and other fish.

Appraisal of processed foods using flying fishes From Geno Prep Cells and Tissue DNA Isolation Kit (GE Healthcare Biosciences) The total DNA was extracted using Total DNA was determined for DNA concentration by measuring absorbance at 260 nm with a spectrophotometer.

  PCR was performed using Thermal Cycler Personal (Takara Bio) using 10 ng of the prepared total DNA as a template. PCR uses SEQ ID NO: 7 and SEQ ID NO: 8 (primer set A) and SEQ ID NO: 11 and SEQ ID NO: 12 (primer set C). The total amount of the reaction solution is 100 μL, and after preheating at 94 ° C. for 2 minutes, A denaturation step at 30 ° C., an annealing step at 55 ° C. for 30 seconds, and an extension step at 72 ° C. for 30 seconds were performed under conditions of 30 cycles. After completion of PCR, 10 μL of the PCR product was taken and subjected to 3% agarose gel electrophoresis.

  The results are shown in FIGS. About 100 bp of PCR products were confirmed for all 17 types of samples by primer set A, which is a primer for detecting flying fish, and primer set C, which is a reaction confirmation primer. Agono-yaki, which is a baked kamaboko, has a high risk of DNA degradation and loss because it undergoes various manufacturing processes such as water exposure, salting, main grinding, molding, heating, and cooling. It has been clarified that the present invention can detect flying fish with high sensitivity even for high-order processed products such as kamaboko.

  Salmon pollock frozen surimi (Nissui second grade) and flying fish surimi (Shimane Prefecture) were salted separately and combined at a predetermined ratio to create an ochano ware. From there, total DNA was extracted using Genomic Prep Cells and Tissue DNA Isolation Kit (GE Healthcare Bioscience). Total DNA was determined for DNA concentration by measuring absorbance at 260 nm with a spectrophotometer.

  PCR was performed using 20 ng of the prepared total DNA as a template and a real-time PCR apparatus (PRISM7000, Applied Bioscience). SYBR green was used as the DNA binding dye. PCR uses SEQ ID NO: 7 and SEQ ID NO: 8 (primer set A) and SEQ ID NO: 11 and SEQ ID NO: 12 (primer set C). The total volume of the reaction solution is 50 μL, and after preheating at 94 ° C. for 10 seconds, 95 A denaturation step at 5 ° C., an annealing step at 57 ° C. for 10 seconds, and an extension step at 72 ° C. for 31 seconds were performed under the conditions of 30 cycles. After completion of PCR, quantification was attempted with dedicated software.

  The results are shown in FIG. The increase curve of the PCR product by primer set A when the flying fish surimi content was 0%, 30%, 70%, and 100% was represented, and the calibration curve of FIG. 9 could be drawn. Moreover, the increase curve of the PCR product by the primer set C was not different from any sample, and it was confirmed that the reaction was performed normally. It has been clarified that the present invention can be applied to the determination of flying fish contained in kamaboko.

It is a schematic diagram of general mitochondrial DNA possessed by eukaryotes. It is the photograph which showed the PCR product amplified with the primer set A from the total DNA of said 11 types of fish species. It is the photograph which showed the PCR product amplified by the primer set C from the total DNA of said 11 types of fish species. RFLP results when PCR products amplified with primer set B were digested with AfaI from the total DNA of photofishfish, sandfishfish, flyingfish, hamatobiuo, sandfishfish, karasutobuo, barnaclefish, walleye pollack, minamidara, jack mackerel, and kraketragis It is. It is the photograph which showed the result of RFLP at the time of digesting the PCR product amplified by the primer set B with MfeI from the total DNA of said 11 types of fish species. It is the photograph which showed the PCR product amplified with the primer set A from the total DNA of the chinono ware which has a flying fish display in the raw material display column of goods. It is the photograph which showed the PCR product amplified with the primer set C from the total DNA of the chinono ware which has a fishfish display in the raw material display column of goods. It is the figure which showed the amplification curve of the PCR product amplified with the primer set A from the total DNA of the chinono ware of different flying fish content. It is drawing which showed the calibration curve calculated | required from the relationship between flying fish content and the number of cycles from FIG.

Claims (5)

A primer set A of a 5'-side primer represented by SEQ ID NO: 7 and a 3'-side primer represented by SEQ ID NO: 8 , or a 5'-primer represented by SEQ ID NO: 9 and a 3'-side primer represented by SEQ ID NO: 10 Primer set B for detecting flying fish species . Amplifying DNA containing a gene encoding mitochondrial 16S ribosomal RNA of flying fish using the primer set A or B shown in claim 1 for a subject suspected of containing flying fish , A method for detecting flying fish in the subject . Using a primer set C including a 5 ′ primer represented by SEQ ID NO: 11 and a 3 ′ primer represented by SEQ ID NO: 12 for a subject suspected to contain flying fish, a mitochondrial 16S ribosome of flying fish The method for detecting flying fishes according to claim 2, wherein DNA containing a gene encoding RNA is amplified. 4. The method for detecting flying fish according to claim 2 or 3, wherein the subject that is thought to contain flying fish is processed food that is labeled as flying fish. A primer set A comprising a 5 ′ primer represented by SEQ ID NO: 7 and a 3 ′ primer represented by SEQ ID NO: 8 , and a 5 ′ primer represented by SEQ ID NO: 11 and a 3 ′ primer represented by SEQ ID NO: 12 Containing primer set C containing , or
A kit for detecting flying fish including a primer set B including a 5 'primer represented by SEQ ID NO: 9 and a 3' primer represented by SEQ ID NO: 10, and a restriction enzyme AfaI or MfeI .