JP5566017B2 - Primer set for giant clam detection - Google Patents

Description

  The present invention relates to a primer set capable of specifically detecting a giant clam with high sensitivity.

  Shrimp and crabs have been included in allergic labeling recommendations (similar to specified raw materials), but have recently been added to allergic labeling mandatory products (specified raw materials). As a result, a detection method with higher accuracy has been desired from the viewpoint of enhancing allergy obligation labeling.

  Methods for detecting crustaceans have been reported to detect tropomyosin, a major shrimp and crab allergen by ELISA (Non-Patent Documents 1 to 3), and PCR methods targeting the 16S rRNA gene. (Patent Document 1). However, all of these methods are methods for detecting shrimp and crabs in general, and it is not enough to detect shrimp and crabs separately from other crustaceans such as krill and amy. In addition, among PCR methods targeting the 16S rRNA gene, there is a method of detecting based on the restriction enzyme fragment length polymorphism (RFLP) of the PCR product, but this method is for specific shrimps and crabs, It is a method for determining what kind of shrimp and crab it is, and it is not possible to distinguish only shrimp from shrimp and crab (Non-Patent Document 4).

  The present inventor has so far devised primer design to further increase specificity and sensitivity, and has established a shrimp detection PCR method (Patent Document 2) and a crab detection PCR method targeting 16S rRNA gene. . However, it has been found that the crab detection method is detected in the same manner as a crab when a sample contains a crab other than a target crab. Sharks are crustaceans like shrimp and crabs and are eaten as they are, mainly as sushi, so it is thought that they are rarely included in processed products like crabs and shrimps. However, in order to ensure the reliability of allergy labeling, it is necessary to avoid false positives by other crustaceans such as giant clams in the method for detecting shrimp and crabs as much as possible.

Jeoung BJ, Reese G, Hauck P, Oliver JB, Daul CB, Lehrer SB.Quantification of the major brown shrimp allergen Pen a 1 (tropomyosin) by a monoclonal antibody-based sandwich ELISA.Journal of allergy and clinical immunology.100, 229 -34 (1997) FA Test EIA-Crustacea "Nissui" Crustacean (shrimp, crab) kit "Maruha" Brzezinski JL. Detection of crustacean DNA and species identification using a PCR-restriction fragment length polymorphism method. Journal of Food Protection. 68, 1866-73 (2005) JP 2006-280281 A JP 2008-128

  An object of the present invention is to provide a simple means for detecting only a giant clam with specific and high sensitivity, as distinguished from shrimp, crab, other crustaceans, seafood and the like.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have selected bases that can distinguish shrimp from crab, shrimp, and other crustaceans in the base sequence of 16S rRNA gene of shrimp, When mixed with a plurality of primers designed from the region including the base sequence before and after the selected base, the clam is distinguished from crab, shrimp, and other crustaceans to detect specifically and with high sensitivity. It was successful and the present invention was completed.

That is, the present invention includes the following inventions.
(1) A primer set for detecting shrimp comprising the following oligonucleotide (a) and oligonucleotide (b):
(a) a mixture of oligonucleotides having the nucleotide sequences shown in SEQ ID NOs: 1 and 2
(b) a mixture of oligonucleotides consisting of the nucleotide sequences shown in SEQ ID NOs: 3 and 4 or an oligonucleotide consisting of the nucleotide sequences shown in SEQ ID NO: 5
(2) A method for detecting a giant clam, characterized in that DNA extracted from a sample is used as a template, PCR is performed using the primer set described in (1), and the presence or absence of an amplification product of a target size is determined as an index.
(3) When using a primer set composed of a mixture of oligonucleotides consisting of the nucleotide sequences shown in SEQ ID NOs: 1 and 2 and a mixture of oligonucleotides consisting of the nucleotide sequences shown in SEQ ID NOs: 3 and 4, the target size is 95 bp to The method for detecting a giant clam according to (2), which is 96 bp.
(4) The target size is 195 bp to 198 bp when a primer set composed of a mixture of oligonucleotides consisting of the nucleotide sequences shown in SEQ ID NO: 1 and 2 and an oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 5 is used. The method for detecting a giant clam according to (2), characterized in that:
(5) A giant clam detection kit comprising the primer set according to (1).

  ADVANTAGE OF THE INVENTION According to this invention, the primer set which can detect a giant clam with specific and high sensitivity is provided. If there is a possibility that a cactus is mixed in the food, a small amount of cactus in the food can be specifically detected with high sensitivity by using the primer set of the present invention. In addition, since the PCR amplification product using the primer set of the present invention is as short as 200 bp or less, even if the DNA is fragmented by food processing, it is highly sensitive for crab, shrimp, and other It can be detected separately from crustaceans.

  The primer set for detecting shrimp of the present invention has a base sequence of a region containing a base common to the shrimp and a base that is common to the shrimp and can be distinguished from crabs, shrimps, and other crustaceans, or a base sequence complementary thereto. Consists of oligonucleotides that can anneal to nucleic acid molecules.

  In this specification, “Scoot” means the classification number 71361 “Squirrel Squillas (Squillidae sp) defined by the Japanese standard product classification [revised June 1992: Statistics Bureau, Ministry of Internal Affairs and Communications, Policy Director (in charge of statistical standards)]. .) ”, And species belonging to the Stomatopoda of the subclass of Hoplocarida, taking into account phylogenetic and morphological features. Further, in the present specification, “shrimp” refers to those belonging to “Nematoda”, “Shrimp lower eye”, “Crayfish lower eye”, and “Shrimp lower eye”, and “crab” refers to “short "Oshita eyes" and those belonging to "Onomidae king crab family". “Other crustaceans” refers to “ami”, “krill”, “barnacles”, etc., and does not include “mantis”.

  The above-mentioned “regions that include bases common to clams and bases that are common to clams and are distinguishable from crabs, shrimps, and other crustaceans” are those of the above clams, crabs, shrimps, and other crustaceans. A plurality of base sequences known as 16S rRNA genes can be identified by aligning and comparing them. Specifically, it compares the base sequences of 16S rRNA genes derived from giant clams, crabs, shrimps, and other crustaceans, and contains a plurality of bases common to giant clams. Identify regions containing bases that can be distinguished from crabs, crabs, and other crustaceans. A base distinguishable from shrimp, crabs, and other crustaceans is placed at the 3 'end of the primer.

  Among the above-mentioned plurality of base sequences used for alignment, any known base sequence may be used as long as it is known as a 16S rRNA gene of giant clam, crab, shrimp, and other crustaceans. Often, such a base sequence can be obtained by searching using a DNA database such as GenBank. For those not disclosed in the database, information can be obtained by obtaining a sample and then decoding the nucleotide sequence using an appropriately designed PCR primer.

  For the alignment (alignment) of the base sequences, alignment software (for example, CLUSTALW, URL: http://www.ddbj.nig.ac.jp/) published on the Internet can be used.

  Next, a primer is designed in the specified region. For primer design, for example, “Forefront of PCR-From Basic Technology to Application” (Protein / Nucleic Acid / Enzyme Special Issue 1996 Kyoritsu Publishing Co., Ltd.) and “Bio-Experiment Illustrated 3 Really Increased PCR: Cell Engineering Attachment” Based on "Experimental note series to see" (Hiroki Nakayama 1996, Shujunsha Co., Ltd.), "PCR technology-Principle and application of DNA amplification" (Henry A Erlich edited by Kuniyuki Kato, Takara Shuzo Co., Ltd.) However, in the case of detection from processed foods, it is possible that DNA is degraded and shortened. From this point of view, a primer that can obtain as short an amplification product as possible is designed. It is preferable.

  Oligonucleotides that serve as primers can be synthesized using methods commonly known in the art as methods for synthesizing oligonucleotides, such as the phosphotriethyl method, phosphodiester method, etc., using a commonly used automatic DNA synthesizer. is there.

  The primer set for detecting the giant clam of the present invention includes the five types of oligonucleotides designed as described above. Specifically, as a forward primer, a mixture of oligonucleotides consisting of the nucleotide sequences shown in SEQ ID NOs: 1 and 2 and The reverse primer is composed of a mixture of oligonucleotides consisting of the base sequences shown in SEQ ID NOs: 3 and 4 or an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 5.

The base sequences shown in the above SEQ ID NOs are as follows:
TTGTATGAATGGTCGGACAAGAT (SEQ ID NO: 1)
TTGTATGAATGGTCCGACAAGAT (SEQ ID NO: 2)
ATCGTCCCTCCATATCATTTAAGCTTTTTT (SEQ ID NO: 3)
ATCGTCCCTCCATATTATTTAAGCTTTTTT (SEQ ID NO: 4)
ATATACTTTTACCGCCCCAGT (SEQ ID NO: 5)

  When PCR amplification is performed using the above primer set, if the sample contains a giant clam, the size specific to the giant clam that is not found in crabs, shrimps and other crustaceans other than the giant clam, that is, the amplification product of the target size Is obtained. Accordingly, the clam can be specifically detected by distinguishing it from crabs, shrimps and other crustaceans only by the presence or absence of the amplification product of the target size.

  The above primer set can also be made into a kit. The kit of the present invention only needs to contain at least the primer set described above, and if necessary, a DNA molecule containing a target sequence that serves as a PCR positive control, a DNA extraction reagent, a PCR buffer, a DNA polymerase, etc. PCR reagents (excluding the primer set), real-time PCR reagents using SYBR Green, detection reagents such as staining agents and electrophoresis gels, and instructions may be included.

  The present invention also provides a method for detecting a giant clam using the above primer set. This method is characterized in that DNA extracted from a sample is used as a template, PCR is performed using the above primer set, and the presence or absence of an amplification product of the target size is detected as an index.

  The sample is not particularly limited as long as it is a food material or product that may contain a giant clam, or a food ingredient or product that may contain a giant clam.

  Extraction of DNA from a sample may be performed by any method known to those skilled in the art as a nucleic acid extraction method, such as phenol / chloroform method, cell lysis with a surfactant, cell lysis with a protease enzyme, and physical lysis with glass beads. It can be performed by a destruction method, a treatment method that repeats freeze-thaw, or the like. Various DNA extraction kits sold by reagent manufacturers may also be used. Depending on the type of sample, it is desirable to perform filtration or homogenization with a membrane filter.

  Next, using the DNA sample obtained by the above operation as a template, PCR is performed using the above primer set to detect the presence or absence of an amplification product of the target size. PCR amplification is not particularly limited except that the above primer set is used, and may be performed according to a conventional method. The composition of the PCR reaction solution and the PCR conditions (temperature cycle, number of cycles, etc.) are, for example, within a range where the total solution volume is 25 to 50 μL, 50 ng of DNA serving as a template, PCR reaction buffer solution, forward primer 0.2 to 0.5 μM, reverse primer 0.2-0.5 μM, DNA polymerase (Taq polymerase, TthDNA polymerase, etc.) 0.025 U / μL, dNTP 200 μM each, 94-96 ° C. 10 minutes × 1 cycle, (94-96 ° C. 30 seconds, 52 ~ 58 ° C 30 seconds, 72 ° C 30 seconds) × 40 cycles, 72 ° C 7 minutes × 1 cycle. This is merely an example, and the composition, reaction temperature, and time of the PCR reaction solution can be appropriately set according to the length of the oligonucleotide sequence that serves as a primer, the base composition, and the like. A series of these PCR operations can be performed using a commercially available PCR kit or PCR apparatus according to the operating instructions.

  Whether or not an amplification product of the target size has been obtained by PCR can be confirmed by using a known method such as agarose gel electrophoresis, DNA hybridization, and real time PCR (including end point and dissociation curve analysis). The size of the amplification product of the target size is the sum of the number of bases of both primers and the number of bases of the region sandwiched between both primers in the 16S rRNA gene region to be detected. For example, when a primer set composed of a mixture of oligonucleotides consisting of the nucleotide sequences shown in SEQ ID NOs: 1 and 2 and a mixture of oligonucleotides consisting of the nucleotide sequences shown in SEQ ID NOs: 3 and 4 is used, the target size is 95 bp. When a primer set composed of a mixture of oligonucleotides consisting of the nucleotide sequences shown in SEQ ID NOs: 1 and 2 and an oligonucleotide consisting of SEQ ID NO: 5 is used, a target size of An amplification product of 195 bp to 198 bp can be obtained. In general processed foods, there is a high possibility that DNA is fragmented in the processing process, but because the target size of the PCR product is 200 bp or less, the shrimp is specifically detected even in foods with a high degree of processing. be able to. Amplification products with a target size of 95 bp to 96 bp or 195 bp to 198 bp have base deletions or insertions in the amplification region between the 3 'end of the forward primer and the 3' end of the reverse primer. Because. From this, there is a possibility that amplification products with target sizes outside the ranges of 95 bp to 96 bp and 195 bp to 198 bp may be obtained from the 16S rRNA gene sequences of the giant clam other than those examined here.

Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.
(Example 1) Synthesis of primer for detecting giant clam
(1) Sequence collection and alignment of 16S rRNA gene region Crab (352 sequences), Shrimp {Earth roots (69 sequences), Shrimp (201 sequences), Crayfish (221 sequences), Shrimp (52) Sequence)}, krill (19 sequences), krill (18 sequences), and giant clam (15 sequences), 16S rRNA gene base sequence information was obtained. The nucleotide sequence information was obtained from the database for the species registered in GenBank, and the nucleotide sequence of the 16S rRNA gene was determined by sequencing for the purchased species. Based on these base sequence information, the sequences of the same region were aligned and compared using analysis software CLUSTAL X (1.8), FROG-Win, SEQ-09.

(2) Primer set design Based on the aligned sequences, the giant clam-specific base was selected. Next, a region suitable for primer design was selected from the homology of the base sequences before and after the selected base. Primers with high clam specificity from these regions were designed according to the following criteria.
<Design criteria>
(a) The 3 'terminal bases of both forward and reverse primers must match the bases of the giant clam DNA sequence.
(b) Both forward and reverse primers should bind / extend to the giant clam DNA sequence, and at least one of them should not bind / extend to the DNA sequence of species other than the giant clam.
(c) Since PCR products are processed products, it is desirable that PCR amplification products be 200 bp or less.
(d) Primer length should be in the range of 20-30base, and Tm value of primer and giant clam DNA sequence should be set between 50-60 ℃.
(e) PCR simulations predict growth of PCR products of target size specific to the giant clam.
(f) Primers should not form dimers or three-dimensional structures.

As a result, 2 kinds of forward primers (F2-1, F2-2) and 3 kinds of reverse primers (R54-1, R54-2, R188), which are expected to specifically detect giant clam on the 16S rRNA gene sequence, were used. Designed. Oligonucleotides (F2-1, F2-2) consisting of base sequences shown in SEQ ID NOs: 1 and 2 as forward primers, and oligonucleotides (R54-1, R54) consisting of base sequences shown in SEQ ID NOs: 3, 4 as reverse primers For -2), the bases (underlined portion of the following sequence) were changed so that they could be combined and extended with various types of giant clam sequences, and they were mixed and used.
Forward primer (F2):
F2-1: TTGTATGAATGGTC G GACAAGAT (SEQ ID NO: 1)
F2-2: TTGTATGAATGGTC C GACAAGAT (SEQ ID NO: 2)
Reverse primer (R54):
R54-1: ATCGTCCCTCCATAT C ATTTAAGCTTTTTT (SEQ ID NO: 3)
R54-2: ATCGTCCCTCCATAT T ATTTAAGCTTTTTT (SEQ ID NO: 4)
Reverse primer (R188):
R188: ATATACTTTTACCGCCCCAGT (SEQ ID NO: 5)

  That is, the primer set for detecting shrimp of the present invention comprises a forward primer F2 (mixture of oligonucleotides consisting of the nucleotide sequences shown in SEQ ID NOs: 1 and 2) and a reverse primer R54 (oligonucleotide consisting of the nucleotide sequences shown in SEQ ID NOs: 3 and 4). Forward primer F2 (mixture of oligonucleotide consisting of the base sequence shown in SEQ ID NO: 1 and oligonucleotide consisting of the base sequence shown in SEQ ID NO: 2) and reverse primer R188 (base shown in SEQ ID NO: 5) Oligonucleotide consisting of a sequence).

(3) Specificity evaluation by BLAST search Blast sequence search was performed for each of the above forward and reverse primers. For invertebrates and non-invertebrate organisms, species were identified for the top 100 sequences with similar sequences to primers. As a result, none of the organisms other than invertebrates had a sequence highly similar to both forward and reverse primers. When targeting invertebrates, two crayfish-derived sequences were hit as sequences with high similarity to the oligonucleotides consisting of the nucleotide sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 5, but the 3 ′ end base The forward and reverse primers were expected to be unlikely to undergo PCR amplification from DNA sequences other than the giant clam.

(4) Anticipation of giant clam amplification using PCR simulation software Two forward primers (F2-1: SEQ ID NO: 1, F2-2: SEQ ID NO: 2) and three reverse primers (R54-1: SEQ ID NO: 3, R54-) 2: PCR simulation using Amplify simulator was performed using all combinations of SEQ ID NO: 4, R188: SEQ ID NO: 5), and amplification of the target product was expected from 14 sequences of 16 sequences of 16S rRNA genes studied (Table 1). For one sequence that was not expected to be amplified (AY947836: Seudosquilla ciliata), a single base insertion was present at the position corresponding to the 3 'side of the oligonucleotide consisting of the nucleotide sequences shown in SEQ ID NOs: 1 and 2, and it was predicted that it was not detected. However, since this single base insertion was not confirmed in other giant clams and other closely related crustaceans, it was judged that the above single base insertion is highly likely to be a sequence smith.

Accession No.及び学名：GenBankで取得
評価：増幅産物が得られる可能性の6段階評価(最大6、最小１)、「-」は増幅産物が得られないものを示す。
予想増幅産物長（bp）：フォワードプライマーとリバースプライマーを含む増幅領域の塩基数を示す。

Accession No. and scientific name: Acquired by GenBank Evaluation: Six-step evaluation of the possibility of obtaining an amplification product (maximum 6, minimum 1), “-” indicates that no amplification product can be obtained.
Expected amplification product length (bp): Indicates the number of bases in the amplification region containing the forward primer and reverse primer.

(5) Amplification prediction of crustaceans other than giant clams using PCR simulation software Each of 16S rRNA gene sequences belonging to crustaceans, cephalopods, seafood, echinoderms (sea urchins, sea cucumbers), etc. It was confirmed whether the 3 'terminal bases of the primers matched, and for the matched species, it was predicted by PCR simulation whether a target size amplification product could be obtained.

(a) Edible shrimp crabs and other crustaceans Crabs (352 sequences), shrimps [Nematoda (69 sequences), Shrimp lower (201 sequences), Crayfish lower (221 sequences), lobster lower (52 sequences)], krill (19 sequences), mami (18 sequences), giant clam (15 sequences), as a result of confirming the match with the 3 'terminal base of the primer, the forward primer matches the king crab, Ami, The reverse primer matched different species of krill and krill from those matched by the forward primer. Next, it was predicted for each of these species whether or not a target size amplification product could be obtained by PCR simulation. As a result, the homology with the primer sequence was low, and the 3 'terminal base of either the forward or reverse primer matched. Therefore, it was expected that no amplification product of the target size could be obtained from these sequences.

(b) Micro-crustaceans Micro-crustaceans, such as the Shrimp, Brachydinae and Agaricus nets, Scarlet subnet, Daphnia magna, Spermoptera, Centipede, Caenidae, Casilla shrimp nets (142) As a result of performing a PCR simulation of the sequence), the forward and reverse primers having the same 3 ′ end base were subjected to PCR simulation. As a result, “W2” was obtained for the two sequences, and a slight amplification product could be obtained. However, as a result of calculating the Tm value between the primer and the similar sequence, it was as low as 30 ° C or lower, so it is expected that primer binding and extension reactions will not occur at the annealing temperature of 54 ° C or 58 ° C in this PCR amplification reaction. Therefore, it was judged that no amplification product was obtained.

(c) closely related species (36 sequences such as hermit crab), other animal species {shellfish (23 sequences), fish (46 sequences), echinoderms (31 sequences), meats (3 sequences)}
In species such as hermit crabs, both the forward and reverse primers matched the 3 'end base, but the homology between the forward primer and similar sequences was low, and it was expected that no amplification product could be obtained. In other animal species, the oligonucleotide having the nucleotide sequence shown in SEQ ID NOs: 1 and 2 did not match the 3 ′ terminal base, and it was expected that no amplification product could be obtained.

  As described above, for the two types of designed primer sets, no prediction was made that amplification products of the target size could be obtained as a result of amplification prediction by Blast search and PCR simulation. It was done.

(Example 2) Specificity and sensitivity evaluation of primers
(1) Extraction of PCR template DNA
[sample]
(i) Mantis
(ii) Shrimp (14 types)
Nematoda: prawn, red shrimp, black tiger, white shrimp, cherry shrimp, shrimp, lower shrimp, shrimp, button shrimp, crayfish
(iii) Crabs (13 types)
Short-tailed Crab: Snow Crab, Crab Crab, Crab Crab, Dungeness Crab, Mars Crab Crab, Crab Crab, Crab Crab Crab Crab, Crab Crab
(iv) Other crustaceans (2 types)
Krill
(v) Cephalopods (4 types)
Octopus, squid, squid, squid
(vi) Shellfish (5 types)
Clams, clams, scallops, turban shells, abalone
(vii) Fish (7 types)
Yellowtail, salmon, cod, mackerel, skipjack, tuna, horse mackerel

[DNA extraction]
After washing each of the above samples with water, weigh 0.1 g of the body (edible part), put it in a 15 ml tube, 2 ml of Buffer G2 (QIAGEN), 100 μl of Proteinase K (QIAGEN), 100 mg / ml RNaseA (QIAGEN) 4 μl) was added, mixed, and incubated at 50 ° C. for 2 hours (stirring was performed several times in the middle). Next, centrifugation (3500 × g, 15 minutes) was performed twice, and the supernatant was recovered. A Genomic Tip 20 / G column (QIAGEN) equilibrated with 1 ml Buffer QBT was set in a 15 ml tube, and the whole supernatant was applied. After washing the column with 3 ml of Buffer QC (QIAGEN), DNA was eluted with 1 ml of Buffer QF (QIAGEN) heated to 50 ° C., subjected to isopropanol precipitation, and dissolved in 50 μl of TE solution. The spectrum of the extracted DNA was measured with an ND-1000 Spectrophotometer (NanoDrop Technologies, Inc), and the DNA concentration and purity were calculated.

(2) Evaluation of primer sensitivity and specificity DNA solution obtained by diluting the DNA extracted from the sample as described above to 20 ng / μl with TE (pH 8.0), and 20 ng / μl of the same DNA solution. Using 2.5 μl of a DNA solution prepared by serial dilution with salmon sperm DNA-containing TE (pH 8.0) buffer as a template for PCR, the primer set for detecting shrimp of the present invention (the oligonucleotide consisting of the nucleotide sequences shown in SEQ ID NOs: 1 and 2) A primer set composed of a mixture and a mixture of oligonucleotides consisting of the nucleotide sequences shown in SEQ ID NOs: 3 and 4, and a mixture of oligonucleotide consisting of the nucleotide sequences shown in SEQ ID NOs: 1 and 2 and the nucleotide sequence shown in SEQ ID NO: 5 PCR was carried out using a primer set composed of oligonucleotides, and the specificity and sensitivity were examined, consisting of the nucleotide sequences shown in SEQ ID NOs: 1 and 2. The Rigonucleotide primer was synthesized as a single primer designated as the G / C mixed base at the 9th base from the 3 'end, and the same was true for the oligonucleotide primer consisting of the base sequences shown in SEQ ID NOs: 3 and 4. The 15th base from the 3 'end was synthesized as a single primer designated as a mixed base of C / T.

  The PCR reaction was performed using a 0.2 ml tube, GeneAmp PCR System 9700 (Applied Biosytems) or GeneAmp PCR System 9600 (PerkinElmer), and the PCR reaction solution composition and PCR reaction conditions (SEQ ID NOs: 1, 2) shown in Table 2 or 3 below. Primer set comprising a mixture of oligonucleotides consisting of the base sequences shown in FIG. 3 and a mixture of oligonucleotides consisting of the base sequences shown in SEQ ID NOs: 3 and 4: An oligo consisting of the base sequence shown in SEQ ID NOs: 1 and 2 at an annealing temperature Primer set composed of oligonucleotide mixture consisting of nucleotide mixture and nucleotide sequence shown in SEQ ID NO: 5 (annealing temperature 58 ° C.).

  As DNA in the table, 20 ng / μl, 20 ng / μl, 200 pg / μl, 2 pg / μl for shrimp, 20 ng / μl DNA solution for shrimp, crab, krill, and mami, 20 ng / μl for other non-crustacean fish and shellfish A DNA solution was used. 200 pg / μl and 2 pg / μl DNA solutions were prepared by serial dilution with 20 ng / μl salmon sperm DNA-containing TE (pH 8.0) buffer. Each reagent in the table was manufactured by Applied Biosystems.

  Next, PCR amplification products were separated by 3% agarose gel electrophoresis and detected. An agarose gel electrophoresis diagram of the PCR amplification product is shown in FIG. According to the primer set of SEQ ID NO: 1, 2 and SEQ ID NO: 3, 4 and the primer set of SEQ ID NO: 1, 2 and SEQ ID NO: 5, in each case, the target is from shrimp, crab, krill, mysid (DNA 50 ng) No size amplification product was obtained (FIG. 1). In addition, amplification products of the target size were not obtained from seafood other than crustaceans (DNA 50 ng) (FIG. 2).

  Under the above conditions, PCR was performed using the primer set of SEQ ID NO: 1, 2 and SEQ ID NO: 3, 4 and the primer set of SEQ ID NO: 1, 2 and SEQ ID NO: 5 using the shrimp DNA as a template. It was confirmed that an amplification product of the target size was obtained from 5 pg of the target giant clam DNA in both primer sets (FIG. 3).

Primer set for detecting shrimp of the present invention for DNA samples derived from crustaceans other than shrimp [shrimp (14 types), crab (13 types), krill, ami] (primer sets of SEQ ID NO: 1, 2 and SEQ ID NO: 3, 4) , And the specificity evaluation test results of the primer sets of SEQ ID NO: 1, 2 and SEQ ID NO: 5 (agarose gel electrophoresis photograph of PCR amplification product) (arrow: band of target amplification product length, M: molecular weight marker, N: Sterile ultrapure water was used as a negative control, P: Pteropia 5pg DNA was used as a positive control). Primer set for detecting shrimp of the present invention for DNA samples derived from craniopods (4 types), shellfish (5 types), and fish (7 types) other than crustaceans (primers of SEQ ID NO: 1, 2 and SEQ ID NO: 3, 4) Set, and specificity evaluation test results (agarose gel electrophoresis photograph of PCR amplification product) of the sequence number 1, 2 and sequence number 5) (arrow: target amplification product length band, M: molecular weight marker, (N: use sterilized ultrapure water as negative control, P: use giant clam 5pg DNA as positive control). Results of sensitivity evaluation test of the primer set for detecting shrimp of the present invention (primer set of SEQ ID NO: 1, 2 and SEQ ID NO: 3, 4 and primer set of SEQ ID NO: 1, 2 and SEQ ID NO: 5) for DNA samples derived from the shrimp An agarose gel electrophoresis photograph of a PCR amplification product is shown (arrow: target amplification product length band, M: molecular weight marker, N: sterile ultrapure water is used as a negative control).

Claims (5)

A primer set for detecting shrimp comprising the following oligonucleotide (a) and oligonucleotide (b):
(a) a mixture of oligonucleotides having the nucleotide sequences shown in SEQ ID NOs: 1 and 2
(b) a mixture of oligonucleotides consisting of the nucleotide sequences shown in SEQ ID NOs: 3 and 4 or an oligonucleotide consisting of the nucleotide sequences shown in SEQ ID NO: 5   A method for detecting a giant clam characterized in that DNA extracted from a sample is used as a template, PCR is performed using the primer set according to claim 1, and the presence or absence of an amplification product of a target size is determined as an index.   When using a primer set composed of a mixture of oligonucleotides consisting of the nucleotide sequences shown in SEQ ID NOs: 1 and 2 and a mixture of oligonucleotides consisting of the nucleotide sequences shown in SEQ ID NOs: 3 and 4, the target size is 95 bp to 96 bp The method for detecting a giant clam according to claim 2, wherein:   The target size is 195 bp to 198 bp when a primer set composed of a mixture of oligonucleotides consisting of the nucleotide sequences shown in SEQ ID NO: 1 and 2 and an oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 5 is used. The method for detecting a giant clam according to claim 2.   A kit for detecting a giant clam, comprising the primer set according to claim 1.

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