JP7013190B2 - A primer set for amplifying a fragment of insect-derived DNA, and a method for identifying an insect species using the primer set. - Google Patents

Description

The present invention relates to a primer set for amplifying a fragment of DNA derived from an insect that can be contained as a foreign substance in food or the like, and a method for identifying the type of insect using the primer set.

In the unlikely event that a foreign substance is mixed in food, it is essential to investigate the cause and prevent recurrence. Promptness and accuracy are important when dealing with insect foreign substances mixed in food. Conventionally, a skilled person has performed rapid identification by observing the morphology with a microscope or the like, and if necessary, verification of the identification result by DNA sequence analysis and identification of a finer species or the like have been performed.

However, identification of insect species by morphological observation has the disadvantages that skillful skills are required, it is difficult to deal with rare foreign insects, and it is difficult to deal with insects that do not retain their original shape due to processing or the like.

On the other hand, in the identification of insect species by DNA sequence analysis, it is possible to identify the insect species by acquiring the base sequence information that can identify the insect species among the DNA molecules derived from the insect foreign body and comparing the sequence with the database. Accurate and detailed identification is possible without the need for skill as in morphological observation. Therefore, in recent years, for the purpose of identifying insect species, an identification method using DNA base sequence information as an objective index has been proposed.

For example, the method of Non-Patent Document 1 (animal DNA barcoding method: Hebert 2003) is obtained by performing a polymerase chain reaction (PCR) that amplifies a region in which a large difference is found between each classification level of an animal gene. This is a method for identifying an animal species by acquiring the base sequence information of an amplified fragment (target amplification product) of the desired fragment length and comparing it with a database (base sequence analysis). The GenBank database is particularly enriched in the first half of the cytochrome c oxidase subunit I (COI) coding gene region, which is the base sequence targeted by this method. Many existing insect species identification methods use this method.

Further, it is considered that the primer pair (forward primer: B_F, reverse primer: HC02198) shown in Non-Patent Document 2 (Shokralla 2015) can identify more insect species than Non-Patent Document 1.

In addition, the primer pairs shown in Non-Patent Document 3 (Hajibabaei 2011), Non-Patent Document 4 (Hajibabaei 2012), and Non-Patent Document 5 (Clarke 2014) (the combination of the forward primer and the reverse primer is LepF1-EPT-long-, respectively. Since univR, BE Forward-BE Reverse, and ls16S_1F-ls16S_1shortR) have short amplified fragment lengths of about 180, 350, and 200 bases, respectively, they can cope with DNA damage.

Other patent documents using the same technique include Patent Documents 1 to 3.

Japanese Unexamined Patent Publication No. 2004-194612 Japanese Unexamined Patent Publication No. 2008-99644 Japanese Patent Application Laid-Open No. 2003-265181

Proceedings of the Royal Society of London. Series B, Biological Sciences, 270, 313-321. (2003) Scientific Reports 5, Article number: 9687 (2015) PLoS ONE 6, e17497. (2011) BMC Ecology 12:28. (2012) Molecular Ecology Resources 14, 1160-1170. (2014)

The present inventors have found that the method described in the above prior art document has the following problems.

In the DNA barcoding method described in Non-Patent Document 1, since the fragment length of the target amplification product by PCR is as long as about 650 bases, it is difficult to deal with damage to DNA derived from insect foreign substances due to heating or the like. In addition, when trying to identify insects that can be mixed in foods and the like, there is a problem that some insect species cannot be amplified.

The method described in Non-Patent Document 2 also has a long fragment length of about 460 bases, so that it is also difficult to deal with damage to DNA derived from insect foreign substances due to heating or the like. In addition, the subjects of the survey are insects in the environment, and it is not known whether they can be applied to insects that can be mixed with foods.

It is unclear whether the methods described in Non-Patent Documents 3, 4 and 5 can be applied to insects in the environment as the subject of investigation and to insects that can be mixed with foods and the like.

Since the fragment length of the target amplification product is longer than 300 bases in the methods described in Patent Documents 1 to 3, it is also difficult to deal with the damage of the insect foreign body-derived DNA due to heating or the like.

The present inventors have a base sequence common to insects that can be mixed with foods and the like in the base sequence used in the DNA bar coding method on the COI coding gene region and the base sequence of the 16S ribosomal RNA (16S) coding gene region. The sequence was found, and each primer pair for worm species identification was designed so that the fragment length of the target amplification product was 100-200 bases or less. Specifically, a primer pair A that amplifies a region of 180 bp in length on the COI coding gene region of an insect, a primer pair B that amplifies a region of 135 bp in length on the COI coding gene region of an insect, and an insect. A primer pair C was designed to amplify a region 200 bp in length on the 16S coding gene region of.

We have found that many insect species can be identified by performing PCR using insect-derived DNA as a template using these primer pairs and analyzing the base sequence of the obtained amplified fragment, and have completed the present invention.

That is, the present invention includes the following inventions.
(1) A primer set for amplifying a fragment of DNA derived from an insect.
Nucleotide sequence of SEQ ID NO: 1 GGTCAACAAATCATAAGATATTGG
A first primer containing a first polynucleotide containing a base sequence identical to or homologous to a continuous base sequence of 10 or more bases from the 3'end at the 3'end.
Nucleotide sequence of SEQ ID NO: 2 GAAGAAATATAAAAAAGCATG
Primer pair A, which comprises a second primer containing a second polynucleotide having the same or homologous base sequence as a continuous base sequence of 10 or more bases from the 3'end at the 3'end.
Nucleotide sequence of SEQ ID NO: 3 GCCAGTTCTAGCAGGAGCTATTAC
A third primer containing a third polynucleotide containing a base sequence identical to or homologous to a continuous base sequence of 10 or more bases from the 3'end at the 3'end.
Nucleotide sequence of SEQ ID NO: 4 TAAACTTCAGGGGTGACCAAAAAAATCA
Primer pair B containing a fourth primer containing a fourth polynucleotide having the same or homologous base sequence as a continuous base sequence of 10 or more bases from the 3'end at the 3'end, and
Nucleotide sequence of SEQ ID NO: 5 ATTACGCTGTTATCCY
(Y is C or T or a mixed base of C and T)
A fifth primer containing a fifth polynucleotide containing a base sequence identical to or homologous to a continuous base sequence of 10 or more bases from the 3'end at the 3'end.
Nucleotide sequence of SEQ ID NO: 6 GACGAGAAGACCCY
(Y is C or T or a mixed base of C and T)
Of these, a primer pair C containing a sixth primer containing a sixth polynucleotide containing a base sequence identical to or homologous to a continuous base sequence of 10 or more bases from the 3'end at the 3'end.
A primer set containing one or more of them.
(2) The primer set of (1), which comprises two or more of the primer pair A, the primer pair B, and the primer pair C.
(3) The primer set of (2), which comprises the primer pair A, the primer pair B, and the primer pair C.
(4) Step 1 in which a nucleic acid amplification reaction is carried out using a DNA derived from an insect as a template and a primer pair contained in any of the primer sets of (1) to (3).
A method for identifying an insect species, which comprises step 2 of analyzing the base sequence of the amplification product of the nucleic acid amplification reaction in step 1.

The primer set of the present invention is useful for amplifying a target region in DNA derived from heat-treated insects such as insect foreign substances mixed in foods and drinks.

The method of the present invention for identifying the type of insect is useful for identifying the type of insect that has been heat-treated.

The confirmation result of the target amplification product by PCR using the primer pair of Non-Patent Document 2-5 and the primer pair A, B, C of the present invention as a template is shown. The results of confirmation of the target amplification product by PCR using the primer pairs of Non-Patent Documents 2 and 4 and the primer pairs A, B, and C of the present invention as templates of DNA samples heated under different conditions are shown. The confirmation result of the target amplification product by PCR using the DNA sample of each insect as a template using primer pair D is shown.

<1. Term>
In the present invention, the "insect" refers to an arthropod phylum that inhabits on land. In addition to insects, it includes myriapoda such as centipedes and spiders. The "insects" in the present invention are insects that may be mixed as foreign substances into foods and drinks and other products, and are flies, booklices, cockroaches, beetles, hemiptera, thrips, lepidoptera, etc. One or more insects belonging to the order Lepidoptera and Scolopendromorpha.

In the present invention, the "DNA derived from an insect" is a DNA derived from an insect body or a part of the insect body. Here, the "insect body or part of the insect body" is typically an example of the insect body or the part of the insect body mixed as a foreign substance in food or drink or other products. "Insects or parts of insects" are parts of insects or insects that cannot be identified by morphological observation because they are deformed, or insects or insects in processed foods and drinks such as heating and other products. It may be a part of the body.

The "animal DNA barcoding region" is a base sequence targeted by the primer pair of Non-Patent Document 1, and is present in the first half of the COI coding gene region.

The "base sequence database" mainly refers to a public or commercial base sequence database (GenBank, etc.) that stores and provides base sequence data of various organisms, and determines various insect-derived DNAs. It may be a database in which the base sequence data obtained by the above is independently accumulated.

In the present invention, the "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), typically DNA. In RNA, thymine (T) can be read as uracil (U). As the DNA, a DNA containing U synthesized by substituting T at an arbitrary position with U can also be used. The polynucleotide may contain a modified nucleotide such as inosine (I) in part.

The polynucleotide may exist as a single strand or may exist as a double strand. When the polynucleotide exists as a double strand, at least one strand may be a polynucleotide having the predetermined characteristics specified below.

The method for producing a polynucleotide is not particularly limited, and the polynucleotide can be produced using a polynucleotide synthesizer.

In the present invention, when "base sequence Y homologous to base sequence X" or "base sequence X and base sequence Y are homologous", a DNA chain consisting of a complementary sequence of base sequence X and base sequence Y It refers to a combination in which a DNA strand consisting of is capable of hybridizing under the annealing conditions of a nucleic acid amplification reaction to form a hydrogen bond sufficient to form a stable double strand, and the base sequences X and Y are partially. It may be different. For example, a polynucleotide consisting of a complementary sequence of the base sequence X and a polynucleotide consisting of the base sequence Y have some mismatches in 10 nucleotides, 1 mismatch in 20 nucleotides, 1 mismatch in 30 nucleotides, and the like. There may be a mismatch. Typically, when the base sequence X is "homologous" to the base sequence Y, it means that the base sequences X and Y satisfy any of the following relationships.

(A) The base sequence Y is a base sequence in which one or several bases are deleted, substituted, added and / or inserted in the base sequence X.
(B) The base sequence Y is a base sequence having 70% or more identity with the base sequence X.
(C) A polynucleotide having a base sequence Y can hybridize with a polynucleotide having a base sequence complementary to SEQ ID NO: X under stringent conditions.
(D) Thymine (T) at an arbitrary position in one of the base sequence X and the base sequence Y is replaced with uracil (U) in the other.

In the above (A), "1 or several" preferably refers to 1 to 5, more preferably 1 to 4, more preferably 1 to 3, particularly preferably 1 or 2, and most preferably. It is one.

In (B) above, the identity value indicates a value calculated with default settings using software (for example, FASTA, DNASIS, and BLAST) that calculates the identity between a plurality of base sequences. For the base sequence identity value, the number of matching bases when a pair of base sequences are aligned so as to maximize the degree of matching is calculated, and the total number of bases in the compared base sequences of the number of matching bases is calculated. Calculated as a percentage of the number. Here, when there is a gap, the above total number of bases is the number of bases counted with one gap as one base. For details on the method of determining identity, see, for example, Altschul et al, Nuc. Acids. Res. 25, 3389-3402, 1977 and Altschul et al, J. et al. Mol. Biol. See 215, 403-410, 1990.

In (B), the identity is more preferably 80% or more, more preferably 90% or more, more preferably 95% or more, more preferably 96% or more, more preferably 97% or more, still more preferably 98% or more. , More preferably 99% or more identity.

In the above (C), the "stringent condition" means a condition in which a so-called specific hybrid is formed and a non-specific hybrid is not formed, for example, Green.
It can be appropriately determined with reference to and Sambrook, Molecular Cloning, 4th Ed (2012), Cold Spring Harbor Laboratory Press. Specifically, stringent conditions can be set by the temperature at the time of Southern hybridization and the salt concentration contained in the solution, and the temperature at the time of the washing step of Southern hybridization and the salt concentration contained in the solution. More specifically, as stringent conditions, for example, in the hybridization step, the sodium concentration is 25 to 500 mM, preferably 25 to 300 mM, and the temperature is 40 to 68 ° C, preferably 40 to 65 ° C. More specifically, hybridization can be performed at 1-7 × SSC, 0.02-3% SDS, and a temperature of 40 ° C-60 ° C. Further, a washing step may be performed after hybridization, and the washing step can be performed, for example, at 0.1 to 2 × SSC, 0.1 to 0.3% SDS, and a temperature of 50 to 65 ° C.

<2. Primer set ＞
The primer set of the present invention is a primer set for amplifying a fragment of DNA derived from an insect, and is one or more, preferably two or more, of primer pair A, primer pair B and primer pair C, more preferably. Includes three.

First, primer pair A will be described.
Primer vs. A
Of the base sequence of SEQ ID NO: 1, the same or homologous base sequence (base) as a continuous base sequence (referred to as base sequence 1') of 10 bases or more, preferably 15 bases or more, more preferably 20 bases or more from the 3'end. The first primer containing the first polynucleotide containing the 3'end (referred to as sequence 1'') and
Of the base sequence of SEQ ID NO: 2, the same or homologous base sequence (base) as a continuous base sequence (referred to as base sequence 2') of 10 bases or more, preferably 15 bases or more, more preferably 20 bases or more from the 3'end. It contains a second primer containing a second polynucleotide containing (referred to as Sequence 2'') at the 3'end.

The first primer will be described.
The base sequence 1'and the base sequence 1'are preferably a region from the 3'end to 3 bases, more preferably a region from the 3'end to 5 bases, and more preferably a region from the 3'end to 8 bases. More preferably, the region from the 3'end to 10 bases (when the length of the base sequence 1'is 15 bases or more, the region from the 3'end to 15 bases, the length of the base sequence 1'is more preferable. When it is 20 bases or more, the region from the 3'end to 20 bases is more preferably the same, and the remaining region on the 5'end side is homologous. The meaning of "homology" is as described above. The base sequence 1'is particularly preferably the entire base sequence of SEQ ID NO: 1. "The first polynucleotide contains the base sequence 1" at the "3'end" means that the entire base sequence of the first polynucleotide consists of only the base sequence 1 ", or the base sequence 1" and the base. This includes both cases including another base sequence connected to the 5'end of sequence 1''. The other base sequence may be any as long as it does not substantially inhibit the nucleic acid amplification reaction, and the number of bases thereof is, for example, 20 bases or less, preferably 10 bases or less, more preferably 5 bases or less, more preferably 1 or less. It is 2 bases. The first polynucleotide is particularly preferably a polynucleotide consisting of the base sequence of SEQ ID NO: 1.

The first primer may consist of the first polynucleotide, and the first polynucleotide is added with a useful chemical structure such as a labeling tag, a labeling substance, and an immobilization tag for detecting an amplification product. It may be the one that has been done.

The second primer will be described.
The base sequence 2'and the base sequence 2'are preferably a region from the 3'end to 3 bases, more preferably a region from the 3'end to 5 bases, and more preferably a region from the 3'end to 8 bases. More preferably, the region from the 3'end to 10 bases (when the length of the base sequence 2'is 15 bases or more, the region from the 3'end to 15 bases, the length of the base sequence 2'is more preferable. When it is 20 bases or more, the region from the 3'end to 20 bases is more preferably the same, and the remaining region on the 5'end side is homologous. The meaning of "homology" is as described above. The base sequence 2'is particularly preferably the entire base sequence of SEQ ID NO: 2. The term "the second polynucleotide contains the base sequence 2" at the "3'end" means that the entire base sequence of the second polynucleotide consists only of the base sequence 2 "and the base sequence 2" and the base. This includes both cases including another base sequence connected to the 5'end of sequence 2''. The other base sequence may be any as long as it does not substantially inhibit the nucleic acid amplification reaction, and the number of bases thereof is, for example, 20 bases or less, preferably 10 bases or less, more preferably 5 bases or less, more preferably 1 or less. It is 2 bases. The second polynucleotide is particularly preferably a polynucleotide consisting of the base sequence of SEQ ID NO: 2.

The second primer may consist of a second polynucleotide, to which a useful chemical structure such as a labeling tag, a labeling substance, an immobilization tag, etc. for detecting an amplification product is added. It may be the one that has been done.

Primer pair A containing the first primer and the second primer can amplify the insect-specific base sequence (non-conserved region) in the COI coding gene region on the mitochondrial genome of the insect, and is common to all insect species. It was designed so that it could be annealed to the possible area (conservation area) of the primer design. The length of the nucleic acid fragment amplified by the nucleic acid amplification reaction using the insect-derived DNA as a template using the primer pair A is mainly 180 bp, and in addition, the insertion / deletion of the sequence sandwiched between the primers and the primer 5 'There is a variation depending on the length of the base sequence connected to the end. The amplified region by primer pair A has a length of a non-conserved region at a level at which a target amplification product can be easily obtained even if the DNA is damaged by processing or the like and an insect species can be identified. In addition, since the base sequence database is enriched in many insect species, the amplified region by primer pair A is suitable for identifying insect species of insect foreign substances based on the base sequence.

Next, primer pair B will be described.
Primer vs. B
Of the base sequence of SEQ ID NO: 3, the same or homologous base sequence (base) as a continuous base sequence (referred to as base sequence 3') of 10 bases or more, preferably 15 bases or more, more preferably 20 bases or more from the 3'end. A third primer containing a third polynucleotide containing (referred to as Sequence 3'') at the 3'end,
Of the base sequence of SEQ ID NO: 4, 10 bases or more, preferably 15 bases or more, more preferably 20 bases or more from the 3'end, and the same or homologous base sequence as the continuous base sequence (base sequence 4'). It contains a fourth primer containing a fourth polynucleotide containing (referred to as Sequence 4'') at the 3'end.

The third primer will be described.
The base sequence 3'and the base sequence 3'are preferably a region from the 3'end to 3 bases, more preferably a region from the 3'end to 5 bases, and more preferably a region from the 3'end to 8 bases. More preferably, the region from the 3'end to 10 bases (when the length of the base sequence 3'is 15 bases or more, the region from the 3'end to 15 bases, the length of the base sequence 3'is more preferable. When it is 20 bases or more, the region from the 3'end to 20 bases is more preferably the same, and the remaining region on the 5'end side is homologous. The meaning of "homology" is as described above. The base sequence 3'is particularly preferably the entire base sequence of SEQ ID NO: 3. The term "the third polynucleotide contains the base sequence 3" at the "3'end" means that the entire base sequence of the third polynucleotide consists only of the base sequence 3 ", or the base sequence 3" and the base. This includes both cases including another base sequence connected to the 5'end of sequence 3''. The other base sequence may be any as long as it does not substantially inhibit the nucleic acid amplification reaction, and the number of bases thereof is, for example, 20 bases or less, preferably 10 bases or less, more preferably 5 bases or less, more preferably 1 or less. It is 2 bases. The third polynucleotide is particularly preferably a polynucleotide consisting of the base sequence of SEQ ID NO: 3.

The third primer may consist of a third polynucleotide, to which a useful chemical structure such as a labeling tag, a labeling substance, an immobilization tag, etc. for detecting an amplification product is added to the third polynucleotide. It may be the one that has been done.

The fourth primer will be described.
The base sequence 4'and the base sequence 4'are preferably a region from the 3'end to 3 bases, more preferably a region from the 3'end to 5 bases, and more preferably a region from the 3'end to 8 bases. , More preferably the region from the 3'end to 10 bases (more preferably the region from the 3'end to 15 bases when the length of the base sequence 4'is 15 bases or more, the length of the base sequence 4'. When it is 20 bases or more, the region from the 3'end to 20 bases is more preferably the same, and the remaining region on the 5'end side is homologous. The meaning of "homology" is as described above. The base sequence 4'is particularly preferably the entire base sequence of SEQ ID NO: 4. The term "the fourth polynucleotide contains the base sequence 4" at the "3'end" means that the entire base sequence of the fourth polynucleotide consists only of the base sequence 4 "and the base sequence 4" and the base. This includes both cases including another base sequence connected to the 5'end of sequence 4''. The other base sequence may be any as long as it does not substantially inhibit the nucleic acid amplification reaction, and the number of bases thereof is, for example, 20 bases or less, preferably 10 bases or less, more preferably 5 bases or less, more preferably 1 or less. It is 2 bases. The fourth polynucleotide is particularly preferably a polynucleotide consisting of the base sequence of SEQ ID NO: 4.

The fourth primer may consist of a fourth polynucleotide, to which a useful chemical structure such as a labeling tag, a labeling substance, an immobilization tag, etc. for detecting an amplification product is added to the fourth polynucleotide. It may be the one that has been done.

Primer pair B containing the third primer and the fourth primer can amplify the insect-specific base sequence (non-conserved region) in the COI coding gene region on the mitochondrial genome of the insect, and is common to all insect species. It was designed so that it could be annealed to the possible area (conservation area) of the primer design. The length of the nucleic acid fragment amplified by the nucleic acid amplification reaction using the insect-derived DNA as a template using primer pair B is mainly 135 bp, and in addition, the insertion / deletion of the sequence sandwiched between the primers and the primer 5 'There is a variation depending on the length of the base sequence connected to the end. The amplification region by primer vs. B has a length of a non-conserved region at a level at which a target amplification product can be easily obtained even if the DNA is damaged by processing or the like and the insect species can be identified. In addition, since the base sequence database is enriched in many insect species, the amplified region by primer vs. B is suitable for identifying the insect species of the insect foreign body based on the base sequence. The amplification region of the COI coding gene by primer pair B does not overlap with the amplification region by primer pair A.

Next, primer vs. C will be described.
Primer vs. C
Of the base sequence of SEQ ID NO: 5, the same or homologous base sequence as the continuous base sequence (base sequence 5') of 10 bases or more, preferably 15 bases or more from the 3'end (base sequence 5''). A fifth primer containing a fifth polynucleotide containing the 3'end, and
Of the base sequence of SEQ ID NO: 6, the same or homologous base sequence as the continuous base sequence (base sequence 6') of 10 bases or more, preferably 12 bases or more from the 3'end (base sequence 6''). Includes a 6th primer containing a 6th polynucleotide comprising 3'end.

Y in SEQ ID NO: 5 is C or T or a mixed base of C and T, and is preferably a mixed base of C and T. When Y is a mixed base of C and T, the fifth polynucleotide is a mixture of a polynucleotide molecule in which Y is C and a polynucleotide molecule in which Y is T.

Similarly, Y in SEQ ID NO: 6 is C or T or a mixed base of C and T, and is preferably a mixed base of C and T. When Y is a mixed base of C and T, the fifth polynucleotide is a mixture of a polynucleotide molecule in which Y is C and a polynucleotide molecule in which Y is T.

The fifth primer will be described.
The base sequence 5'and the base sequence 5'are preferably a region from the 3'end to 3 bases, more preferably a region from the 3'end to 5 bases, and more preferably a region from the 3'end to 8 bases. , More preferably the region from the 3'end to 10 bases (more preferably the region from the 3'end to 15 bases when the length of the base sequence 5'is 15 bases or more) is the same, and the remaining 5 'The terminal regions are homologous. The meaning of "homology" is as described above. The base sequence 5'is
Particularly preferred is the entire base sequence of SEQ ID NO: 5. The phrase "the fifth polynucleotide contains the base sequence 5" at the "3'end" means that the entire base sequence of the fifth polynucleotide consists of only the base sequence 5 ", or the base sequence 5" and the base. It includes both cases including another base sequence connected to the 5'end of the sequence 5''. The other base sequence may be any as long as it does not substantially inhibit the nucleic acid amplification reaction, and the number of bases thereof is, for example, 20 bases or less, preferably 10 bases or less, more preferably 5 bases or less, more preferably 1 or less. It is 2 bases. The fifth polynucleotide is particularly preferably a polynucleotide consisting of the base sequence of SEQ ID NO: 5.

The fifth primer may consist of the fifth polynucleotide, and the fifth polynucleotide is added with a useful chemical structure such as a labeling tag, a labeling substance, and an immobilization tag for detecting an amplification product. It may be the one that has been done.

The sixth primer will be described.
The base sequence 6'and the base sequence 6'are preferably a region from the 3'end to 3 bases, more preferably a region from the 3'end to 5 bases, and more preferably a region from the 3'end to 8 bases. , More preferably the region from the 3'end to 10 bases (more preferably the region from the 3'end to 12 bases when the length of the base sequence 6'is 12 bases or more) is the same, and the remaining 5 'The terminal regions are homologous. The meaning of "homology" is as described above. The base sequence 6'is particularly preferably the entire base sequence of SEQ ID NO: 6. The 6th polynucleotide "contains the base sequence 6" at the "3'end" means that the entire base sequence of the 6th polynucleotide consists only of the base sequence 6 "and the base sequence 6" and the base. This includes both cases including another base sequence connected to the 5'end of sequence 6''. The other base sequence may be any as long as it does not substantially inhibit the nucleic acid amplification reaction, and the number of bases thereof is, for example, 20 bases or less, preferably 10 bases or less, more preferably 5 bases or less, more preferably 1 or less. It is 2 bases. The sixth polynucleotide is particularly preferably a polynucleotide consisting of the base sequence of SEQ ID NO: 6.

The sixth primer may consist of the sixth polynucleotide, and the sixth polynucleotide is added with a useful chemical structure such as a labeling tag, a labeling substance, and an immobilization tag for detecting an amplification product. It may be the one that has been done.

Primer pair C containing the 5th primer and the 6th primer can amplify the insect-specific base sequence (non-conserved region) in the 16S coding gene region on the mitochondrial genome of the insect, and is common to all insect species. It was designed so that it could be annealed to the possible area (conservation area) of the primer design. The length of the nucleic acid fragment amplified by the nucleic acid amplification reaction using the insect-derived DNA as a template using the primer pair C is mainly 200 bp, and in addition, the insertion / deletion of the sequence sandwiched between the primers and the primer 5 'There is a variation depending on the length of the base sequence connected to the end. The amplification region by primer vs. C has a length of a non-conserved region at a level at which a target amplification product can be easily obtained even if the DNA is damaged by processing or the like and the insect species can be identified. In addition, since the base sequence database is enriched in many insect species, the amplified region by primer vs. C is suitable for identifying the insect species of the insect foreign body based on the base sequence.

Each base sequence of SEQ ID NOs: 1 to 6 is designed so that the number of mismatches is constant or less, preferably 7 bases or less, and more preferably 5 bases or less in many insect species to be detected.

The above-mentioned primer pair A, primer pair B and primer pair C are logically designed so that a predetermined region of the COI coding gene region or the 16S coding gene region on the worm mitochondrial genome can be amplified by a nucleic acid amplification reaction, respectively. In addition to the above, the PCR reaction conditions were enthusiastically examined for the primer pair that was judged to be the best form by devising the base sequence of the primers, and finally the actual performance evaluation test was conducted to obtain the intended sensitivity and specificity. It is confirmed that it is demonstrating its sexuality. It is well known that even a well-designed primer probe may not possess the required sensitivity and specificity in theory. For example, the primer pair D consisting of SEQ ID NO: 4 and SEQ ID NO: 7 is a primer pair designed in the DNA barcoding region at the research stage, but when the nucleic acid amplification reaction is actually performed, some cockroaches are found. Since no target amplification product was obtained in insects, it was not suitable as a primer pair for insect species identification. As described above, it is not always possible to secure the required specificity by simply designing the primer logically.

The primer set of the present invention contains one or more, preferably two or more, and more preferably three of the above-mentioned primer pair A, primer pair B and primer pair C. When the primer set contains two primer pairs, the remaining one of the primer pairs is obtained even if the nucleic acid fragment cannot be amplified because one of the amplified regions in the insect-derived DNA to be detected is cleaved by processing such as heat treatment. Since the nucleic acid fragment in the target region can be amplified by the above, the possibility of unidentification can be reduced. When the primer set contains three primer pairs, even if one or two of the amplified regions in the insect-derived DNA to be detected are cleaved by processing such as heat treatment, the remaining two or one of the primer pairs are used. Since the nucleic acid fragment in the target region can be amplified, the possibility of unidentification can be reduced.

<3. How to identify the type of insect>
The method of the present invention for identifying the type of insect is
Step 1 of performing a nucleic acid amplification reaction using a DNA derived from an insect as a template and a primer pair contained in the primer set of the present invention.
It is characterized by including step 2 for analyzing the base sequence of the amplification product of the nucleic acid amplification reaction in step 1.

In this embodiment of the present invention, the base sequence of the amplification product of the nucleic acid amplification reaction in step 1 is analyzed in step 2 and searched in the base sequence database to identify the type of insect that is the origin of DNA. Here, "identifying" the type of insect refers to estimating which biological taxon the insect that originated the DNA belongs to. Here, the "taxon group" refers to a taxon used for classification of organisms in a general biological field, and refers to, for example, a taxon at the eye level, family level, genus level, or species level.

As the DNA used as a template in step 1, DNA extracted from the insect sample to be examined can be used. The amount of the insect sample used may be a small amount, for example, about 0.1 mg. For the extraction of DNA from the insect sample, an existing method such as a method using a DNAy Blood & Tissue kit (QIAGEN) can be appropriately used.

The nucleic acid amplification reaction in step 1 can be carried out according to a general PCR (polymerase chain reaction) method. The DNA polymerase used for PCR may be any thermostable DNA polymerase. It is possible to use a commercially available DNA polymerase. Further, the primer concentration, the number of cycles, the temperature, the time, the composition of the buffer solution and the like can be appropriately selected according to the DNA polymerase to be used, the concentration of each primer and the like.

In an embodiment in which the primer set comprises two or three of primer pair A, primer pair B and primer pair C, step 1 can be performed on a primer pair basis.

In the nucleic acid amplification reaction, when the template DNA has a target base sequence of the primer pair to be used, a nucleic acid fragment containing the target base sequence is generated as an amplification product.

In step 2, the base sequence of the amplified product can usually be analyzed using an automatic base sequence determining machine (for example, 310 Genetic Analyzer manufactured by Thermo Fisher Scientific Co., Ltd.).

The method for identifying the type of insect that originated the DNA from the base sequence of the amplification product obtained by the analysis in step 2 is not particularly limited. For example, the base sequence of the amplification product can be searched in a base sequence database, and as a result of the search, the insect species having the most identical base sequence can be identified as the insect species from which the DNA originated. The base sequence database can be searched by, for example, the GenBank registered sequence search system (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch) using blastn or BOLD systems (http: // www). .boldsystems.org/index.php/IDS_OpenIdEngine). Alternatively, the base sequence of the amplification product obtained by the analysis in step 2 is compared with the corresponding base sequence of a known insect species, and when the sequence identity is equal to or higher than a certain threshold value, the insect that is the origin of DNA. Can be identified as the known insect species.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the present invention is not limited to these examples.

Example 1: Obtaining a sequence used for primer design and primer design For each of the COI coding gene region and the 16S coding gene region, the base sequence information of various insects was collected, and three types of primer pairs for identifying foreign substances were designed.

<Sample preparation>
As model samples of foreign substances that can be mixed in food, 24 samples shown in Table 1 below were obtained from the surrounding environment. After crushing 0.1 mg-3 mg of a part of the insects with Biomasher II (Nippi Co., Ltd.), add 20 μL of Proteinase K solution, stir with a touch mixer, and heat in a constant temperature water bath adjusted to 55 ° C for 1-3 hours. did. After that, DNA extraction was performed according to the protocol of DNeasy Blood & Tissue kit (QIAGEN).

<PCR>
Using the above 24 kinds of insect DNA as a template, PCR was performed to obtain sequence information of the COI coding gene region by the animal DNA barcoding method of Non-Patent Document 1. As a primer pair, a combination of a forward primer consisting of the base sequence of SEQ ID NO: 1 and a reverse primer consisting of the base sequence of SEQ ID NO: 4 was used.

The reaction solution composition was 1 × PCR Buffer II (Thermo Fisher Scientific), 4.0 mM MgCl ₂ , 0.2 mM dNTP Mix (dATP, dGTP, dCTP, dTTP), 1.0 μM forward primer, 1.0 μM.
With a reverse primer, 1.25 U of AmpliTaq GOLD DNA polymerase, 2 μL of extracted DNA solution was added to prepare a 50 μL solution.

The PCR cycle is 95 ° C., 10 minutes of denaturation, then 95 ° C., 1 minute of denaturation → 40 ° C., 1 minute of annealing → 72 ° C., 1 minute and 30 seconds of primer extension reaction cycle, and finally 40 cycles. An elongation reaction was carried out at 72 ° C. for 10 minutes.

<Confirmation of presence or absence of PCR amplification product and fragment length>
A part of each PCR reaction solution after the amplification reaction was subjected to an automatic electrophoresis apparatus MultiNA (Shimadzu Corporation). The fragment length of the amplified DNA was confirmed, and it was confirmed that the target amplification product was obtained.

<Nucleotide sequence analysis of PCR amplification product>
The PCR amplification product was purified from the PCR reaction solution from which the target amplification product was obtained, using the MinElute PCR purification Kit (QIAGEN). The base sequence analysis was commissioned to Eurofin Genomics Co., Ltd. The nucleotide sequence of each PCR amplification product was determined by the direct sequencing method.

As a result, 17 sequences were obtained from 24 samples as shown in Table 1. At the eye level and above, it was divided into 8 categories. An "array ID" was assigned to each of the 17 sequences for convenience. When the sequence IDs are the same, it indicates that the determined sequences are the same.

<Nucleotide sequence analysis of PCR amplification product>
A base sequence having high identity with the base sequence determined above was searched using a base sequence database. NCBI's blastn (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch) was used for the search. Table 1 shows the accession numbers of the base sequences having the highest identity with each of the base sequences determined above.

<Primer design>
For the design of the primer for identifying an insect foreign body on the COI coding gene region, 17 sequences of the sequences IDs 1 to 17 obtained above were used. The 16S coding gene region was obtained from GenBank for 14 insect species different at the eye level as shown in Table 2. A region with high storage stability was selected from each, and the position and base sequence of the primer were adjusted. The designed primer pairs are named A, B, and C, respectively, and the sequences are shown in Table 3. In primer pair C, "Y" indicates a mixed base of C and T. That is, in this embodiment, in primer vs. C, the polynucleotide molecule consisting of the base sequence of SEQ ID NO: 5 constituting the forward primer is a mixture of the polynucleotide molecule of Y = C and the polynucleotide molecule of Y = T. The polynucleotide molecule consisting of the base sequence of SEQ ID NO: 6 constituting the reverse primer is also a mixture of the polynucleotide molecule of Y = C and the polynucleotide molecule of Y = T.

Primer vs. A SEQ ID NO: 1 and primer vs. B SEQ ID NO: 4 are the same as the forward primer and reverse primer used in the DNA barcoding method described in Non-Patent Document 1, respectively.

SEQ ID NO: 2 can be annealed to a base sequence complementary to the base sequence of SEQ ID NO: 2 on the COI coding gene region of each insect. SEQ ID NO: 1 and SEQ ID NO: 2 are used as a primer-to-A forward primer and a reverse primer, respectively.

SEQ ID NO: 3 can be annealed to a base sequence complementary to the base sequence of SEQ ID NO: 3 on the COI coding gene region of each insect. SEQ ID NO: 3 and SEQ ID NO: 4 are used as a primer-to-B forward primer and a reverse primer, respectively.

SEQ ID NOs: 5 and 6 can be annealed to a base sequence complementary to each of the base sequences of SEQ ID NOs: 5 and 6 on the 16S coding gene region of each insect. SEQ ID NO: 5 and SEQ ID NO: 6 are used as a primer-to-C forward primer and a reverse primer, respectively.

Example 2: Identification of a foreign substance sample Three types of primer pairs A, B, and C designed in Example 1 and a primer pair described in Non-Patent Document 1 (a primer consisting of the base sequence of SEQ ID NO: 1 and SEQ ID NO: 2). The ability to identify foreign substances was compared for the combination with a primer consisting of the base sequence of.

<Sample preparation>
As a model sample of foreign substances that can be mixed in food, insect samples of 38 species of 7 eyes shown in Table 4 were obtained from Sumika Techno Service Co., Ltd. DNA extraction was performed in the same manner as in Example 1.

<PCR>
The composition of the reaction solution is common to primer pairs A, B, and C, and 1 × HotStarTaq DNA Master Mix (QIAGEN), 0.5 μM forward primer, 0.5 μM reverse primer, add 2 μL of the extracted DNA solution to a 20 μL solution. It was prepared to be.

The PCR cycle consisted of 45 cycles of denaturation at 95 ° C. for 15 minutes, denaturation at 95 ° C. for 30 seconds → annealing for 1 minute → extension reaction at 72 ° C. for 30 seconds. The annealing temperature was set to 40 ° C. for both primer-to-A and B-based PCR and 50 ° C. for primer-to-C PCR.

In addition, the animal DNA barcoding method of Non-Patent Document 1 was performed as a comparison target of the primer pair prepared in Example 1. The PCR conditions were the same as in Example 1.

<Confirmation of presence or absence of PCR amplification product and fragment length>
It was carried out in the same manner as in Example 1. For apple kakumonhamaki, konaga, chanokokakumonhamaki, and kashinoshimameiga, PCR amplification products obtained by setting the annealing temperature of the PCR cycle using primer vs. C to 58, 58, 58, and 54 ° C., respectively, were used. ..

<Nucleotide sequence analysis of PCR amplification product>
The base sequence was obtained from the PCR product in the same manner as in Example 1.
First, NCBI's blastn (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch) was used for the search in the base sequence database. If the sequence match rate was less than 100%, BOLD systems (http://www.boldsystems.org/index.php/IDS_OpenIdEngine) were also added to the sequence database search. The insect species with the highest nucleotide sequence match rate was identified as the insect species that originated the DNA.

For the target amplification products obtained from each sample with each primer pair, if the insect species identified by searching in the nucleotide sequence database matched the name of the obtained insect species, it was determined that the identification results matched. Further, when 2/3 of the results of the primer pairs A, B, and C were matched, it was determined that the identification results of A + B + C were matched.

<Experimental results>
Table 5 shows the percentages of the target amplification products obtained for the 38 insect species and the percentages of which the identification results matched the sample names by base sequence analysis.

The proportions of the identification results of each of the primer pairs A, B, and C that matched the sample name were 87%, 92%, and 74%, which were higher than those of 71% or less according to the primer pair of Non-Patent Document 1. In addition, by combining the primer pairs A, B, and C, the agreement rate between the identification result and the sample name was further increased to 92%. That is, it was confirmed that the identification accuracy was improved by combining the three types of methods.

The three species (Hiratachatate, Shirahoshikamemushi, and Angoumois grain moth) that could not be identified by primer pair A + B + C have different causes that could not be identified, but all of them may be identifiable if the GenBank database is enriched.

Hirata chatate could be identified correctly with primer vs. C. However, when each of the primer pairs A and B was used, there was a sequence with a higher concordance rate in the heterologous sequence even though the sequence of the corresponding species was present in the GenBank database.

In primer pair A, a plurality of base sequences were mixed and the sequence of the stink bug could not be determined. For primer vs. B, sequences of higher concordance rate were present for heterologous sequences, even though the sequences of the relevant species were present in the GenBank database. Although the base sequence could be determined by primer vs. C, the sequence of the corresponding species did not exist in the GenBank database.

Angoumois was correctly identified with primer pair A. However, in Primer vs. B, there were sequences of higher concordance rate in heterologous sequences, even though the sequences of the relevant species were present in the GenBank database. Although the base sequence could be determined by primer vs. C, the sequence of the corresponding species did not exist in the GenBank database.

Since these three species had different eyes, the method of the present invention does not tend to be unsuitable for identification in a particular class of insects.

In addition, as insect samples whose sample names are not clear, we obtained samples that were presumed to be spiders and centipedes from morphological observation, and tried to identify them by the same procedure. Primer vs. C was used for the spider sample, and primer pairs A, B, and C were used for the centipede sample. As a result, one clear target amplification product was obtained from each primer pair from spider and centipede samples. As a result of searching the base sequence of each target amplification product by blastn and confirming the matching rate with the base sequence that matched at the highest level, the spider sample matched the base sequence of European red-headed centipede by 93%, and the centipede sample showed that of Tobizumukade. It was 80-96% consistent with the base sequence. For insect samples whose sample names are not clear, it was confirmed that spiders and centipedes can also be identified by the method of the present invention, although the correctness of the identification results cannot be verified.

Example 3: Confirmation of presence / absence of amplification for various insect species in Non-Patent Document 2-5 Three types of primer pairs A, B, C designed in Example 1 and Non-Patent Documents 2, 3, 4, 5 The ability to identify foreign substances was compared for the primer pairs described in.

As the insect DNA sample, 7 samples different at the eye level shown in Table 6 were used from the model samples of the foreign substances shown in Table 4 of Example 2. All of them are samples in which the identification results and the sample names match in any of the primer pairs A, B, and C in Example 2.

The sequences of the primer pairs described in Non-Patent Documents 2, 3, 4, and 5 are shown in Table 7. In SEQ ID NOs: 8, 10 and 11, "I" represents inosine. In SEQ ID NOs: 8, 10, 11 and 12, "R" indicates a mixed base of A and G, and the polynucleotide molecule constituting each primer shown in SEQ ID NOs: 8, 10, 11 and 12 is R at each position. Is a mixture of a polynucleotide molecule with R = A and a polynucleotide molecule with R = G. In SEQ ID NOs: 8 and 10, "Y" indicates a mixed base of C and T, and the polynucleotide molecule constituting each primer represented by SEQ ID NOs: 8 and 10 is a poly in which Y = C for Y at each position. It is a mixture of a nucleotide molecule and a polynucleotide molecule of which Y = T.

<PCR>
The reaction solution composition of the primer pair of Non-Patent Documents 2, 3 and 4 is 1 × PCR Buffer, 2.0 mM MgCl ₂ , 0.2 mM dNTP Mix (dATP, dGTP, dCTP, dTTP), 0.2 μM forward primer, 0. . With 2 μM reverse primer, 2 U of Platinum Taq and 2 μL of extracted DNA solution were added to prepare a 25 μL solution.

The PCR cycle of the primer pair of Non-Patent Documents 2, 3 and 4 is a cycle of denaturation at 95 ° C. for 5 minutes, denaturation at 94 ° C. for 40 seconds → annealing for 1 minute → extension reaction of the primer at 72 ° C. for 30 seconds. Was carried out for 35 cycles, and finally an extension reaction was carried out at 72 ° C. for 5 minutes. The annealing temperature was set to 46 ° C. for both Non-Patent Documents 2 and 4 and 43.5 ° C. for Non-Patent Document 3.

The PCR reaction solution of the primer pair of Non-Patent Document 5 is 1 × PCR Buffer II (Thermo Fisher Scientific), 2.0 mM MgCl ₂ , 0.25 mM dNTP Mix (dATP, dGTP, dCTP, dTTP), 0.5 μM forward primer. , 0.5 U of AmpliTaq GOLD DNA polymerase with 0.5 μM reverse primer, 1 μL of extracted DNA solution was added to prepare a 10 μL solution.

The PCR cycle of the primer pair of Non-Patent Document 5 is a cycle of denaturation at 94 ° C. for 10 minutes, denaturation at 94 ° C. for 30 seconds → 45 ° C., annealing for 30 seconds → extension reaction of the primer at 72 ° C. for 45 seconds. A 35 cycle was carried out, and finally an extension reaction was carried out at 72 ° C. for 10 minutes.

<Experimental results>
The results of gel electrophoresis of the PCR amplification product are shown in FIG. Only the target amplification product was obtained from all 7 samples by PCR using the primer pairs of Non-Patent Documents 2 and 4. In PCR using the primer pair of Non-Patent Document 3, no target amplification product was obtained in 3 samples of Hiratachatate, Azuki bean weevil, and Western flower thrips out of 7 samples. In addition, in PCR using the primer pair of Non-Patent Document 5, not only the target amplification product was not obtained in Hiratachatate, but also the non-target amplification product was obtained in Thrips palmi Karny, Brown marmorated stink, Thrips palmi Karny, and Mediterranean Flour Moth. It was considered that there is a high possibility that sequence information could not be obtained even by base sequence analysis for all of these 5 samples. On the other hand, in PCR using primer pairs A, B, and C, only the target amplification product was obtained for one sample of Western flower thrips, except that the target amplification product was not obtained by PCR using primer pair A. Obtained.

From this result, it was considered that the primer pairs of Non-Patent Documents 3 and 5 could not cope with worm foreign substances that could be mixed from foods as compared with the primer pairs A, B and C of the present invention.

Regarding the primer pairs of Non-Patent Documents 2 and 4 in which the target products were obtained in all 7 samples, it was confirmed whether the foreign matter samples that had been continuously heated could be amplified.

Example 4: Confirmation of amplification of heat-processed foreign matter sample A model foreign matter sample heat-processed for the three types of primer pairs A, B, and C designed in Example 1 and the primer pairs of Non-Patent Documents 2 and 4. We compared the ability to identify foreign substances with insects.

<Preparation of DNA sample>
As the heated insect DNA sample, 30 μL of the brown marmorated stink bug DNA sample used in Example 3 was dispensed into a 1.5 mL tube and heated at 121 ° C. for 5 minutes, 10 minutes, 20 minutes or 30 minutes, respectively. A sample was used.

As the heat-processed insect foreign body sample, the limbs of the brown marmorated stink bug, which had been mixed in advance with the retort pouch white porridge prepared as a model, were used. Specifically, one limb (3 mg) of brown marmorated stink bug was sealed in a retort pouch together with 15 g of white rice and 135 g of water, and heated at 118 ° C. for 25 minutes to prepare the product. Two bags of samples were prepared. After cooling, the limbs were taken out from each sample, washed with water, and DNA was extracted by the same method as in Example 2 to obtain a DNA sample.

<PCR>
PCR was performed by the method of Example 2 using the primer pairs A, B, and C of the present invention.
PCR was performed by the method of Example 3 using the primer pairs of Non-Patent Documents 2 and 4.

<Experimental results>
FIG. 2 shows the confirmation results by gel electrophoresis of the amplified product by PCR using the primer pairs of Non-Patent Documents 2 and 4 and the primer pairs A, B, and C of the present invention, respectively.

The target amplification product was obtained by PCR using the primer pair of Non-Patent Document 2 only when a DNA sample heated at 121 ° C. for 5 minutes or 10 minutes was used as a template. The target amplification product was obtained by PCR using the primer pair of Non-Patent Document 4 only when a DNA sample heated at 121 ° C. for 5 minutes was used as a template. On the other hand, in PCR using the primer pairs A, B, and C of the present invention, a target amplification product was obtained regardless of which DNA sample was used as a template. From this result, the PCR using the primer pairs A, B, and C of the present invention is different from the PCR using the primer pairs of Non-Patent Documents 2 and 4, and the target amplification product can be obtained even with the heat-treated DNA. confirmed.

Example 5: Confirmation of Primer vs. D Specificity The following SEQ ID NOs: 4 and SEQ ID NOs indicate that even a well-designed primer may not possess the required sensitivity and specificity. It is shown by a primer pair D consisting of 7.

<Primer design>
The primer pair obtained by adjusting the 17 sequences of the 24 animal DNA barcoding regions obtained in Example 1 in the same manner as in Example 1 was named D. Each primer sequence of primer vs. D is as follows.

<Sample>
The DNA solutions of Samples 10, 11 (all cockroaches), 18 (Lepidoptera), and 24 (Hymenoptera) described in Example 1 were used.

<PCR>
The reaction solution composition was the same as the reaction solution composition of PCR using the primer pairs A, B, and C in Example 2. The temperature of the annealing and extension reaction was set to 40 ° C or 45 ° C.

<Confirmation of presence or absence of PCR amplification product and fragment length>
It was carried out in the same manner as in Example 1.

<Experimental results>
As a result, as shown in FIG. 3, in PCR using primer vs. D, a target amplification product was obtained when the DNA of the sample 18 of the order Lepidoptera and the sample 24 of the order Hymenoptera were used as templates, but the sample of the order Cockroach was obtained. Of these, when the DNA of sample 10 was used as a template, no target amplification product was obtained. The two samples 10 and 11 of the order Cockroach had the same sequence of animal DNA barcoding regions, but no results were obtained on one of them. It was judged.

It has been confirmed that the primer pairs A, B, and C of the present invention are not only logically designed but also further devised to obtain the intended target amplification product.

The present invention can be used to identify insect species of insect foreign substances mixed in foods and drinks and other products.

Claims (4)

A primer set for amplifying fragments of DNA derived from insects.
A first primer comprising a first polynucleotide, or consisting of the first polynucleotide and a tag for labeling, a labeling substance or a tag for immobilization.
The base sequence of the first polynucleotide consists of a base sequence 1 ″ or another base sequence of 10 bases or less connected to the base sequence 1 ″ and the 5 ′ end of the base sequence 1 ″. ,
The base sequence 1 ″ is the base sequence 1 ′ of SEQ ID NO : 1.
GGTCAACAAATCATAAGATATTGG
Is the same as, or
In the base sequence 1'', the region from the 3'end to 10 bases is the same as the region from the 3'end to 10 bases in the base sequence 1', and the remaining region on the 5'end side is the base sequence 1. With the first primer , which is a base sequence in which one or two bases are deleted, substituted, added and / or inserted in the remaining 5'terminal region of' .
A second primer consisting of a second polynucleotide, or consisting of the second polynucleotide and a tag for labeling, a labeling substance or a tag for immobilization.
The base sequence of the second polynucleotide consists of a base sequence 2 ″ or another base sequence of 10 bases or less connected to the base sequence 2 ″ and the 5 ′ end of the base sequence 2 ″. ,
The base sequence 2'' is the base sequence 2': of SEQ ID NO: 2.
GAAGAAAATTATAACAAAGCATG
Is the same as, or
In the base sequence 2'', the region from the 3'end to 10 bases is the same as the region from the 3'end to 10 bases in the base sequence 2', and the remaining region on the 5'end side is the base sequence 2. Primer pair A, including a second primer , which is a base sequence in which one or two bases are deleted, substituted, added and / or inserted in the remaining 5'terminal region of' .
A third primer consisting of a third polynucleotide, or consisting of the third polynucleotide and a tag for labeling, a labeling substance, or a tag for immobilization.
The base sequence of the third polynucleotide consists of a base sequence 3 ″ or another base sequence of 10 bases or less connected to the 5 ′ end of the base sequence 3 ″ and the base sequence 3 ″. ,
The base sequence 3 ″ is the base sequence 3 ′ of SEQ ID NO : 3.
GCCAGTTCAGCAGGACTATTAC
Is the same as, or
In the base sequence 3'', the region from the 3'end to 10 bases is the same as the region from the 3'end to 10 bases in the base sequence 3', and the remaining region on the 5'end side is the base sequence 3'. A third primer , which is a base sequence in which one or two bases are deleted, substituted, added and / or inserted in the remaining 5'terminal region of' .
A fourth primer consisting of a fourth polynucleotide, or consisting of the fourth polynucleotide and a tag for labeling, a labeling substance, or a tag for immobilization.
The base sequence of the fourth polynucleotide consists of a base sequence 4'' or another base sequence of 10 bases or less connected to the 5'end of the base sequence 4'' and the base sequence 4''. ,
The base sequence 4 ″ is the base sequence 4 ′ of SEQ ID NO : 4.
TAAACTTCAGGTGACCAAAAAAATCA
Is the same as, or
In the base sequence 4'', the region from the 3'end to 10 bases is the same as the region from the 3'end to 10 bases in the base sequence 4', and the remaining region on the 5'end side is the base sequence 4. Primer pair B, including a fourth primer , which is a base sequence in which one or two bases are deleted, substituted, added and / or inserted in the remaining 5'terminal region of' , and
A fifth primer consisting of the fifth polynucleotide, or the fifth polynucleotide and a tag for labeling, a labeling substance, or a tag for immobilization.
The base sequence of the fifth polynucleotide consists of a base sequence 5 ″ or another base sequence of 10 bases or less connected to the 5 ′ end of the base sequence 5 ″ and the base sequence 5 ″. ,
The base sequence 5 ″ is the base sequence 5 ′ of SEQ ID NO : 5.
ATTACGCTGTATTCCCY
(Y is C or T or a mixed base of C and T)
Is the same as, or
In the base sequence 5'', the region from the 3'end to 10 bases is the same as the region from the 3'end to 10 bases in the base sequence 5', and the remaining region on the 5'end side is the base sequence 5 A fifth primer , which is a base sequence in which one or two bases are deleted, substituted, added and / or inserted in the remaining 5'terminal region of' .
A sixth primer consisting of the sixth polynucleotide, or the sixth polynucleotide and a tag for labeling, a labeling substance, or a tag for immobilization.
The base sequence of the sixth polynucleotide consists of a base sequence 6'' or another base sequence of 10 bases or less connected to the 5'end of the base sequence 6'' and the base sequence 6''. ,
The base sequence 6'' is the base sequence 6': of SEQ ID NO: 6.
GACGAGAAGACCCY
(Y is C or T or a mixed base of C and T)
Is the same as, or
In the base sequence 6'', the region from the 3'end to 10 bases is the same as the region from the 3'end to 10 bases in the base sequence 6', and the remaining region on the 5'end side is the base sequence 6 Primer vs. C containing a sixth primer , which is a base sequence in which one or two bases are deleted, substituted, added and / or inserted in the remaining 5'terminal region of' .
A primer set containing one or more of them. The primer set according to claim 1, which comprises two or more of the primer pair A, the primer pair B, and the primer pair C. The primer set according to claim 2, wherein the primer pair A, the primer pair B, and the primer pair C are included. Step 1 of performing a nucleic acid amplification reaction using a DNA derived from an insect as a template and using a primer pair contained in the primer set according to any one of claims 1 to 3.
A method for identifying an insect species, which comprises step 2 of analyzing the base sequence of the amplification product of the nucleic acid amplification reaction in step 1.

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