JP2014000091A - Primer sequence for detecting animal-derived dna - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for efficiently designing and acquiring a primer capable of detecting an animal-derived DNA, and the primer acquired by the method, which is specific and can be detected at high sensitivity.SOLUTION: A method which detects a primer pair for detecting a pig specific DNA which is a combination of primers in a predetermined arrangement, and a component derived from specific animal species or group in a sample, comprises the steps of: amplifying a DNA fragment by a PCR method in which the predetermined primer pair is used and the DNA in the sample is used as a template; and detecting the amplified DNA fragment. The specific animal species or group is a pig.

Description

  The present invention relates to a primer sequence for animal-derived DNA detection and a method for efficiently designing and obtaining the primer sequence.

  At present, meat-and-bone meal derived from bovine spongiform encephalopathy (BSE) cattle is added to the feed, and cattle fed with the feed suffer from BSE, which is a problem. And it is pointed out that a disease similar to BSE may exist in various livestock. Therefore, since the occurrence of BSE, it has become necessary to develop a technology for detecting sensitive animal-derived DNA using advanced technology, which has become an urgent administrative matter in particular. Furthermore, disguise of meat types and the like has become a problem recently, and means for easily confirming authenticity such as display of processed meat products is also required.

  For detection of animal-derived DNA, conventionally, immunological techniques and gene detection techniques using nuclear genes have been used. Examples of the immunological technique include an ELISA method and an immunoblot method. An example of a gene detection technique using a nuclear gene is a PCR method. However, because many samples to be detected are heat-treated, there is a high possibility that protein denaturation or nucleic acid fragmentation has occurred, and because cattle feed contains many plant-derived components, it is derived from animals. There are many problems with animal-derived DNA detection methods currently being performed, such as the need for trace analysis of components. Thus, there is an urgent need to develop a highly sensitive and effective detection method that can be performed on a sample after heat treatment.

  So far, methods for detecting animal species have been developed using an animal-specific DNA sequence derived from the ATP synthase subunit 8 gene (atp8 gene) present in the animal mitochondrial genome as a primer pair (Patent Documents 1 and 2). ). These utilize the fact that the homologous sequence of the atp8 gene does not exist in the plant (rice) mitochondrial genome, and can detect a very small amount of animal genes in plant-based feed. Other groups have also developed primers that can be used for meat type discrimination and meat-and-bone meal detection (Non-Patent Documents 1 and 2).

  These primers were developed after trial and error in which the specificity and detection sensitivity were individually confirmed for a huge number of combinations of the position of the DNA to be amplified, the length of the primer, the base, and the like. Therefore, it is not easy to obtain a novel primer that is specific and sensitive to a specific range of animal species.

JP 2003-164287 A JP 2005-323587 A

Lahiff, S. et al., Mol. Cell Probes, 15, 27-35, 2001 Tartaglia, M. et al., J. Food Prot., 61, 513-518, 1998 Rice, P. et al., Trends in Genetics, 16, 276-277, 2000 Imaizumi et al., Int. J. Legal Med., 121, 184-191, 2007 Dalmasso, A. et al., Mol. Cell Probes, 18, 81-87, 2004 Marco T. et al., J. Anim. Sci., 76, 2207-2208, 1998 Jane M. et al., Environ. Sci. Technol., 41, 3277-3283, 2007 Shinoda, N. et al., J. Food Prot., 71, 2257-2262, 2008

  The present invention provides a method for efficiently designing and obtaining a primer capable of detecting animal-derived DNA, and a specific and highly sensitive detectable primer obtained by such a method. With the goal.

The present invention provides a method for obtaining a primer sequence for DNA detection derived from a specific animal species or group,
(A) determining a DNA sequence search target region;
(B) obtaining a list of DNA sequences of the specific animal species or group for the region and multiple aligning the DNA sequences of the specific animal species or group;
(C) obtaining a consensus sequence from the multiple-aligned DNA sequence;
(D) obtaining a list of DNA sequences of animal or plant species or groups different from the specific animal species or group for the region, and multiple aligning the DNA sequences of the different animal or plant species or groups and the consensus sequence;
(E) comparing the bases between tandem columns within a range of arbitrary base lengths in the DNA sequence subjected to multiple alignment in step (d), and scoring the consensus sequence in the range, wherein the scoring , For a base different from the consensus sequence, adding a weight set according to the distance from the 5 ′ or 3 ′ end of the range, the presence or absence of a gap, and the type of base;
(F) selecting a consensus sequence having a high score from the consensus sequence scored in the step (e);
(G) correcting the base of the consensus sequence selected in step (f), wherein the correction adjusts the base length so that the Tm value of the consensus sequence in the range is at an arbitrary temperature; And substituting bases so as not to form homodimers with the regulated consensus sequence;
(H) selecting, for the modified consensus sequence, a consensus sequence to be paired so that the distance between the consensus sequences in the region is an appropriate base length; and (i) the selected consensus Confirming specificity and detection sensitivity for the pair of sequences;
including.

  In one embodiment, the weight set according to the distance from the 5 'or 3' end is heavier toward the 5 'or 3' end.

  In another embodiment, the weight set according to the presence or absence of the gap is heavier as the absolute value of the gap difference is larger.

  In a further embodiment, the weight set according to the type of the base is heavier as the base is less prone to misannealing with the base in the consensus sequence.

  In one embodiment, the search target region is mitochondrial DNA.

  In a particular embodiment, the particular animal species or group is a ruminant, pig, whale dolphin, deer, sheep or goat.

  In one embodiment, the above steps (b) to (d) are performed using the sequence analysis program EMBOSS (The European Molecular Biology Open Software Suite).

  In an embodiment, the steps (b) to (g) can be automatically performed as a series of programs.

  The present invention also provides a combination of the sequence of SEQ ID NO: 1 of the sequence listing and the sequence of SEQ ID NO: 2 of the sequence listing; a combination of the sequence of SEQ ID NO: 3 of the sequence listing and the sequence of SEQ ID NO: 4 of the sequence listing; A ruminant-specific DNA detection primer which is a combination of the sequence of SEQ ID NO: 5 and the sequence of SEQ ID NO: 6 of the sequence listing; or a combination of the sequence of SEQ ID NO: 7 of the sequence listing and the sequence of SEQ ID NO: 8 of the sequence listing Offer a pair.

  The present invention also provides a combination of the sequence of SEQ ID NO: 9 and the sequence of SEQ ID NO: 10 of the sequence listing; the combination of the sequence of SEQ ID NO: 11 of the sequence listing and the sequence of SEQ ID NO: 12 of the sequence listing; A combination of the sequence of SEQ ID NO: 13 and the sequence of SEQ ID NO: 14 in the sequence listing; or a combination of the sequence of SEQ ID NO: 15 in the sequence listing and the sequence of SEQ ID NO: 16 in the sequence listing I will provide a.

  The present invention is also a combination of the sequence of SEQ ID NO: 52 and the sequence of SEQ ID NO: 53 of the sequence listing; or the combination of the sequence of SEQ ID NO: 52 of the sequence listing and the sequence of SEQ ID NO: 54 of the sequence listing. Provided is a primer pair for detecting whale / dolphins-specific DNA.

  The present invention also provides a deer-specific primer pair for detecting DNA, which is a combination of the sequence of SEQ ID NO: 55 of the sequence listing and the sequence of SEQ ID NO: 56 of the sequence listing.

  The present invention also provides a primer pair for detecting sheep-specific DNA, which is a combination of the sequence of SEQ ID NO: 57 of the sequence listing and the sequence of SEQ ID NO: 58 of the sequence listing.

  The present invention also provides a primer pair for detecting goat-specific DNA, which is a combination of the sequence of SEQ ID NO: 59 of the sequence listing and the sequence of SEQ ID NO: 60 of the sequence listing.

  The present invention further provides a method for detecting a component derived from a specific animal species or group in a sample, and a DNA fragment is amplified by a PCR method using any of the primer pairs described above, using the DNA in the sample as a template. And the step of detecting the amplified DNA fragment, wherein the particular animal species or group is a ruminant, pig, whale, dolphin, deer, sheep or goat.

  In one embodiment, the sample is raw meat, raw fish, processed meat food, processed fish food, processed food containing food, processed fish containing food, blood, body hair, body fluid, milk, processed milk, meat and bone meal, bone meal, It is selected from the group consisting of fish meal, fish solubles, broth, and feeds containing these, fertilizers, pet foods, and feed additives.

  According to the method of the present invention, primers for detecting DNA derived from an arbitrary range of animal species or groups (groups) can be designed and obtained more efficiently. The method of the present invention provides very sensitive ruminant, pig, whale, dolphin, deer, sheep or goat specific primer pairs for DNA detection. For example, ruminants, pigs, whales, dolphins, deer, sheep, or goat-specific primer pairs obtained by the method of the present invention can be used to detect trace ruminants, pigs, whales, dolphins, deer in feed. The meat-and-bone meal derived from sheep or goat can be detected with high sensitivity.

Electricity after PCR using a ruminant-specific DNA detection primer pair (pair 1: combination of 5D2 (SEQ ID NO: 1) and 3D5 (SEQ ID NO: 2)) using DNA derived from various animals as a template It is an electrophoretic photograph. Electricity after PCR using ruminant-specific DNA detection primer pair (pair 2: combination of 5K4 (SEQ ID NO: 3) and 3K4 (SEQ ID NO: 4)) using DNA derived from various animals as a template It is an electrophoretic photograph. After performing PCR using ruminant-specific DNA detection primer pairs (pair 1: combination of 5D2 (SEQ ID NO: 1) and 3D5 (SEQ ID NO: 2)) with varying concentrations of DNA derived from various ruminants It is an electrophoresis photograph of. Electrophoresis photographs showing the results of DNA detection for various feeds using ruminant-specific DNA detection primer pairs (pair 1: combination of 5D2 (SEQ ID NO: 1) and 3D5 (SEQ ID NO: 2)). is there. PCR was performed on the mixed feed containing various concentrations of beef bone meal using a ruminant-specific primer pair for DNA detection (pair 1: combination of 5D2 (SEQ ID NO: 1) and 3D5 (SEQ ID NO: 2)). It is a later electrophoretic photograph. Pig-specific primer pair for DNA detection (pair I: combination of PigATP6-5.3 (SEQ ID NO: 9) and PigATP6-3.9 (SEQ ID NO: 10); and pair IV: PIG 5J (SEQ ID NO: 15) and PIG 3J (SEQ ID NO: 16) is an electrophoresis photograph after performing PCR using DNA derived from pigs of various breeds as a template. PCR was performed using DNA from various animals as a template using a primer pair for detecting pig-specific DNA (pair I: combination of PigATP6-5.3 (SEQ ID NO: 9) and PigATP6-3.9 (SEQ ID NO: 10)). It is a later electrophoretic photograph. PCR was carried out by changing the concentration of pig-derived DNA using a primer pair for detecting pig-specific DNA (pair I: combination of PigATP6-5.3 (SEQ ID NO: 9) and PigATP6-3.9 (SEQ ID NO: 10)). It is a later electrophoretic photograph. PCR using a pair of pig-specific DNA detection primers (pair I: combination of PigATP6-5.3 (SEQ ID NO: 9) and PigATP6-3.9 (SEQ ID NO: 10)) with various concentrations of pork bone and bone meal It is the electrophoresis photograph after performing. A pair of primers for detecting whales, dolphins-specific DNA (pair I: combination of whale51 (SEQ ID NO: 52) and whale31 (SEQ ID NO: 53); and pair II: whale51 (SEQ ID NO: 52) and whale32 (SEQ ID NO: 54) Is a electrophoresis photograph after performing PCR using DNA derived from whales and dolphins of various varieties as a template. A pair of primers for detecting whales, dolphins-specific DNA (pair I: combination of whale51 (SEQ ID NO: 52) and whale31 (SEQ ID NO: 53); and pair II: whale51 (SEQ ID NO: 52) and whale32 (SEQ ID NO: 54) Is an electrophoresis photograph after performing PCR using DNA derived from various animals as a template. It is the electrophoresis photograph after performing PCR using DNA derived from various animals as a template using a deer specific DNA detection primer pair (combination of deer54 (SEQ ID NO: 55) and deer33 (SEQ ID NO: 56)). . It is an electrophoretic photograph after PCR using sheep-specific DNA detection primer pair (combination of sheep53 (SEQ ID NO: 57) and sheep33 (SEQ ID NO: 58)) using DNA derived from various animals as a template. . It is the electrophoresis photograph after performing PCR which used DNA derived from various animals as a template using the primer pair for goat specific DNA detection (combination of goat55 (sequence number 59) and goat35 (sequence number 60)). .

  In order to detect a component derived from a specific animal species or group in a sample, a method for detecting a DNA sequence specific to each animal species or group is performed. Examples of such a method include Southern blotting and PCR (polymerase chain reaction), but the PCR method is preferably used in that it can be detected even with a small amount of DNA sample and the accuracy is improved. Yes. In PCR, a pair of primers containing a DNA sequence specific to the target animal species is used to detect an amplified DNA fragment. The method of the present invention is a method for efficiently designing and obtaining primers for use in this PCR.

The method for obtaining a primer sequence for DNA detection derived from a specific animal species or group of the present invention comprises the following steps:
(A) determining a DNA sequence search target region;
(B) obtaining a list of DNA sequences of the specific animal species or group for the region and multiple aligning the DNA sequences of the specific animal species or group;
(C) obtaining a consensus sequence from the multiple-aligned DNA sequence;
(D) obtaining a list of DNA sequences of animal or plant species or groups different from the specific animal species or group for the region, and multiple aligning the DNA sequences of the different animal or plant species or groups and the consensus sequence;
(E) comparing the bases between tandem columns within a range of arbitrary base lengths in the DNA sequence subjected to multiple alignment in step (d), and scoring the consensus sequence in the range, wherein the scoring , For a base different from the consensus sequence, adding a weight set according to the distance from the 5 ′ or 3 ′ end of the range, the presence or absence of a gap, and the type of base;
(F) selecting a consensus sequence having a high score from the consensus sequence scored in the step (e);
(G) correcting the base of the consensus sequence selected in step (f), wherein the correction adjusts the base length so that the Tm value of the consensus sequence in the range is at an arbitrary temperature; And substituting bases so as not to form homodimers with the regulated consensus sequence;
(H) for the selected consensus sequence, selecting a consensus sequence to be paired so that the distance between the consensus sequences in the region is an appropriate base length; and (i) the selected consensus Confirming specificity and detection sensitivity for the pair of sequences;
including.

  In the present invention, the species or group of animals to be detected is not particularly limited. Extensive groups such as mammals, birds, fish; a range of classes or groups such as ruminants, rodents, whales, dolphins; cattle, goats, sheep, deer, pigs, horses, rabbits, rats, mice, It may be a specific range of genera, species, or groups such as humans, whales; or species or breeds such as Japanese Black cattle breeds, dairy cows, red cattle, middle Yorkshire (pigs), Berkshire (pigs), minipigs. Depending on the purpose of detection, the range of the target animal can be set as appropriate.

  The method of the present invention can be performed using various genetic analysis tools. In each step, an appropriate analysis program can be selected and used. For example, in the present invention, steps (b) to (d) can be performed using a command of the sequence analysis program EMBOSS (The European Molecular Biology Open Software Suite). Alternatively, a sequence analysis program capable of automatically and continuously performing a series of operations from steps (b) to (g) may be independently set up.

  Here, EMBOSS is a completely new open source analysis software package developed for a molecular biology user community (for example, EMBnet) (Non-patent Document 3). By using this software package, automatic processing of data written in various formats, transparent sequence data retrieval from the web, and the like are possible. The EMBOSS includes 150 or more programs (applications). For example, sequence alignment; fast database search by sequence pattern; protein motif identification, including domain analysis; EST analysis; nucleic acid sequence pattern analysis such as CpG island identification; simple species-specific repeat identification; Analysis of codon usage in small genomes; rapid sequence pattern identification in large sequence sets. In addition, since the EMBOSS package includes an extended library and various sequence analysis packages and tools are seamlessly integrated, it is possible to efficiently perform a target analysis and the like.

  Hereinafter, the method of the present invention will be described in detail in the order of steps.

Step (a):
First, in step (a), a DNA sequence search target region is determined. As the search target region, any region of the DNA sequence of the specific animal species or group of interest may be selected. In consideration of obtaining a list of DNA sequences of various animals in the following steps (b) and (d), DNA regions whose DNA sequences have been revealed for many animal species or groups are preferred. In the present invention, mitochondrial DNA is preferably used because it has a higher copy number than nuclear genomic DNA and is circular and resistant to denaturation. On mitochondrial DNA, ATP synthase, cytochrome c oxidase, etc., which are essential for biosynthesis of enzymes necessary for oxidative phosphorylation (electron transport system), are encoded. The entire mitochondrial DNA (that is, the entire region) may be selected as the region of the DNA sequence to be searched, or only a part may be selected.

Step (b):
Next, with respect to the region of the DNA sequence selected in the above step (a), a list of DNA sequences of the specific animal species or group of interest is obtained, and the obtained DNA sequences of the specific animal species or group are multiple aligned. .

  DNA sequences are available from various databases available to those skilled in the art. Examples of the database include GenBank (NCBI), DDBJ, and EMBL. Obtain as many DNA sequences as possible from the database and use them as a DNA sequence list. When a DNA sequence is acquired as an ID number of a database, a DNA sequence list can be easily acquired by a command set in EMBOSS.

  The DNA sequence list is then multiple aligned. Here, multiple alignment refers to aligning a plurality of sequences based on homology. As a program for performing multiple alignment, there are Clustal (ClustalW, ClustalX) and the like. These programs are also included in EMBOSS.

Step (c):
Next, in step (c), a consensus sequence is obtained from the multiple aligned DNA sequences. A consensus sequence can be created based on the homology between DNA sequences using, for example, a command (for example, CONS) in EMBOSS.

  If necessary, two types of consensus sequences are created for the purpose of more accurately determining primer design. One is a strict consensus sequence and the other is a loose consensus sequence. In this case, a strict consensus sequence is used to specify the degree of mismatch described in detail below, but the actual primer is obtained based on the loose consensus sequence. The acquisition of two types of consensus sequences will be described according to the following example.

  Take for example the case of creating a consensus sequence for ruminants. For example, cattle, sheep, and goat DNA sequences, respectively,

Assume that In this case, when compared in columns,

It becomes. Note that the loose consensus sequence is replaced with the most bases in the column. On the other hand, in the strict consensus sequence, if there is even one different base in the column, N is displayed.

The degree of inconsistency is specified according to the allowable number of N in the strict consensus sequence in an arbitrary range of, for example, 17 bases. For example, when defining three levels of inconsistency, in order from the highest inconsistency,
Discrepancy 1 is “when N is 0 in the first to fifth bases from the 5 ′ end and N is within 6 in the sixth to 17th bases from the 5 ′ end”,
The mismatch degree 2 is “when N is 0 in the 1st to 7th bases from the 5 ′ end and N is within 3 in the 8th to 17th bases from the 5 ′ end”,
Inconsistency 3 is defined as “when N is 0 in the 1st to 9th bases from the 5 ′ end and N is within 6 in the 10th to 17th bases from the 5 ′ end”. The In this case, any number of N in the 18th to 25th bases from the 5 ′ end is allowed. Normally, the mismatch degree 2 is designated.

Step (d):
Next, for the region of the DNA sequence selected in the step (a), a list of DNA sequences of animal or plant species or groups different from a specific animal species or group is obtained, and the DNA sequences of the different animal or plant species or groups and the Multiple alignment with consensus sequence.

  An animal or plant species or group different from a specific animal species or group is not particularly limited. It is preferred to obtain a DNA sequence list for a diverse range of animals and plants, preferably including animal species or groups that are relatively close to a particular animal species or group. The DNA sequence list acquisition and the multiple alignment method are the same as in the above step (b). For example, it can be performed using a command in EMBOSS.

Step (e):
In step (e), bases between columns are compared within an arbitrary base length in the DNA sequence subjected to multiple alignment in step (d). Any base length can be a base length suitable as a primer. Usually, the length is 15 to 30 bases, preferably 17 to 25 bases. The above consensus sequence is scored. In the range of arbitrary base lengths at various positions of the consensus sequence, the bases of the consensus sequence and the bases of the multiple aligned DNA sequences are compared between the columns. When compared, differences such as base matches or mismatches, gaps, etc. are found for each base.

  The found differences are then scored against the consensus sequence. Scoring is performed by adding weights set according to the distance from the 5 'or 3' end of this range, the presence or absence of a gap, and the type of base for a base different from the consensus sequence. In the present invention, the “weight” is set so as to be heavier as it is difficult to anneal with DNA other than the target DNA (that is, DNA of an animal species or group different from the specific animal species or group to be detected). For example, when the base on the terminal side of the primer is different or when a gap is present on the terminal side, annealing is difficult (in other words, mis-annealing is difficult to occur). Therefore, a weight is appropriately set as a score according to the position from the end. In particular, since the influence of the 5 bases at the end is large, it is preferable that only the 5 bases at the end are subject to scoring.

  For the score when the bases are different, for example, the Xth weight score from the end is set as the total score of the X + 1th and X + 2th from the end. When scoring is performed on the last 5 bases, if the 5th base from the end is different, the score 1 is score 4 because the 4th from the end is the sum of the 5th and 6th. In the third case from the end, the sum of the fourth and fifth is 3, and in the second case, 5, and the first (end) is 8.

  Regarding the score when there is a gap, for example, when only the terminal 5 bases are subject to scoring, the absolute value of the gap difference (that is, the gap difference) is 1, 2, 3, 4, and 5 The scores are set to 3, 5, 7, 9, and 11, respectively.

  For a base G or C that is unlikely to cause misannealing with a base in the consensus sequence, the base score is increased 1.5 times.

  Hereinafter, a specific example will be described. The consensus sequence of ruminants such as cattle, sheep, goats and deer and the DNA sequence of pigs and horses are compared for a certain range (in the box) as follows.

  First, the ruminant consensus sequence and the pig sequence are compared in tandem. For example, when scoring is performed on the 3 ′ end side, the first is different from the 3 ′ end and the base is C, so the score is 8 × 1.5 = 12. Since the 4th from the 3 'end is different and the base is C, the score is 2 × 1.5 = 3. Therefore, the sum of the scores 12 and 3 is a score of 15 for the swine sequence.

  On the other hand, in the comparison between the ruminant consensus sequence and the horse sequence, the second and fifth positions including the gap from the 3 'end are different and the base is C, so the total score is 9. Since there is a 1 base gap in the equine sequence, a score of 3 is added. Therefore, the score of 9 and 3 is the sum, and the score of this consensus sequence is 12 in comparison with the horse sequence.

  The score is 15 in comparison with the pig sequence, but is 12 in comparison with the horse sequence, so the lowest score of 12 compared is the score of the ruminant consensus sequence in this range.

  Thus, the lowest score among the various scores obtained by comparison with each sequence is the score of the consensus sequence in the certain range. Therefore, if even one sequence exists, the score is 0.

  Even in the case of a score of 0, there are cases in which only a few types of DNA sequences cause a high score in comparison with sequences excluding them. A primer based on such a consensus sequence can be used as a primer for that pair only if a high-scoring primer is used for one of the primer pairs. Therefore, a score based on a comparison of sequences other than the causative DNA sequence may be used with conditions, so it is preferable to record it with an annotation. Such scoring can be done by creating any suitable program. Annotated records can also be set to be automatically recorded.

Step (f):
A consensus sequence having a high score is selected from the consensus sequences scored in the step (e). For example, when the above score is set for the terminal 5 bases, a sequence having a score of 1 or more, preferably a score of 5 or more, more preferably a score of 10 or more is selected. If either the 5 'or 3' side score is high, it can be a candidate for either the forward primer or the reverse primer, and if both have high scores, it is a candidate for both the forward primer or the reverse primer. obtain. Also, as described above, sequences that can be used conditionally can be selected as well. Such a selection can also be made by creating any appropriate program.

Step (g):
Next, the base of the consensus sequence selected in the above step (f) is corrected. The correction adjusts the base length so that the Tm value of the consensus sequence in this range is at an arbitrary temperature, and replaces the base so as not to form a homodimer in the adjusted consensus sequence.

  The Tm value (primer melting temperature) of the selected consensus sequence is generally calculated by the nearest base pair method. The Tm value can be set to an arbitrary value, but is preferably set near the annealing temperature normally used in PCR. For example, it is set to about 55 ° C. Tm values increase with longer sequences and also with GC rich sequences. Therefore, bases are added or deleted in the terminal sequence opposite to the scoring target, and the Tm value is corrected so as to approach the set value.

  Furthermore, a consensus sequence with a regulated base length may form a homodimer depending on the sequence. Therefore, by modifying the base at a position not subject to the above scoring, correction is made so as not to form a homodimer. In the case of the above example, the base in the region on the 5 'end side of the region paired as a homodimer is changed. Such correction of the consensus sequence can be performed by creating any appropriate program.

Step (h):
In step (h), for the corrected consensus sequence, a consensus sequence to be paired is selected so that the distance between the consensus sequences in the region is an appropriate base length. For example, when the sample to be detected using the obtained primer is a meat-derived product after heat treatment, the DNA is cut and is relatively short. Therefore, the base length of the DNA amplified by the primer can be preferably about 50 to 500 bp, more preferably about 60 to 300 bp, and still more preferably about 70 to 200 bp. Accordingly, the distance between consensus sequences selected as candidates may also be preferably about 50 to 500 bp, more preferably about 60 to 300 bp, still more preferably about 70 to 200 bp, and particularly preferably about 75 to 170 bp. In this case, since the specificity of either the forward primer or the reverse primer only needs to be high, the sequences selected as a pair need not necessarily be the consensus sequence selected in the step (f).

Step (i):
In step (i), the specificity and detection sensitivity of the pair of consensus sequences selected in step (h) are confirmed. This consensus sequence is a candidate primer.

  The specificity is preferably confirmed by performing PCR on the actually extracted DNA. Alternatively, it is preferable to confirm the specificity in advance by a program. Examples of the program for confirming such specificity include BLAST and FASTA. Both BLAST and FASTA refer to an algorithm for performing sequence alignment of a DNA base sequence or a protein amino acid sequence by bioinformatics, and a program that implements the algorithm.

  Primer candidates whose specificity has been confirmed in advance can be obtained by applying primer pairs to various DNAs extracted from the target animal and other animals and plants by a method commonly used by those skilled in the art (for example, using a commercially available DNA extraction kit). Specificity is confirmed. The candidate primer DNA sequence is synthesized by a commonly used method. In general, nucleotides are extended on a support using an automated DNA synthesizer, followed by deprotection and cleavage from the support. Subsequently, the target primer can be obtained by purification by a commonly used method (for example, column chromatography).

  For the various DNAs, PCR is performed using the selected primer candidate combinations, and DNA fragments in the region sandwiched between the primers are amplified. PCR is performed under the conditions that are usually performed, and appropriate conditions are set for each primer pair. For example, after heat denaturation at 95 ° C. for 9 minutes, denaturation: 92 ° C., 30 seconds to 1 minute; annealing: 40 to 65 ° C., 30 seconds to 2 minutes; extension: 72 ° C., 30 seconds to 2 minutes. 30 to 50 cycles, preferably 35 to 45 cycles, and finally, the reaction is completed at 72 ° C. for 5 minutes to complete the PCR. For example, AmpliTaq GOLD polymerase is used as the DNA polymerase. The size of the PCR product (DNA fragment) amplified by the primer pair varies depending on the number of bases between the selected primer pair. Next, the PCR product is subjected to, for example, agarose gel electrophoresis, polyacrylamide gel electrophoresis, or the like according to the size of the DNA fragment under conditions that allow the DNA fragment to be separated.

  DNA fragments on the electrophoresed gel can be detected by detection means commonly used by those skilled in the art such as ethidium bromide staining, fluorescence detection, Southern hybridization, and can be confirmed by DNA sequencing.

  A combination of candidate primers confirmed to have high specificity is then examined for detection sensitivity. The detection sensitivity can be confirmed by performing PCR as described above on various concentrations of DNA extracted from the target animal species (group). The detection limit of the DNA of the primer pair thus obtained can be preferably 10 pg per PCR tube, more preferably 1 pg, and even more preferably 0.1 pg.

  For example, ruminant-derived DNA detection primer pairs and swine-specific DNA detection primer pairs can be obtained by performing the above-described steps on the mitochondrial DNA sequence list obtained from GenBank according to the above method.

  As a specific example, the ruminant-specific DNA detection primer pair of the present invention is a combination of the sequence of SEQ ID NO: 1 of the sequence listing and the sequence of SEQ ID NO: 2 of the sequence listing; A combination with the sequence of SEQ ID NO: 4 in the sequence listing; a combination of the sequence with SEQ ID NO: 5 in the sequence listing; and the sequence of SEQ ID NO: 6 in the sequence listing; Is a combination. These primer pairs are detectable for DNA from cattle, deer, sheep, and goats, but from mammals other than ruminants (humans, whales, pigs, rabbits, etc.), poultry, and seafood It does not detect DNA. Also, DNA is not detected in feeds that do not contain ruminant-derived raw materials.

  As yet another specific example, the primer pair for detecting pig-specific DNA of the present invention is a combination of the sequence of SEQ ID NO: 9 and the sequence of SEQ ID NO: 10 in the sequence listing; And a sequence of SEQ ID NO: 12 in the sequence listing; a combination of a sequence of SEQ ID NO: 13 in the sequence listing and a sequence of SEQ ID NO: 14 in the sequence listing; or a sequence of SEQ ID NO: 15 in the sequence listing and SEQ ID NO: 16 in the sequence listing It is a combination with the array of In any of these primer pairs, pig-derived DNA can be detected regardless of the breed, and can be detected when pork-derived meat-and-bone meal is used as a sample. On the other hand, DNA derived from mammals other than pigs (human, whale, cow, rabbit, etc.), poultry, and seafood is not detected. Moreover, DNA is not detected also about the feed which does not contain the raw material derived from a pig.

  As another specific example, the primer pair for detecting whale dolphins-specific DNA of the present invention is a combination of the sequence of SEQ ID NO: 52 and the sequence of SEQ ID NO: 53 in the sequence listing; It is a combination of the sequence of 52 and the sequence of SEQ ID NO: 54 in the sequence listing. These primer pairs are detectable for DNA from whales and dolphins, but for DNA from mammals other than whales and dolphins (humans, cows, pigs, rabbits, etc.), poultry, and seafood Is not detected. Moreover, DNA is not detected also about the feed which does not contain the raw material derived from a whale and dolphins.

  As yet another specific example, the primer pair for detecting deer-specific DNA of the present invention is a combination of the sequence of SEQ ID NO: 55 in the sequence listing and the sequence of SEQ ID NO: 56 in the sequence listing. In any of these primer pairs, DNA derived from deer can be detected regardless of the variety, and can be detected when meat-and-bone meal derived from deer is used as a sample. On the other hand, DNA derived from mammals other than deer (human, whale, cow, rabbit, etc.), poultry, and seafood is not detected. Moreover, DNA is not detected also about the feed which does not contain the raw material derived from a deer.

  As another specific example, the primer pair for detecting sheep-specific DNA of the present invention is a combination of the sequence of SEQ ID NO: 57 in the sequence listing and the sequence of SEQ ID NO: 58 in the sequence listing. In any of these primer pairs, DNA derived from sheep can be detected regardless of the breed, and can be detected when meat-and-bone meal derived from sheep is used as a sample. On the other hand, DNA derived from mammals other than sheep (human, whale, cow, deer, goat, rabbit, etc.), poultry, and seafood is not detected. Also, DNA is not detected in feeds that do not contain sheep-derived ingredients.

  As yet another specific example, the primer pair for detecting goat-specific DNA of the present invention is a combination of the sequence of SEQ ID NO: 59 in the sequence listing and the sequence of SEQ ID NO: 60 in the sequence listing. In any of these primer pairs, DNA derived from goat can be detected regardless of the breed, and can be detected when meat-and-bone meal derived from goat is used as a sample. On the other hand, it does not detect DNA derived from mammals other than goats (human, whale, cow, deer, sheep, rabbit, etc.), poultry, and seafood. Moreover, DNA is not detected also about the feed which does not contain the goat-derived raw material.

  The primer pairs thus obtained can detect the presence of DNA from the animal species or group of interest in various samples. That is, using the primer pair obtained by the above method, using the DNA in the sample as a template, the DNA fragment is amplified by the PCR method, and the amplified DNA fragment is detected.

  Samples to be detected include raw meat, raw fish, processed meat foods, processed fish foods, processed food products, processed food products, blood, hair, body fluids, milk, processed milk products, meat and bone meal, bone meal , Fish meal, fish soluble, dashi salmon, feed containing these, fertilizer, pet food, and feed additives. Extraction of DNA from a sample is performed by a method commonly used by those skilled in the art depending on the primer pair used. For example, when a primer pair designed from mitochondrial DNA is used, mitochondrial DNA is extracted. Extraction of mitochondrial DNA from a sample is performed as follows, for example. A sample of about 50 mg to 500 mg (for example, 50 mg for raw meat, 100 to 500 mg for a dry powder sample) is suspended in about 10 times the amount of buffer solution, crushed by a bead crushing method, and then commercially available tissue cell mitochondria. Extraction is performed using a DNA extraction kit (for example, manufactured by Wako Pure Chemical Industries, Ltd.). Such a kit collects highly purified mitochondrial DNA with less contamination of nuclear genomic DNA in tissue cells, and performs DNA extraction and concentration operations by centrifugation, precipitation collection, and the like. In this way, mitochondrial DNA with a low contamination level of a large amount of nuclear genomic DNA present in the sample can be extracted more efficiently. It will be apparent to those skilled in the art that DNA extraction methods are not limited to those described herein, and that other methods may be utilized.

  The amount of primer used is not particularly limited, but generally it is preferably used at about 0.4 μM.

  In this way, if DNA derived from the target animal species is present in the sample, the amplified DNA fragment can be detected on the gel. The detection limit may vary depending on various factors such as the type and combination of primer pairs used, the amount of sample, PCR conditions, detection methods and the like. If appropriate conditions are selected, the presence of DNA can be detected with high sensitivity even in a small amount of sample. For example, if an appropriate condition is selected, when the sample is a mixed feed containing bovine-derived meat-and-bone meal, a primer pair for ruminant-specific DNA detection is used and at least 0.01% by mass of beef-and-bone meal is mixed. Even it can be detected.

(Example 1: Acquisition of ruminant-specific DNA detection primer)
(Example 1-1: Design of ruminant-specific DNA detection primer)
The region of the DNA sequence to be searched was defined as mitochondrial DNA. A list of 20 ID numbers for ruminant mitochondrial DNA sequences was obtained from GenBank (V00654, AY526085, AY676768, AY6767681, AB074964, AF492351, AY676857, AY126696, DQ985076, EF035447, AB245426B, AB245426B, AB245426 , AY702618, AF533344, AY225986, AF527537, and AF010406). In a computer in which EMBOSS was installed, a list of 20 kinds of ID numbers was input to obtain a DNA sequence, and multiple alignment was performed to create a consensus sequence for ruminants. Here, two types of consensus sequences were created: strict and loose.

  The degree of inconsistency is set to 3 levels, “N is 0 in the 1st to 7th bases from the 5 ′ end, and N is within 3 in the 8th to 17th bases from the 5 ′ end. When the discrepancy degree 2 was designated as “case”, the following scoring was not performed for those not corresponding to this.

  Next, for mitochondrial DNA sequences other than ruminants, a list of 14 types of ID numbers was obtained from GenBank (AF034253 (pig), X79547 (horse), AF347015 (human), J01415 (human), AB032554 (sardine), AB201258). (Whale), U20753 (cat), U96639 (dog), AY172335 (mouse), AP002932 (Saury), X14848 (rat), AP003322 (chicken), EF126369 (salmon), and AB18502 (tuna)). A DNA sequence was obtained by inputting these 14 types of ID numbers, and multiple alignment was performed with the previously generated consensus sequence.

  Next, the bases between the columns in the multiple aligned DNA sequences were compared. The terminal 5 bases were targeted for scoring, and the scoring weight was set as follows. The case where the fifth base from the terminal is different was scored 1, the fourth was score 2, the third was score 3, the second was score 5, and the first (terminal) was score 8. For the gap, the absolute value of the gap difference was 1, 2, 3, 4, and 5, and the scores were set to 3, 5, 7, 9, and 11, respectively. Due to the stability of annealing, the score was increased 1.5 times for G or C bases. Scoring with such settings determined the score for each consensus sequence. In addition, among the consensus sequences with a score of 0, those that were caused by only a few DNA sequences were scored based on a comparison of sequences other than the causative DNA sequences with annotations in parentheses.

  Next, for the bases of the consensus sequence, the base length was adjusted by setting the Tm value to 55 ° C., and the homodimer formation was automatically corrected.

  Among the obtained sequences, consensus sequences having a score of 12 or more (5 types as forward primer candidates and 6 types as reverse primer candidates) are exemplified in Table 1 below. In Table 1, for reverse primers, the consensus sequence is shown in the upper part and the sequence as the primer is shown in parentheses in the lower part.

(Example 1-2: Confirmation of specificity of ruminant-specific DNA detection primer)
Specificity of the consensus sequence (score 8 or higher) obtained in Example 1-1 was confirmed by BLAST. Among these, the sequence | arrangement as a primer with favorable specificity was synthesize | combined using the DNA automatic synthesizer. Cattle, deer, sheep, goat, human, horse, pig, rabbit, rat, mouse, whale, bird, salmon, flatfish, horse mackerel, cod, mackerel, saury, rainbow trout, bonito, sardine, tuna, crab, clam, shrimp, And from each meat sample of squid, mitochondrial DNA was prepared as follows. Each meat sample was suspended in 10 times the amount of buffer solution (10 mM Tris-HCl, pH 7.5, 20 mM EDTA, pH 7.5), crushed by the bead crushing method, and then mtDNA, a commercially available tissue cell mitochondrial DNA extraction kit. DNA was extracted using an extractor CT kit (manufactured by Wako Pure Chemical Industries).

PCR was performed on various primer pairs using each DNA sample as a template. PCR conditions are as follows: reaction buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl ₂ , 0.001% (W / V) gelatin); heat at 95 ° C. for 9 minutes After denaturation, the reaction of denaturation: 92 ° C., 30 seconds; annealing: 55 ° C., 30 seconds; extension: 72 ° C., 30 seconds was repeated 45 cycles, and finally, the reaction was carried out at 72 ° C. for 5 minutes. After completion of the reaction, agarose gel electrophoresis was performed, and ethidium bromide staining was performed to detect a PCR product (DNA fragment). As a result, when the combination of the forward primer and the reverse primer shown in Table 2 below was used, very high specificity and detection sensitivity could be obtained.

  Table 2 lists the GenBank AY526085 bovine mitochondrial DNA sequences for reference for the sites amplified by these primer pairs. The portion amplified by pair 1 (a combination of 5D2 and 3D5) is a region near or partially overlapping the amplification region described in Non-Patent Documents 4 and 5, but the amplified fragment and the primer sequence Both are different. Thus, by designing according to the method of the present invention, in the vicinity of a region known to be amplified for the same purpose even though the region to be amplified is not specified in advance. As a result, a new primer pair with higher sensitivity could be obtained. In addition, there has been no report of a portion amplified by other primer pairs as a region that is species-specifically amplified so far.

  Next, PCR was performed using DNA derived from various animals as a template when these primer pairs were used. Representative electrophoretic photographs are shown in FIG. 1 (pair 1: combination of 5D2 and 3D5) and FIG. 2 (pair 2: combination of 5K4 and 3K4), and the results are summarized in Table 3. As can be seen from FIGS. 1 and 2 and Table 3, these primer pairs were found to be able to detect only ruminants.

(Example 1-3: Confirmation of detection sensitivity of ruminant-specific DNA detection primer pair)
For the mitochondrial DNA of cattle, deer and sheep, the concentration of the extracted DNA was measured in advance. Then, 1.0 ng, 0.1 ng, 0.01 ng, 0.001 ng (1.0 pg), 0.0001 ng (0.1 pg), and 0.00001 ng (0.01 pg) DNA was added per PCR tube, PCR was performed using pair 1 primer pair (combination of 5D2 and 3D5). An electrophoresis photograph is shown in FIG. As a result, even in the case of containing 0.1 pg of DNA, the electrophoresis band had a strong signal intensity and could be detected with very high sensitivity.

(Example 2: Detection of ruminant-specific DNA in various feeds)
The detection of ruminant-specific DNA was investigated for various fish meals, chicken meal, feather meal, pork chicken meal, and livestock formula feed. Take 100 mg, suspend in 10 times the amount of buffer as in Example 1-2 above, crush by bead crushing method, and then use mitochondrial DNA using a commercially available tissue cell mitochondrial DNA extraction kit (Wako Pure Chemical Industries). Extracted. Using this DNA as a template, PCR was performed under the same conditions as in Example 1-2 using a ruminant-specific DNA detection primer pair (pair 1: combination of 5D2 and 3D5). The results are shown in FIG. A 201 bp band was not detected in the feed containing no ruminant-derived component (lanes 1 to 12 and 14 to 18), but a 201 bp band could be detected for the mixed feed in lane 13 containing the bovine-derived component. Also, samples containing various concentrations of beef and bone meal were prepared by adding beef and bone meal to the blended feed. Mitochondrial DNA was extracted from these samples in the same procedure as in Example 1-2, and the detection sensitivity was confirmed using a pair 1 primer pair (combination of 5D2 and 3D5). As a result, it was found that detection was possible up to 0.01% (w / w) addition (FIG. 5).

(Example 3: Acquisition of pig-specific DNA detection primer)
(Example 3-1: Design of primers for detecting pig-specific DNA)
A region for the DNA sequence to be searched for was mitochondrial DNA, and a primer for detecting pig-specific DNA was designed by the same procedure as in Example 1-1. A list of 40 ID numbers for porcine mitochondrial DNA sequences was obtained from GenBank. A list of 33 types of ID numbers was also obtained for mitochondrial DNA of animal species other than pigs. A list of ID numbers is shown in Table 4 below.

  Based on the list of ID numbers, a primer for detecting pig-specific DNA was designed in the same manner as in Example 1-1. Table 5 below illustrates consensus sequences (12 types of forward primer candidates and 5 types of reverse primer candidates) having a score of 15 or more from the obtained sequences. In Table 5, with respect to the reverse primer, the consensus sequence is shown in the upper part, and the sequence as the primer is shown in parentheses in the lower part.

(Example 3-2: Confirmation of specificity of pig-specific DNA detection primer)
Specificity of the consensus sequence (score 8 or higher) obtained in Example 3-1 was confirmed by BLAST. Among these, the sequence | arrangement as a primer with favorable specificity was synthesize | combined using the DNA automatic synthesizer. Mitochondrial DNA was prepared from 6 types of pork meat samples by the same procedure as in Example 1-2. PCR was performed in the same manner as in Example 1-2, using these extracted DNA and commercially available porcine mitochondrial DNA (manufactured by Texas Genomics Japan) as a template. As a result, when a combination of forward primer and reverse primer shown in Table 6 below was used, very high specificity and detection sensitivity could be obtained (see FIG. 6).

  Table 6 lists the GenBank AF034253 porcine mitochondrial DNA sequence for reference for the sites amplified by these primer pairs. Non-patent documents 1 and 6 describe the portion amplified by pair I (combination of PigATP6-5.3 and PigATP6-3.9) and the portion amplified by pair III (combination of PIG 5H and PIG 3H). The amplified fragment and the primer sequence are different, although the region is near or partially overlapping with the amplified region. The portion amplified by pair IV (a combination of PIG 5J and PIG 3J) is in the vicinity of the amplification region described in Non-Patent Document 7, but the amplified fragment and the primer sequence are completely different. In addition, the part amplified by pair II (a combination of PIG 5G and PIG 3G) has not been reported as a species-specific amplified region so far.

  Next, PCR was performed using these primer pairs with DNAs derived from various pig breeds as templates. A typical electrophoresis photograph is shown in FIG. In any primer pair, porcine DNA could be detected regardless of the breed.

  Next, cattle, sheep, goats, horses, deer, rabbits, rats, mice, humans, whales, pigs, chickens, quail, aigamo, sardines, horse mackerel, tuna, skipjack, saury, salmon, cod, flounder, rainbow trout, crabs, Mitochondrial DNA was prepared from shrimp, squid, clam, corn, and mixed feed samples by the same procedure as in Example 1-2 above, and PCR was performed using various pig-specific primer pairs. In all of the four types of primer pairs, only porcine DNA as a positive control was detected, and the other samples were not detected. FIG. 7 illustrates an electrophoretic photograph when Pair I (a combination of PigATP6-5.3 and PigATP6-3.9) is used.

(Example 3-3: Confirmation of detection sensitivity of primer pair for detecting pig-specific DNA)
Using porcine mitochondrial DNA (manufactured by Texas Genomics Japan, Inc .: concentration 10 ng / μL), 1.0 ng, 0.1 ng, 0.01 ng, 0.001 ng, and 0.0001 ng of DNA were added per PCR tube. Similarly, PCR was performed to examine detection sensitivity for various primer pairs. As a result, when Pair I (a combination of PigATP6-5.3 and PigATP6-3.9) is used, even when 0.0001 ng of DNA is contained, the electrophoresis band has a high signal intensity and can be detected with very high sensitivity. (FIG. 8). Similarly, in the case of Pair III (combination of PIG 5H and PIG 3H), detection was possible even when 0.0001 ng of DNA was contained. Furthermore, in the case of pair IV (a combination of PIG 5J and PIG 3J), detection was possible up to the case of containing 0.1 ng of DNA.

(Example 4: Detection of pig-specific DNA in feed)
Various amounts of pork bone and bone meal were added to the blended feed to prepare samples containing various concentrations of pork bone and bone meal. Mitochondrial DNA was extracted from these samples by the same procedure as in the above example and detected using the above primer pair. As a result, when Pair I (combination of PigATP6-5.3 and PigATP6-3.9) was used, detection was possible even when it contained 0.01% (w / w) pork bone and bone meal (FIG. 9). ). In addition, in the case of Pair III (combination of PIG 5H and PIG 3H), detection is possible up to 0.1% (w / w) addition, and in the case of Pair IV (combination of PIG 5J and PIG 3J) , 1.0% (w / w) addition was detectable.

(Example 5: Acquisition of primers for detecting DNA specific to whales and dolphins)
(Example 5-1: Design of primers for detecting DNA specific to whales and dolphins)
The region for the DNA sequence to be searched for was mitochondrial DNA, and primers for detecting whale / dolphins-specific DNA were designed in the same manner as in Example 1-1. A list of nine ID numbers for the mitochondrial DNA sequence of whales and dolphins was obtained from GenBank. A list of 18 ID numbers was also obtained for mitochondrial DNA of animal species other than whales and dolphins. A list of ID numbers is shown in Table 7 below.

  Based on the list of these ID numbers, primers for detecting a whale / dolphins-specific DNA were designed in the same manner as in Example 1-1. Table 8 below illustrates consensus sequences (13 types as forward primer candidates and 8 types as reverse primer candidates) having a score of 10 or more from the obtained sequences. In Table 8, for the reverse primer, the consensus sequence is shown in the upper part, and the sequence as the primer is shown in parentheses in the lower part.

(Example 5-2: Confirmation of specificity of primer for detecting DNA specific to whales and dolphins)
Specificity of the consensus sequence obtained in Example 5-1 was confirmed by BLAST. Among these, the sequence | arrangement as a primer with favorable specificity was synthesize | combined using the DNA automatic synthesizer. Mitochondrial DNA was prepared from meat samples of five kinds of whales and dolphins by the same procedure as in Example 1-2. PCR was carried out in the same manner as in Example 1-2, using these extracted DNAs as templates. As a result, when the combination of the forward primer and the reverse primer shown in Table 9 below was used, very high specificity and detection sensitivity could be obtained.

  Table 9 lists the mitochondrial DNA sequence of GenBank's AJ554040 minke whale (Balaenoptera acutorostrata) for the sites amplified by these primer pairs. Amplified by pair I (combination of whale51 (SEQ ID NO: 52) and whale31 (SEQ ID NO: 53)) and amplified by pair II (combination of whale51 (SEQ ID NO: 52) and whale32 (SEQ ID NO: 54)) This portion is different from those described in any conventional literature in both the amplified fragment and the primer sequence.

  Next, using these primer pairs, PCR was performed using DNAs derived from various species of whales and dolphins as templates. A typical electrophoresis photograph is shown in FIG. In any primer pair, whale / dolphins DNA could be detected regardless of species.

  Then, from each sample of cattle, deer, sheep, goat, horse, pig, rabbit, rat, mouse, chicken, horse mackerel, cod, mackerel, saury, skipjack, sardine, tuna, hairy crab, shrimp, squid, and human, the above Mitochondrial DNA was prepared by the same procedure as in Example 1-2, and PCR was performed using two types of primer pairs specific to whales and dolphins. In each of the two types of primer pairs, only whale DNA as a positive control was detected (lane P), and the other samples were not detected. In the case of using pair I (combination of whale51 (sequence number 52) and whale31 (sequence number 53)) and pair II (combination of whale51 (sequence number 52) and whale32 (sequence number 54)) in FIG. An electrophoretic photograph is illustrated.

(Example 6: Acquisition of deer-specific DNA detection primer)
(Example 6-1: Design of deer-specific DNA detection primer)
A region for the DNA sequence to be searched for was mitochondrial DNA, and a deer-specific DNA detection primer was designed in the same procedure as in Example 1-1. A list of 10 ID numbers for the deer mitochondrial DNA sequence was obtained from GenBank. A list of 21 types of ID numbers was also obtained for mitochondrial DNA of animal species other than deer. A list of ID numbers is shown in Table 10 below.

  Based on the list of ID numbers, deer-specific DNA detection primers were designed in the same manner as in Example 1-1. Among the obtained sequences, consensus sequences having a score of 10 or more (eight types as forward primer candidates and three types as reverse primer candidates) are exemplified in Table 11 below. In Table 11, the consensus sequence is shown in the upper part of the reverse primer, and the primer sequence is shown in the parentheses in the lower part.

(Example 6-2: Confirmation of specificity of deer-specific DNA detection primer)
Specificity of the consensus sequence obtained in Example 6-1 was confirmed by BLAST. Among these, the sequence | arrangement as a primer with favorable specificity was synthesize | combined using the DNA automatic synthesizer. Mitochondrial DNA was prepared from two types of deer meat samples by the same procedure as in Example 1-2. PCR was carried out in the same manner as in Example 1-2, using these extracted DNAs as templates. As a result, when a combination of forward primer and reverse primer shown in Table 12 below was used, very high specificity and detection sensitivity could be obtained.

  In Table 12, the mitochondrial DNA sequence of GenBank DQ985076 Cervus nippon taiouanus is described with reference to the sites amplified by this primer pair. The amplified portion by the combination of deer54 (SEQ ID NO: 55) and deer33 (SEQ ID NO: 56) is different from those described in any conventional literature in both the amplified fragment and the primer sequence.

  Next, mitochondrial DNA was prepared from various animal and plant-derived samples by the same procedure as in Example 1-2, and PCR was performed using deer-specific primer pairs. A typical electrophoresis photograph is shown in FIG. When this primer pair was used, deer DNA was detected along with deer DNA as a positive control regardless of the breed (lanes 5 and 6), and other samples were not detected.

(Example 7: Acquisition of sheep-specific DNA detection primer)
(Example 7-1: Design of sheep-specific DNA detection primer)
A region for the DNA sequence to be searched for was mitochondrial DNA, and a sheep-specific DNA detection primer was designed in the same procedure as in Example 1-1. A list of two ID numbers for the mitochondrial DNA sequence of sheep was obtained from GenBank. A list of 29 ID numbers was also obtained for mitochondrial DNA of animal species other than sheep. A list of ID numbers is shown in Table 13 below.

  Based on the list of ID numbers, sheep-specific DNA detection primers were designed in the same manner as in Example 1-1. Among the obtained sequences, consensus sequences having a score of 10 or more (6 types as forward primer candidates and 2 types as reverse primer candidates) are exemplified in Table 14 below. In Table 14, for the reverse primer, the consensus sequence is shown in the upper part, and the sequence as the primer is shown in parentheses in the lower part.

(Example 7-2: Confirmation of specificity of sheep-specific DNA detection primer)
Specificity of the consensus sequence obtained in Example 7-1 was confirmed by BLAST. Among these, the sequence | arrangement as a primer with favorable specificity was synthesize | combined using the DNA automatic synthesizer. Mitochondrial DNA was prepared from one kind of sheep meat sample by the same procedure as in Example 1-2. PCR was performed in the same manner as in Example 1-2, using this extracted DNA as a template. As a result, when the combination of the forward primer and the reverse primer shown in Table 15 below was used, very high specificity and detection sensitivity could be obtained.

  Table 15 lists the mitochondrial DNA sequences of GenBank AF010406 sheep for the sites amplified by this primer pair. The amplified portion by the combination of sheep53 (SEQ ID NO: 57) and sheep33 (SEQ ID NO: 58) is different from those described in any conventional literature in both the amplified fragment and the primer sequence.

  Next, mitochondrial DNA was prepared from various animal and plant-derived samples by the same procedure as in Example 1-2, and PCR was performed using sheep-specific primer pairs. A typical electrophoresis photograph is shown in FIG. Using this primer pair, only sheep were detected (lanes 7 and P) and no other samples were detected.

(Example 8: Acquisition of goat-specific DNA detection primer)
(Example 8-1: Design of primers for detecting goat-specific DNA)
A region for the DNA sequence to be searched for was mitochondrial DNA, and a goat-specific DNA detection primer was designed by the same procedure as in Example 1-1. A list of one ID number for goat mitochondrial DNA sequences was obtained from GenBank. A list of 30 ID numbers was also obtained for mitochondrial DNA of animal species other than goats. A list of ID numbers is shown in Table 16 below.

  Based on the list of ID numbers, a goat-specific DNA detection primer was designed in the same manner as in Example 1-1. Among the obtained sequences, consensus sequences having scores of 15 or more (13 types of forward primer candidates and 15 types of reverse primer candidates) are exemplified in Table 17 below. In Table 17, for the reverse primer, the consensus sequence is shown in the upper part and the sequence as the primer is shown in parentheses in the lower part.

(Example 8-2: Confirmation of specificity of primer for detecting goat-specific DNA)
Specificity of the consensus sequence obtained in Example 8-1 was confirmed by BLAST. Among these, the sequence | arrangement as a primer with favorable specificity was synthesize | combined using the DNA automatic synthesizer. Mitochondrial DNA was prepared from one kind of goat meat sample by the same procedure as in Example 1-2. PCR was performed in the same manner as in Example 1-2, using this extracted DNA as a template. As a result, when the combination of the forward primer and the reverse primer shown in Table 18 below was used, very high specificity and detection sensitivity could be obtained.

  Table 18 lists the mitochondrial DNA sequence of GenBank's AF533441 goat (Capra hircus) for the sites amplified by this primer pair. The portion amplified by the combination of goat55 (SEQ ID NO: 59) and goat35 (SEQ ID NO: 60) is different from those described in any conventional literature in both the amplified fragment and the primer sequence.

  Subsequently, mitochondrial DNA was prepared from various animal and plant-derived samples by the same procedure as in Example 1-2, and PCR was performed using a goat-specific primer pair. A typical electrophoresis photograph is shown in FIG. Using this primer pair, only goats were detected (lanes 8 and P) and no other samples were detected.

  According to the present invention, primers for detecting DNA derived from an arbitrary range of animal species or groups can be designed and acquired more efficiently. For example, a primer designed for detection of ruminants can detect minute amounts of meat-and-bone meal derived from ruminants in feed with high sensitivity. A primer designed for detection of pigs, deer, sheep or goats can detect a small amount of meat-and-bone meal derived from these animals with high sensitivity. Therefore, it is useful for preventing BSE generation and mixing of inappropriate raw materials. Furthermore, a primer designed for detection of whales and dolphins can detect a minute amount of meat-and-bone meal derived from whales and dolphins with high sensitivity. Moreover, the reliability of a test result is securable by using these primers of this invention as a positive control primer at the time of the test | inspection of the animal origin DNA in meat-and-bone meal. Moreover, these primers of the present invention can also be used for authenticity confirmation such as display of processed meat products.

Claims (3)

  A combination of a primer comprising the sequence of SEQ ID NO: 9 and a primer comprising the sequence of SEQ ID NO: 10 of the sequence listing; a primer comprising the sequence of SEQ ID NO: 11 of the sequence listing and a primer comprising the sequence of SEQ ID NO: 12 of the sequence listing A combination of a primer comprising the sequence of SEQ ID NO: 13 and a primer comprising the sequence of SEQ ID NO: 14 of the sequence listing; or a primer comprising the sequence of SEQ ID NO: 15 of the sequence listing and SEQ ID NO: 16 of the sequence listing A primer pair for detecting pig-specific DNA, which is a combination with a primer comprising the sequence of   A method for detecting a component derived from a specific animal species or group in a sample, comprising amplifying a DNA fragment by a PCR method using the primer pair according to claim 1 and using the DNA in the sample as a template, and Detecting the amplified DNA fragment, wherein the particular animal species or group is a pig.   The sample is raw meat, raw fish, processed meat food, processed fish food, processed food containing food, processed fish containing food, blood, hair, body fluid, milk, milk processed food, meat and bone meal, bone meal, fish meal, fish soluble, The method according to claim 2, wherein the method is selected from the group consisting of dashi koji, and feed, fertilizer, pet food, and feed additives containing them.

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