CN111187844B - Gene bar code detection kit and detection method for bark beetle insects - Google Patents
Gene bar code detection kit and detection method for bark beetle insects Download PDFInfo
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- CN111187844B CN111187844B CN202010045283.7A CN202010045283A CN111187844B CN 111187844 B CN111187844 B CN 111187844B CN 202010045283 A CN202010045283 A CN 202010045283A CN 111187844 B CN111187844 B CN 111187844B
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Abstract
The invention discloses a gene bar code detection kit for bark beetles, which comprises a primer DRI/DYI reagent and a gene bar code sequence disc; primer DRI sequence: 5' -AGGAGCATCTGTTGACTT-3, primer DYI sequence: 5'-ATAAACTTCTGGGTGTCC-3'. The invention also discloses a method for detecting the bark beetle insects by using the detection kit, which uses the DNA of the sample to be detected as a template to carry out PCR amplification reaction, and the PCR product is sequenced to obtain a gene sequence; comparing the sequence obtained by sequencing the bark beetles to be detected with a standard gene bar code sequence in a gene bar code sequence disk, and judging whether the target species is the target species. The gene bar code detection kit for the bark beetles is a high-efficiency, accurate and convenient molecular detection technology for the bark beetles, can distinguish 9 insect species of the bark beetles at a molecular level, and can meet the use requirement.
Description
Technical Field
The invention relates to the technical field of detection of bark beetles, in particular to a bark beetle insect gene bar code detection kit and a detection method.
Background
The insect of the bark beetle is an important forest pest, most of which are dead trees, and hosts comprise spruce, fir, masson pine, chinese pine and the like. All insect states can be spread along with the long-distance transportation of host timber and wooden packaging materials, so that the damage is caused. At present, species identification research of the bark beetle genus is still mainly focused on morphological identification, and the traditional insect morphological identification method has certain limitation that the complete adult is required to be used as a morphological research object, so that morphological structures of kindred species, larvae and pupae and incomplete insect bodies are difficult to identify. With the start of molecular biology research, more and more technologies can be used for species identification of the bark beetle insects. With the improvement of the technological level, in recent years, many people begin to perform molecular identification on insects by using molecular biological means. The molecular biology means generally needs less sample quantity, has the advantages of rapidness, accuracy and the like, and can provide a reliable reference for pest identification.
The mitochondrial DNA (mtDNA) of insects is a closed double-chain annular genetic material, has high stability, is generally 15.4-16.3 kb in size, exists in mitochondria in a high copy number, has a faster evolution rate than a nucleus, is maternal inheritance, does not generate mutation such as gene recombination inversion translocation and the like in the inheritance process, and has very important significance in researches such as classification of insect kindred species and identification among species. Furthermore, it is a very important marker object in the identification of new species of insects and even of multiple species. Wang Yinzhu et al (2010) determined and analyzed mtDNA COI gene sequences of 10 species of bark beetle insects (Platypodidae) from different countries, found that the 10 species of bark beetles have obvious differences in gene sequences, and provided a molecular basis for accurate identification of bark beetle insects. Feng et al (2012) analyzed mtDNA COI gene sequences of 31 important fly insects (Muscidae), studied the phylogenetic evolution and classification identification of fly insects, and showed that the sequence can be used as the basis for the molecular identification of fly insects. Pan Chengying et al (2006) determined the mtDNA COI gene sequences of 7 locusts and examined the feasibility of the COI gene as a DNA barcode to identify locusts species. The study of halon et al (2012) showed that the use of DNA barcodes based on mtDNA COI gene fragments for the classification and identification of bark beetles is viable.
DNA barcode technology is a new technology for identifying species using one or several standard, readily amplified DNA fragments with significantly greater differences between species than within species, the concept originally proposed by canadian Hebert. DNA barcode technology has many incomparable advantages over traditional categorical identification technologies: (1) The operation is simple, and people lacking knowledge of species classification and identification can also identify the species through standard technical procedures; (2) The method is not limited by the development stage and morphological characteristics of individuals, and the identification requirement can be met as long as the individuals have complete target DNA fragments; (3) The technology can be used as an auxiliary means of traditional taxonomies, helps traditional taxonomies to correct the previous classification conclusion, and solves the problem that morphological classification cannot be solved. Due to the many advantages of DNA barcode technology, among others, more and more researchers have added to the research lines of DNA barcode technology, making this area one of the hot spots for biological research (Hebert, 2006). However, at present, the research on the gene bar code rapid detection technology of the bark beetle genus has no related system report at home and abroad. Therefore, the accurate and reliable detection technology of the bark beetle insect bar code is established, which not only has important significance for the quarantine departments of China, but also supplements the gene resources of China, especially the bark beetle insect resources, makes a certain contribution to China in terms of competing for limited resources, and provides a new method for classifying and identifying bark beetles.
Disclosure of Invention
The invention aims to overcome the defects of the prior art, and provides a gene bar code detection kit and a detection method for the bark beetles, which can be used for rapidly and efficiently detecting the bark beetles.
In order to solve the technical problems, the invention provides a gene bar code detection kit for bark beetles, which at least comprises a gene bar code sequence disc and a primer DRI/DYI reagent; the primer DRI/DYI sequence is as follows:
primer DRI sequence: 5' -AGGAGCATCTGTTGACTT-3 of the total weight of the plant,
primer DYI sequence: 5'-ATAAACTTCTGGGTGTCC-3'.
Further, 45 standard bar code sequences are stored in the gene bar code sequence disc, and the detailed sequences are shown in SEQ ID NO.1 to SEQ ID NO. 45.
Further, also packageThe preparation method comprises the following steps: taq buffer, mgCl 2 dNTP mix, taq DNA polymerase, ddH 2 O, DNA standard.
Further, 9 types of bark beetles can be rapidly detected, and the 9 types of bark beetles are specifically: the preparation method comprises the following steps of enabling the bark beetle to be a fir bark beetle, a spruce bark beetle, a Betula alnoides, a Mei Yun Maoxiao bark beetle, a Kidney point bark beetle, a Pinus bark beetle and a Pinus bark beetle.
The invention also provides a method for detecting the codling insects by using the codling insect gene bar code detection kit, which comprises the following steps:
extracting DNA of a sample to be detected;
taking the DNA of a sample to be detected as a template, performing PCR amplification reaction by using the primer, performing agarose gel electrophoresis on an amplified product, and placing the gel in an imaging system for photographing; the photo shows that the bark beetle and the standard DNA sample have clear amplified bands at the 316bp position, namely the target band;
sequencing a PCR product to obtain a gene sequence;
comparing the sequence obtained by sequencing the bark beetles to be detected with a standard gene bar code sequence in a gene bar code sequence disk, and judging whether the target species is the target species.
Further, a GenMagBio animal cell tissue/cell genome DNA magnetic bead extraction kit is used for extracting a sample to be detected.
Further, the PCR amplification reaction system comprises: the total volume was 50. Mu.L containing 5. Mu.L of 10X Taq buffer with KCl, 2mmol/L MgCl 2 200. Mu. Mol/L dNTP mix, 2U Taq DNA polymerase, 3. Mu.L DNA template, 1. Mu. Mol/L primer.
Further, the PCR amplification reaction procedure is: pre-denaturation at 95 ℃ for 2min before circulation; denaturation at 95℃for 30s, annealing at 52℃for 30s, extension at 72℃for 45s, and 35 cycles; extending at 72 deg.C for 10min after circulation is completed, and preserving at-20deg.C.
Further, the specific method for comparing the sequence obtained by sequencing the to-be-detected bark beetle with the standard gene bar code sequence in the gene bar code sequence disc comprises the following steps:
a: 100% similar to the standard gene bar code sequence, and identifying the same as the type;
b: if the similarity with any standard barcode sequence is not 100%, the gene sequence of the bark beetle to be detected is translated into amino acid according to an invertebrate codon, the genetic distance is calculated by using a P-distance model on the amino acid sequence corresponding to the standard gene barcode sequence, and the standard gene barcode type with the genetic distance of less than 2% from the gene sequence of the bark beetle of unknown type is the target type; if the genetic distances are all more than 2%, the unknown types of the bark beetles are judged not to be in the range of the 9 bark beetles.
The invention has the beneficial effects that: compared with the prior art, the gene bar code detection kit for the bark beetles is an efficient, accurate and convenient detection technology for the bark beetles, can distinguish 9 insect species of bark beetles at a molecular level, and has the following characteristics:
(1) The method is simple to operate, and a person lacking knowledge of species classification and identification can also identify Pi Duke species through standard technical procedures;
(2) The method is not limited by the development stage and morphological characteristics of individuals, and the identification requirement can be met as long as the individuals have complete target DNA fragments;
(3) The target gene is short enough and is only 316bp, so that the requirement of experiments on specimen quality is reduced, and the amplification efficiency is high;
(4) The inter-species variation is large and the intra-species variation is small to distinguish between different species; sufficient system evolution information is included to locate the position of species in the classification system. Can meet the use requirement.
Drawings
FIG. 1 is an electrophoretogram of a sample to be tested, wherein 1 spruce bark beetle, 2 fir bark beetle, 3 kidney point bark beetle, 4 American Yun Maoxiao bark beetle, 5 larch bark beetle, 6 white pine bark beetle, 7 light shoulder beetle.
Detailed Description
The invention is further described below with reference to the accompanying drawings. The following examples are only for more clearly illustrating the technical aspects of the present invention, and are not intended to limit the scope of the present invention.
Example 1
The gene bar code detection kit for bark beetles can be used for rapidly detecting 9 bark beetles (shown in table 1), and at least comprises more than 20 doses of primer DRI/DYI reagent and 1 gene bar code sequence disc; the primer DRI/DYI sequence is as follows:
DRI sequence: 5'-AGGAGCATCTGTTGACTT-3' the number of the individual pieces of the plastic,
DYI sequence: 5'-ATAAACTTCTGGGTGTCC-3'.
The inventory of gene bar code sequences includes 9 total 45 standard bar code sequences, the detailed sequences are shown in SEQ ID NO. 1-45.
TABLE 1 bark beetle species names
| Sequence number | Chinese name | Latin name |
| 1 | Bark beetle of fir | Dryocoetes striatus |
| 2 | Bark beetle of spruce | Dryocoetes hectographus |
| 3 | Betula alnoides | Dryocoetes betulae |
| 4 | America Yun Maoxiao bark beetle | Dryocoetes affaber |
| 5 | Bark beetle | Dryocoetes autogruphus |
| 6 | Bark beetle | Dryocoetes villosus |
| 7 | Mixed point bark beetle | Dryocoetes confusus |
| 8 | Bark beetle | Dryocoetes luteus |
| 9 | Bark beetle of pine | Dryocoetes pini |
Preferred kit components may also include the following reagents in amounts of 20 or more: taq buffer, mgCl 2 dNTP mix, taq DNA polymerase, ddH 2 O, DNA standard.
Example 2
Selecting the bark beetles of Picea, the bark beetles of Abies, the bark beetles of Kidney, the bark beetles of America Yun Maoxiao, the bark beetles of larch, the bark beetles of white pine and the beetles of Alternaria glabra. Seven samples were used as samples to be tested. The procedure for detection using the kit of example 1 was as follows:
1) Total DNA extraction
The extraction of DNA is carried out by using a GenMagBio animal cell tissue/cell genome DNA magnetic bead extraction kit. The specific process is as follows:
directly cutting the foot and chest of the sample to be tested, putting 30mg of the foot and chest of the sample to be tested into a 2mm test tube, soaking and preserving the sample in absolute ethyl alcohol, washing and soaking the sample for 4-5 times by using sterile distilled water, and discarding the water; the dried specimens were soaked in sterile distilled water for 3 hours and then blotted dry. Placing into a 2mL centrifuge tube, vibrating and grinding (30 times/s) for 30s in a MM400 ball mill, adding 180 mu L of Lysis Buffer and 20 mu L of protease K, vibrating and mixing uniformly, standing overnight at normal temperature, vibrating and warm-bathing at 55 ℃ for 3-5h, centrifuging at 12000rpm for 10min, taking the supernatant, adding 200 mu L of Binding Buffer and 200 mu L of absolute ethyl alcohol, fully mixing uniformly, adding 20 mu L of magnetic beads, gently reversing and mixing uniformly for 10min. Placing the centrifuge tube on a magnetic frame, sucking and discarding the liquid in the tube, retaining the magnetic beads, placing the Wash Buffer I500 uL in the magnetic frame after reversing and uniformly mixing for 2min, discarding the liquid in the tube, washing the Wash Buffer II twice again, placing the centrifuge tube on the magnetic frame, slowly adding the Wash Buffer III, and removing the liquid in the tube after 1 min. Add 20. Mu.L of Elutation Buffer, elute DNA adsorbed on the beads in a water bath at 55deg.C for 10min, store at 4deg.C for use.
2) Sequence amplification and determination
PCR reaction system: the total volume was 50. Mu.L containing 5. Mu.L of 10X Taq buffer with KCl, 2mmol/L MgCl 2 200 mu mol/L dNTP mix, 2U Taq DNA polymerase, 3 mu L DNA template, and 1 mu mol/L primers; PCR reaction procedure: pre-denaturation at 95 ℃ for 2min before circulation; denaturation at 95℃for 30s, annealing at 52℃for 30s, extension at 72℃for 45s, and 35 cycles; extending at 72 deg.C for 10min after circulation is completed, and preserving at-20deg.C. Mixing 8 mu L of a sample to be detected with 2 mu L of 6 multiplied by Glycerol DNA loading Buffer, loading the sample, taking DNA Marker DL100 as a molecular weight Marker, carrying out constant pressure 120V at room temperature, carrying out electrophoresis with 3% agarose gel for 45min, carrying out EB staining with 0.5% mu mol/L, detecting the sample by a gel imaging system, and carrying out photographing analysis; as shown in the electrophoresis chart in figure 1, the band with the size of 316bp is amplified in 7 samples, four types of bark beetles, larch bark beetles and positive samples, the band is not amplified in bark beetles and Alternaria glabra, and the 2 types of the band are not in bark beetles insects, so that the method meets the practical requirement and is accurate in elimination. The amplified products were subjected to two-way sequencing using PCR primers as sequencing primers, which were done by Nanjing Jinsri Biotechnology Co.
3) Gene alignment
Comparing a sequence result obtained by sequencing of the unknown types of the bark beetles with a standard gene bar code sequence in a gene bar code sequence disc by using the existing comparison software and computer technology, and operating the result in the following mode:
a: 100% similar to the standard gene bar code sequence, and identifying the same as the type;
b: if the similarity with any standard barcode sequence is not 100%, the gene sequence of the bark beetle of the unknown species is translated into amino acid according to an invertebrate codon, the genetic distance is calculated with the amino acid sequence of the standard gene barcode by using a P-distance model, and the standard gene barcode species with the genetic distance of less than 2% with the gene sequence of the bark beetle of the unknown species is the target species; if the genetic distances are all greater than 2%, it is indicated that the unknown species of bark beetles are not within the 9 species of bark beetles.
The obtained 5 sequences are sequentially input into a gene barcode sequence disc for calculation and comparison, and the result is as follows:
1) The similarity of the bark beetle standard barcode genes in the sequence disc is 100%, and the identification result is accurate.
2) The similarity between the fir bark beetle sequence and the fir bark beetle standard bar code gene in the sequence disc is 100%, and the identification result is accurate.
3) The similarity between the nephritis diversifolia sequences and the nephritis diversifolia standard bar code genes in the sequence disc is 98-100%, the genetic distance between the nephritis diversifolia sequences and the nephritis diversifolia standard bar code genes in the sequence disc is 0.6%, the nephritis diversifolia sequences and the nephritis diversifolia standard bar code genes in the sequence disc are identified as the nephritis diversifolia according to the result identification rule, and the identification result is accurate.
4) The similarity between the America Yun Maoxiao moth sequence and the America Yun Maoxiao moth standard bar code gene in the sequence disc is 100%, and the identification result is accurate.
5) The genetic distance between the bark beetle of larch and the bark beetle standard bar code gene in the sequence disc is 43.7-50.3 percent, which is far more than 2 percent, and the bark beetle is not 9 species according to the result identification rule.
All the results are consistent with the types of the tested samples, and the results are consistent with the requirements, so that the kit can accurately identify the 4 types of bark beetles.
The foregoing is merely a preferred embodiment of the present invention, and it should be noted that modifications and variations could be made by those skilled in the art without departing from the technical principles of the present invention, and such modifications and variations should also be regarded as being within the scope of the invention.
Sequence listing
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caactttctc tttttacttg atctgttaaa attacagcca tcctgcttct tctatctcta 180
cctgttctag caggaggaat tactatatta ttaacagatc gaaatatcaa tacatcattt 240
tttgacccag ccggcggggg ggatccaatt ttataccaac acttattttg attttttgga 300
catccagaag tttaca 316
<210> 15
<211> 316
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 15
ggtgcgtctg ttgacctagc tatttttagt cttcacataa caggagtctc ctcaatttta 60
ggggccatca actttatctc aacaattatc aatatacacc ccgcaggaat aaaacctgaa 120
caactttctc tttttacttg atctgttaaa attacagcca tcctgcttct tctatctcta 180
cctgttctag caggaggaat tactatatta ttaacagatc gaaatatcaa tacatcattt 240
tttgacccag ccggcggggg ggatccaatt ttataccagc acttattttg attttttgga 300
catccagaag tttaca 316
<210> 16
<211> 316
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 16
ggtgcatctg ttgacctagc tatttttagt ctacacataa caggagtctc ctcaatttta 60
ggggccatca actttatctc aacaattatc aatatacacc ctgcaggaat aaaacctgaa 120
caactttctc tttttacttg atctgttaaa attacagcca tcctgcttct tctatctcta 180
cctgttctag cgggaggaat tactatatta ttaacagatc gaaatattaa tacatcattt 240
tttgacccag ccggcggagg ggacccaatt ttataccagc acttattttg attttttgga 300
cacccagaag tttaca 316
<210> 17
<211> 316
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 17
ggtgcatctg ttgatctagc tatttttagt cttcacatat caggagtctc ctcaatttta 60
ggggctatca actttatctc aacaattatc aatatacacc ctgcaggaat aaaacctgaa 120
caactttctc tttttacttg atctgttaaa attacagcca ttctgcttct tttgtctctt 180
cctgttctgg caggaggaat tactatatta ttaacagatc gaaatatcaa tacatcattt 240
tttgacccag ccggcggagg ggatccaatt ttataccaac acttattttg attttttgga 300
catccagaag tttaca 316
<210> 18
<211> 316
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 18
ggtgcatctg ttgatctagc tatttttagt cttcacatat caggagtctc ctcaatttta 60
ggggctatca actttatctc aacaattatc aatatacacc ctgcaggaat aaaacctgaa 120
caactttctc tttttacttg atctgttaaa attacagcca ttctgcttct tttgtctctt 180
cctgttctgg caggaggaat tactatatta ttaacagatc gaaatatcaa tacatcattt 240
tttgacccag ccggcggagg ggatccaatt ttataccaac acttattttg attttttgga 300
catccagaag tttaca 316
<210> 19
<211> 316
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 19
ggtgcatctg ttgatctagc tatttttagt cttcacatat caggagtctc ctcaatttta 60
ggggctatca actttatctc aacaattatc aatatacacc ctgcaggaat aaaacctgaa 120
caactttctc tttttacttg atctgttaaa attacagcca ttctgcttct tttgtctctt 180
cctgttctgg caggaggaat tactatatta ttaacagatc gaaatatcaa tacatcattt 240
tttgacccag ccggcggagg ggatccaatt ttataccaac acttattttg attttttgga 300
catccagaag tttaca 316
<210> 20
<211> 316
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 20
ggagcatctg ttgatttagc tatttttagt cttcatatat ctggagtatc ttcaatytta 60
ggagctatta actttatttc aacaattatt aatatgcacc ccgcagggat aaaacctgaa 120
caactatctc tttttacttg atctgtcaaa attacagcaa ttttacttct tctttctcta 180
cccgtactag caggaggaat caccatatta ttaacagatc gaaatattaa tacatcattt 240
tttgatccag ccggaggagg cgacccaatc ttatatcaac accttttctg attctttgga 300
cacccagaag tttata 316
<210> 21
<211> 316
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 21
ggagcatctg ttgacttagc tatttttagt cttcatatat ctggagtatc ttcaatttta 60
ggagctatta actttatttc aacaattatt aatatacacc ccgcaggaat aaaacctgaa 120
caactatctc tttttacttg atctgtcaaa attacagcaa ttttacttct tctttctcta 180
cccgtactag caggaggaat caccatatta ttaacagatc gaaatattaa tacatcattt 240
tttgatccag ccggaggagg cgacccaatc ttatatcaac accttttctg attttttgga 300
cacccagaag tttata 316
<210> 22
<211> 316
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 22
ggagcatctg ttgacttagc tatttttagt cttcatatat ctggagtatc ttcaatttta 60
ggagctatta actttatttc aacaattatt aatatacacc ccgcaggaat aaaacctgaa 120
caactatctc tttttacttg atctgtcaaa attacagcaa ttttacttct tctttctcta 180
cccgtactag caggaggaat caccatatta ttaacagatc gaaatattaa tacatcattt 240
tttgatccag ccggaggagg cgacccaatc ttatatcaac accttttctg attttttgga 300
cacccagaag tttata 316
<210> 23
<211> 316
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 23
ggagcatctg ttgatttagc tatttttagt cttcatatat ctggagtatc ttcaatytta 60
ggagctatta actttatttc aacaattatt aatatgcacc ccgcagggat aaaacctgaa 120
caactatctc tttttacttg atctgtcaaa attacagcaa ttttacttct tctttctcta 180
cccgtactag caggaggaat caccatatta ttaacagatc gaaatattaa tacatcattt 240
tttgatccag ccggaggagg cgacccaatc ttatatcaac accttttctg attctttgga 300
cacccagaag tttata 316
<210> 24
<211> 316
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 24
ggagcttctg tagatttagc tatttttagt cttcacatat caggagtctc ctcaatctta 60
ggggccatta actttatttc aacaattatt aatatacacc ccgcaggaat aaaatctgaa 120
caactatctc tttttacttg atctgttaaa attacagcaa ttttacttct tttatctcta 180
cctgttctag caggaggaat tactatacta ttaacagatc gaaatattaa tacatcattt 240
tttgatccag ctgggggagg agacccaatt ttataccagc acttattttg attttttgga 300
cacccagaag tttata 316
<210> 25
<211> 316
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 25
ggagcttctg tngatttngc tatttttagc cttcatatat caggagtctc ctcaatctta 60
ggggccatta actttatttc aacaattatt aatatacacc ccgcaggaat aaaatctgag 120
caactatccc tttttacttg atctgttaaa attacagcaa ttttactcct tttatctcta 180
cctgttctag caggaggaat tactatatta ttaacagatc gaaatattaa cacatcattt 240
tttgatccag ctggaggagg ggacccaatt ttataccagc acttattttg attttttggg 300
cacccagaag tctata 316
<210> 26
<211> 316
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 26
ggagcctctg ttgacttagc aatttttagt ttacatatat ctggggtttc ctcaatttta 60
ggggcaatta attttatctc aacagttatc aatatgcacc ccgcaggaat aaaacctgaa 120
caactctcac tctttacatg agcagtaaaa attacagcca ttcttctctt gttatccctc 180
cctgtgttgg cggggggaat tactatacta ttaacagatc gaaatattaa tacatcattt 240
tttgaccctg ctgggggagg agatcccatc ctataccaac acctattttg attctttggc 300
cacccagaag tatata 316
<210> 27
<211> 316
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 27
ggagcttctg tngatttngc tatttttagc cttcatatat caggagtctc ctcaatctta 60
ggggccatta actttatttc aacaattatt aatatacacc ccgcaggaat aaaatctgag 120
caactatccc tttttacttg atctgttaaa attacagcaa ttttactcct tttatctcta 180
cctgttctag caggaggaat tactatatta ttaacagatc gaaatattaa cacatcattt 240
tttgatccag ctggaggagg ggacccaatt ttataccagc acttattttg attttttggg 300
cacccagaag tctata 316
<210> 28
<211> 316
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 28
ggagcttctg tagatttagc tatttttagt cttcacatat caggagtctc ctcaatctta 60
ggggccatta actttatttc aacaattatt aatatacacc ccgcaggaat aaaatctgaa 120
caactatctc tttttacttg atctgttaaa attacagcaa ttttacttct tttatctcta 180
cctgttctag caggaggaat tactatacta ttaacagatc gaaatattaa tacatcattt 240
tttgatccag ctgggggagg agacccaatt ttataccagc acttattttg attttttgga 300
cacccagaag tttata 316
<210> 29
<211> 314
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 29
gacaccttta tgtagtggcc catttccact ttttatctat ggggggtatt cgcaattctt 60
gcaggaattg tactgattcc ccctattcac aggactaact cttaataata agtacctaaa 120
aattcaattc tactctatat ttattggagt aaacttaact ttttttcccc agcacttctt 180
agggctaaga ggtatacctc gacgatactc agactaccct gatgcttaca ttttattatt 240
gtatcttcaa ttggaagatt aatctccctc attagagtgt tttattttat ttttatttta 300
tgagaaagat tcgc 314
<210> 30
<211> 316
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 30
ggggcctctg ttgacttagc aatttttaga ctccatatat ctggtgtatc atcaatctta 60
ggggcaatca acttcatttc aacaattatt aacatgcacc ccaaaggaat aaaacctgaa 120
caactgtccc tttttacatg agcagtaaaa atcactgcaa tccttctttt actatcactt 180
cctgttttag ccggaggaat tactatatta ttaactgatc gaaatattaa tacatcattt 240
tttgacccag ctggaggtgg tgatcctatc ttataccaac atttattttg gttttttggg 300
caccctgaag tttata 316
<210> 31
<211> 316
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 31
ggggcctctg ttgacttagc aatttttaga ctccatatat ctggtgtatc atcaatctta 60
ggggcaatca acttcatttc aacaattatt aacatgcacc ccaaaggaat aaaacctgaa 120
caactgtccc tttttacatg agcagtaaaa atcactgcaa tccttctttt actatcactt 180
cctgttttag ccggaggaat tactatatta ttaactgatc gaaatattaa tacatcattt 240
tttgacccag ctggaggtgg tgatcctatc ttataccaac atttattttg gttttttggg 300
caccctgaag tttata 316
<210> 32
<211> 316
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 32
ggagcctctg ttgatttagc aatctttaga cttcacatat ctggagtatc atcaatttta 60
ggagcaatta attttatttc aacaattatt aatatacacc ccgcaggaat aaaacctgaa 120
cagctttctc tttttacctg agcagtaaaa attacagcag tccttctttt attatcccta 180
cccgttctag caggaggtat caccatatta ttaacagacc gaaatattaa tacatcattt 240
tttgacccag ctggaggagg agacccgatt ttatatcaac atttattttg attttttggg 300
caccctgaag tatata 316
<210> 33
<211> 316
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 33
ggagcctctg ttgatttagc aatctttaga cttcacatat ctggagtatc atcaatttta 60
ggagcaatta attttatttc aacaattatt aatatacacc ccgcaggaat aaaacctgaa 120
cagctttctc tttttacctg agcagtaaaa attacagcag tccttctttt attatcccta 180
cccgttctag caggaggtat caccatatta ttaacagacc gaaatattaa tacatcattt 240
tttgacccag ctggaggagg agacccgatt ttatatcaac atttattttg attttttggg 300
caccctgaag tatata 316
<210> 34
<211> 316
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 34
ggagcatctg ttgacttagc aatctttaga cttcacatat ctggagtatc atcaatttta 60
ggagcaatta attttatttc aacaattatt aatatacacc ccgcaggaat aaaacctgaa 120
cagctttctc tttttacctg agcagtaaaa attacagcag tccttctttt attatcccta 180
cccgttctag caggaggtat caccatatta ttaacagacc gaaatattaa tacatcattt 240
tttgacccag ctggaggagg agacccgatt ttatatcaac atttattttg attttttgga 300
cacccagaag tttata 316
<210> 35
<211> 316
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 35
ggagcctctg ttgatttagc aatctttaga cttcacatat ctggagtatc atcaatttta 60
ggagcaatta attttatttc aacaattatt aatatacacc ccgcaggaat aaaacctgaa 120
cagctttctc tttttacctg agcagtaaaa attacagcag tccttctttt attatcccta 180
cccgttctag caggaggtat caccatatta ttaacagacc gaaatattaa tacatcattt 240
tttgacccag ctggaggagg agacccgatt ttatatcaac atttattttg attttttggg 300
caccctgaag tatata 316
<210> 36
<211> 316
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 36
ggagcatcag tagatctagc catttttagt cttcatatag caggaatttc ctctatcctc 60
ggggcagtta actttatttc aacaattaac aacatacacc cagcaggaat aaagcctgaa 120
caactttctt tatttacttg agctgtaaaa attacagcaa ttcttctttt attatccctc 180
ccagttctag caggaggaat tactatacta ttaactgatc gaaatatcaa tacctcattt 240
tttgatcctg caggaggagg ggatccaatt ctatatcaac acttattctg attttttggc 300
catccagaag tataca 316
<210> 37
<211> 288
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 37
ggagcatcag tagatctagc catttttagt cttcatatag caggaatttc ctctatcctc 60
ggggcagtta actttatttc aacaattatc aacatacacc cagcaggaat aaagcctgaa 120
caactttctt tatttacttg agctgtnaaa attacagcaa ttcttctttt attatccctc 180
ccagttctag caggaggaat tactatacta ttaactgatc gaaatatcaa tacctcattt 240
tttgatcctg caggaggagg agatccaatt ctatatcaac acttattc 288
<210> 38
<211> 288
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 38
ggagcatcag tagatctagc catttttagt cttcatatag caggaatttc ctctatcctc 60
ggggcagtta actttatttc aacaattaac aacatacacc cagcaggaat aaagcctgaa 120
caactttctt tatttacttg agctgtaaaa attacagcaa ttcttctttt attatccctc 180
ccagttctag caggaggaat tactatacta ttaactgatc gaaatatcaa tacctcattt 240
tttgatcctg caggaggagg ggatccaatt ctatatcaac acttattc 288
<210> 39
<211> 288
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 39
ggagcatcag tagatctagc catttttagt cttcatatag caggaatttc ctctatcctc 60
ggggcagtta actttatttc aacaattaac aacatacacc cagcaggaat aaagcctgaa 120
caactttctt tatttacttg agctgtaaaa attacagcaa ttcttctttt attatccctc 180
ccagttctag caggaggaat tactatacta ttaactgatc gaaatatcaa tacctcattt 240
tttgatcctg caggaggagg ggatccaatt ctatatcaac acttattc 288
<210> 40
<211> 288
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 40
ggaacatcag tagatctagc catttttagt cttcatatag caggaatttc ctctatcctc 60
ggggcagtta actttatttc aacaattatc aacatacacc cagcaggaat aaagcctgaa 120
caactttctt tatttacttg agctgtaaaa attacagcaa ttcttctttt attatccctc 180
ccagttttag cgggaggaat tactatacta ttaactgatc gaaatatcaa tacctcattt 240
tttgatcctg caggaggagg agatccaatt ctatatcaac acttattc 288
<210> 41
<211> 288
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 41
ggagcatcag tagatctagc catttttagt cttcatatag caggaatttc ctctatcctc 60
ggggcagtta actttatttc aacaattatc aacatacacc cagcaggaat aaagcctgaa 120
caactttctt tatttacttg agctgtaaaa attacagcaa ttcttctttt attatccctc 180
ccagttctag cnggaggaat tactatacta ttaactgatc gaaatatcaa tacctcattt 240
tttgatcctg caggaggagg agatccaatt ctatatcaac acttattc 288
<210> 42
<211> 288
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 42
ggagcatcag tagatctagc catttttagt cttcatatag caggaatttc ctctatcctc 60
ggggcagtta actttatttc aacaattaac aacatacacc cagcaggaat aaagcctgaa 120
caactttctt tatttacttg agctgtaaaa attacagcaa ttcttctttt attatccctc 180
ccagttctag caggaggaat tactatacta ttaactgatc gaaatatcaa tacctcattt 240
tttgatcctg caggaggagg ggatccaatt ctatatcaac acttattc 288
<210> 43
<211> 288
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 43
ggagcatcag tagatctagc catttttagt cttcatatag caggaatttc ctctatcctc 60
ggggcagtta actttatttc aacaattatc aacatacacc cagcaggaat aaagcctgaa 120
caactttctt tatttacttg agctgtaaaa attacagcaa ttcttctttt attatccctc 180
ccagttctag caggaggaat tactatacta ttaactgatc gaaatatcaa tacctcattt 240
tttgatcctg caggaggagg ggatccaatt ctatatcaac acttattc 288
<210> 44
<211> 288
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 44
ggagcatcag tagatctagc catttttagt cttcatatag caggaatttc ctctatcctc 60
ggggcagtta actttatttc aacaattatc aacatacacc cagcaggaat aaagcctgaa 120
caactttctt tatttacttg agctgtaaaa attacagcaa ttcttctttt attatccctc 180
ccagttctag caggaggaat tactatacta ttaactgatc gaaatatcaa tacctcattt 240
tttgatcctg caggaggagg ggatccaatt ctatatcaac acttattc 288
<210> 45
<211> 316
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 45
ggagcatcag tagatctagc catttttagt cttcatatag caggaatttc ctctatcctc 60
ggggcagtta actttatttc aacaattaac aacatacacc cagcaggaat aaagcctgaa 120
caactttctt tatttacttg agctgtaaaa attacagcaa ttcttctttt attatccctc 180
ccagttctag caggaggaat tactatacta ttaactgatc gaaatatcaa tacctcattt 240
tttgatcctg caggaggagg ggatccaatt ctatatcaac acttattctg attttttggc 300
catccagaag tataca 316
Claims (7)
1. A gene bar code detection kit for bark beetles is characterized by at least comprising a gene bar code sequence disc and a primer DRI/DYI reagent; the primer DRI/DYI sequence is as follows:
primer DRI sequence: 5' -AGGAGCATCTGTTGACTT-3 of the total weight of the plant,
primer DYI sequence: 5'-ATAAACTTCTGGGTGTCC-3'; the gene bar code sequence has 45 standard bar code sequences, and the detailed sequences are shown in SEQ ID NO.1 to SEQ ID NO. 45.
2. The kit for detecting the gene bar code of the bark beetle insects according to claim 1, which is characterized by further comprising the following reagents: taq buffer, mgCl 2 dNTP mix, taq DNA polymerase, ddH 2 O, DNA standard.
3. The kit for detecting the gene bar code of the codling insect according to claim 1, wherein 9 codling species can be detected rapidly, and the 9 codling species are specifically: the preparation method comprises the following steps of enabling the bark beetle to be a fir bark beetle, a spruce bark beetle, a Betula alnoides, a Mei Yun Maoxiao bark beetle, a Kidney point bark beetle, a Pinus bark beetle and a Pinus bark beetle.
4. A method for detecting the bark beetle insects by using a bark beetle insect gene bar code detection kit, which is characterized by comprising the following steps:
extracting DNA of a sample to be detected;
performing PCR amplification reaction by using the primer of claim 1 and taking DNA of a sample to be detected as a template, performing agarose gel electrophoresis on an amplified product, and placing the gel in an imaging system for photographing; the photo shows that the bark beetle and the standard DNA sample have clear amplified bands at the 316bp position, namely the target band;
sequencing a PCR product to obtain a gene sequence;
comparing the sequencing sequence of the bark beetles to be detected with the standard gene bar code sequence in the gene bar code sequence disk of claim 1 to judge whether the target species is the target species;
the specific method for comparing the sequence obtained by sequencing the bark beetles to be detected with the standard gene bar code sequence in the gene bar code sequence disk comprises the following steps:
a: 100% similar to the standard gene bar code sequence, and identifying the same as the type;
b: if the similarity with any standard barcode sequence is not 100%, the gene sequence of the bark beetle to be detected is translated into amino acid according to an invertebrate codon, the genetic distance is calculated by using a P-distance model on the amino acid sequence corresponding to the standard gene barcode sequence, and the standard gene barcode type with the genetic distance of less than 2% from the gene sequence of the bark beetle of unknown type is the target type; if the genetic distances are all more than 2%, the unknown types of the bark beetles are judged not to be in the range of the 9 bark beetles.
5. The method for detecting the bark beetle insect gene bar code detection kit as defined in claim 4, wherein the sample to be detected is extracted by using a GenMagBio animal cell tissue/cell genome DNA magnetic bead extraction kit.
6. The method for detecting the codling insect gene bar code detection kit of the codling insect according to claim 4, wherein the PCR amplification reaction system is as follows: the total volume was 50. Mu.L containing 5. Mu.L of 10X Taq buffer with KCl, 2mmol/L MgCl 2 200. Mu. Mol/LdNTP mix, 2U Taq DNA polymerase, 3. Mu.L DNA template, 1. Mu. Mol/L primer.
7. The method for detecting the codling insect gene bar code detection kit of the codling insect according to claim 4, wherein the PCR amplification reaction procedure is as follows: pre-denaturation at 95 ℃ for 2min before circulation; denaturation at 95℃for 30s, annealing at 52℃for 30s, extension at 72℃for 45s, and 35 cycles; extending at 72 deg.C for 10min after circulation is completed, and preserving at-20deg.C.
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| CN111748638A (en) * | 2020-07-28 | 2020-10-09 | 南京海关动植物与食品检测中心 | Specific primer, kit and method for identifying ips insects based on DNA bar code |
| CN113025724B (en) * | 2021-03-10 | 2023-01-17 | 华南农业大学 | Dual PCR primer, method and kit for identifying small pissodes punctatus |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103146811A (en) * | 2012-12-28 | 2013-06-12 | 北京林业大学 | Method to detect dendroctonus valens and detection kit |
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