CN111748638A - Specific primer, kit and method for identifying ips insects based on DNA bar code - Google Patents
Specific primer, kit and method for identifying ips insects based on DNA bar code Download PDFInfo
- Publication number
- CN111748638A CN111748638A CN202010735848.4A CN202010735848A CN111748638A CN 111748638 A CN111748638 A CN 111748638A CN 202010735848 A CN202010735848 A CN 202010735848A CN 111748638 A CN111748638 A CN 111748638A
- Authority
- CN
- China
- Prior art keywords
- ips
- sequence
- kit
- dna
- seq
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a specific primer, a kit and a method for identifying ips insects based on DNA bar codes, belonging to the technical field of molecular biology. The kit comprises a specific primer and a DNA barcode sequence. The DNA barcodes comprise a ips typographus barcode, a ips typographus barcode and a ips protocolatous barcode; the sequences are shown in SEQ ID NO.3 to SEQ ID NO. 11; the sequence of the specific primer is as follows: j2246: 5 '-GCTTTTGGAGTYYTAGG-3'; n2723: 5 '-GAAWGTTAAATTTACTC-3'. The method is applied to identifying the ips insects carried by the spruce plants, can make up the defects of morphological identification, is not limited by the insect states of pests, and can quickly and accurately identify the ips insects intercepted by plant quarantine.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and relates to a DNA bar code technology for identifying and identifying species by using a short DNA fragment, in particular to a specific primer, a kit and a method for identifying ips insects based on a DNA bar code.
Background
The ips insect carried by the spruce plant from canada is a dangerous pest, canada is a main import country of raw wood in China, and once the ips is imported into China through the port, the ips insect can cause serious harm to the ecological environment. The existing detection method mainly takes the form as the main part, but the forms of the ips insects are very similar, mostly related species and difficult to distinguish, and the identification accuracy is greatly reduced due to the old Chinese identification materials, so that a lot of inconvenience is brought to the quarantine work of related entry ports. Molecular methods such as PCR, fluorescent quantitative PCR, PCR-RFLP, DNA barcode and the like are increasingly used as auxiliary identification means for morphological identification, but the same method and the same target gene fragment cause deviation of results. A DNA bar code detection kit aiming at important pests carried by wood species can solve the problem of shortage of port front-line personnel, greatly reduce the time consumed by identification work and improve the identification efficiency.
Disclosure of Invention
Aiming at the problems in the prior art, the technical problem to be solved by the invention is to provide a specific primer for identifying the ips insects based on a DNA bar code; the invention aims to solve another technical problem of providing a kit for identifying ips insects based on DNA bar codes; the last technical problem to be solved by the invention is to provide a method for identifying ips insects based on DNA bar codes.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
in a first aspect, a specific primer is provided, the specific primer comprises a primer J2246 and a primer N2723,
the sequence of the primer J2246 is shown as SEQ ID NO. 1: 5 '-GCTTTTGGAGTYYTAGG-3';
the sequence of the primer N2723 is shown as SEQ ID NO. 2: 5 '-GAAWGTTAAATTTACTC-3'.
Also provides the application of the specific primer in identifying the ips insects.
In a second aspect, there is provided a kit for identifying an insect of the genus ips, the kit comprising a DNA barcode sequence and the specific primers of claim 1.
Furthermore, the kit also comprises a DNA standard sample, PremixTaq and ddH2O。
Further, the ips insect is an ips insect carried by a picea plant from canada.
Further, the ips insects include the ips typographus, the ips meretrix and the ips protocoioides.
Further, the DNA barcode sequences include the dendroctonus pissodes barcode sequence, the platyhalodon pissodes barcode sequence, and the dendroctonus pissodes barcode sequence; the ips typographus bar code sequence is shown in SEQ ID NO.3-SEQ ID NO. 4; the ips typographus bar code sequence is shown in SEQ ID NO.5 to SEQ ID NO. 8; the code sequence of the ips typographus is shown as SEQ ID NO. 9; the cissus macrantha barcode sequence is shown in SEQ ID NO.10 to SEQ ID NO. 11.
Also provides an application of the kit in identifying the ips insects.
In a third aspect, a method for identifying ips insects based on the kit is provided, which comprises the following steps:
1) extracting DNA of a species to be detected;
2) carrying out PCR amplification on the DNA of the species to be detected by using the specific primer;
3) and after sequencing the PCR amplification product, comparing a sequence result obtained by sequencing with the DNA barcode sequence to identify whether the species to be detected is the ips insect.
Further, in the step 3), comparing the sequence result obtained by sequencing with the DNA barcode sequence until the similarity reaches 100%, and determining that the species to be detected is a bark beetle insect;
if the comparison similarity between the sequence result obtained by sequencing and the DNA barcode sequence is less than 100%, translating the sequence result of the species to be detected into amino acid according to invertebrate codons, calculating the genetic distance with the amino acid corresponding to the DNA barcode sequence by using a P-distance model, wherein the genetic distance is less than or equal to 2%, and then the species to be detected is one of ips typographus, ips meretrix and ips maximus; and if the genetic distance is more than 2%, the species to be detected is not in the range of ips typographus, ips meretrix, ips magnus.
Has the advantages that: compared with the prior art, the invention has the advantages that: the invention provides a specific primer and a kit for identifying ips insects carried by spruce plants from Canada based on DNA bar codes, which are applied to identifying ips insects carried by spruce plants from Canada, can make up the defect of morphological identification, are not limited by the pest state, and can quickly and accurately identify the ips insects intercepted and quarantined by port plants.
Drawings
FIG. 1 is a diagram showing the result of electrophoresis of PCR products of ips insects and mixed counterfeit thereof; 1: bark beetles of the Mecanus spruce; 2: bark beetles of white spruce; 3: balancing the weight of bark beetles; 4: bark beetles of the Tortoise genus; 5: (iii) the kidney punctum beetle; 6: anoplophora glabripennis; positive control: other reaction conditions are unchanged, and the DNA standard sample is used as template DNA; negative control: other reaction conditions were unchanged, and water was used instead of the template DNA.
Detailed Description
The invention is further described with reference to specific examples. The molecular biological experiments, which are not specifically described in the following examples, can be performed by methods listed in molecular cloning, a laboratory manual (third edition) J. SammBruker, or methods conventional in the art, or according to kits and product instructions.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1
A DNA bar code detection kit for ips insects carried by spruce plants from Canada can rapidly detect 4 ips (shown in Table 1), and comprises at least 20 times of primers J2246/N2723 reagent and 1 gene bar code sequence tray; the sequence of the primer J2246/N2723 is as follows:
primer J2246 sequence: 5'-GCTTTTGGAGTYYTAGG-3'
Primer N2723 sequence: 5 '-GAAWGTTAAATTTACTC-3'.
The gene bar code sequence disk contains 9 standard bar code sequences in total, which are shown as SEQ ID No.3-SEQID No. 11.
TABLE 1 Dendroctonus species name carried on the Canadian Picea plant
| Serial number | Name of Chinese | Latin |
| 1 | Bark beetle of spruce | Ips borealis |
| 2 | White spruceBark beetle | Ipsperturbatus |
| 3 | Bark beetle with balanced weight | Ipsplastographus |
| 4 | Bark of Tolygodium | Ipstridens |
The preferred kit components can also comprise the following reagents in an amount of more than 20 times: PremixTaq, ddH2O, DNA standards.
Example 2
Selecting 6 samples of ips typographus, ips protodentalis, ips nephridioides and anoplophora glabripennis as samples to be detected. The kit of example 1 was used for detection, the procedure was as follows:
1) DNA extraction
Extraction of DNA was performed using a GenMagBio animal cell tissue/cell genomic DNA magnetic bead extraction kit. The specific process is as follows:
directly shearing 30mg of feet and chest of a specimen to be tested, putting the feet and chest into a 2mL test tube, soaking the specimen in absolute ethyl alcohol, washing the specimen with sterile distilled water for 4-5 times, and discarding water; and (5) soaking the dry specimen in sterile distilled water for 3h, and then sucking water. Placing in a 2mL centrifuge tube, shaking and milling (30 times/s) in an MM400 ball mill for 30s, adding 180 mu L lysine Buffer and 20 mu L protease K, shaking and mixing uniformly, standing overnight at normal temperature, shaking and bathing at 55 ℃ for 3-5h, centrifuging at 12000rpm for 10min, taking supernatant, adding 200 mu L Binding Buffer and 200 mu L absolute ethyl alcohol, mixing uniformly, adding 20 mu L magnetic beads, and mixing by gentle inversion for 10 min. And placing the centrifugal tube in a magnetic frame, sucking away liquid in the tube, keeping magnetic beads, reversing and uniformly mixing the Wash Buffer I and 500uL for 2min, placing the centrifugal tube in the magnetic frame, discarding the liquid in the tube, washing the Wash Buffer II twice in the same way, still placing the centrifugal tube on the magnetic frame, slowly adding the Wash Buffer III and 550uL, and moving away the liquid in the tube after 1 min. Add 60. mu.L of Elution Buffer, elute the DNA adsorbed on the magnetic beads in a water bath at 55 ℃ for 10min, and store at 4 ℃ for further use.
2) Sequence amplification and determination
And (3) PCR reaction system: the total volume is 25 μ L, which contains 10 μ L of LPremixTaq, 1 μ L of LDNA template, and ddH of each primer 1 μmol/L and 12 μ L2O; PCR reaction procedure: pre-denaturation at 95 ℃ for 5min before circulation; denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 45s, and 35 cycles; after circulation, the product is extended at 72 deg.C for 10min and stored at-20 deg.C. Taking a 5 mu LPCR product to load a sample, taking a DNA Marker DL2000 as a molecular weight Marker, carrying out constant pressure of 150V at room temperature, carrying out electrophoresis for 30min by using 3% agarose gel, staining by using 0.5% mu mol/L EB, detecting the sample by using a gel imaging system, and carrying out photographing analysis; as shown in FIG. 1, the electrophoresis chart shows that the 478bp bands are amplified in 4 kinds of ips typographus and the positive samples in 6 samples, the bands are not amplified in the kidney point beetles and the anoplophora glabripennis, and the 2 kinds of ips typographus are not in the insects of the genus ips, so that the method is in line with the fact and accurate in elimination. The product of the amplified band was subjected to bidirectional sequencing using PCR primers as sequencing primers, and completed by Nanjing Kingsler Biotech Co.
3) Gene alignment
By utilizing the existing comparison software and computer technology, the sequence result obtained by sequencing the unknown species bark beetle carried by the picea plant from Canada is compared with the standard gene barcode sequence in the gene barcode sequence disk, and the result operates according to the following mode:
a: if the similarity of the standard gene barcode sequence is 100%, the standard gene barcode sequence is determined as the type;
b: if the similarity of the gene sequence of the ips typographus and any standard barcode sequence does not reach 100 percent, translating the gene sequence of the ips typographus into amino acid according to an invertebrate codon, calculating a genetic distance with the amino acid sequence of the standard gene barcode by using a P-distance model, and obtaining a standard gene barcode type with the genetic distance less than or equal to 2 percent of the gene sequence of the ips typographus, namely a target type; if the genetic distance is more than 2 percent, the unknown species of the ips typographus is out of the range of the 4 species of ips typographus.
The obtained 4 sequences were input into a gene barcode sequence tray in sequence for calculation and comparison, and the results were as follows:
1) the similarity between the ips typographus dentatus sequence and the ips typographus dentatus standard bar code gene in the sequence disc is 100%, and the identification result is accurate.
2) The similarity between the ips typographus ips sequence and the ips typographus ips standard barcode gene in the sequence disc is 100%, and the identification result is accurate.
3) The similarity of the mean weight ips sequence and the mean weight ips standard barcode gene in the sequence plate is 98-100%, the genetic distance with the mean weight ips standard barcode gene in the sequence plate is 0.6%, the result is identified as the mean weight ips according to the result identification rule, and the identification result is accurate.
4) The similarity between the protocya brevicaulis sequence and protocya brevicaulis standard bar code gene in the sequence disk is 100%, and the identification result is accurate.
The result is in line with the sample type of the test, and the result is in line with the requirement, which shows that the kit can accurately identify the 4 kinds of ips typographus.
Sequence listing
<110> Nanjing customs animal, plant and food detection center
<120> specific primer, kit and method for identifying ips insects based on DNA bar code
<160>11
<170>SIPOSequenceListing 1.0
<210>1
<211>17
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>1
gcttttggag tyytagg 17
<210>2
<211>17
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>2
gaawgttaaa tttactc 17
<210>3
<211>765
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>3
ccaggatttg nactaatttc ccatattatt agccaagaaa gaggnaaaaa agaagctttt 60
ggagttctag gtataattta tgcaataaca gcaattggtc ttctaggttt tgtagtttga 120
gctcatcata tatttacagt aggaatagat gttgataccc gagcctactt cacctcagcc 180
actataatta ttgctgtacc tacaggaatt aaaattttta gttgactagc tactttccat 240
ggagcacaaa tttcttttaa tccttcaaga ctttgatctt taggatttat ttttcttttc 300
actttaggag gtctaactgg agttattctc gctaattctt ctattgatat tattttacat 360
gacacttatt atgtagttgc tcattttcat tatgtactct caataggagc tgtttttgct 420
attatcgcag gaattgtaca atgatttcca ttatttacag gtttaacatt aaataaaaaa 480
tacttaaaaa ttcacttttt tactatattt gctggagtaa atttaacatt ctttccgcaa 540
cactttctag gtttaagagg ratacctcga cgttactcag actaccctga tgcttatcta 600
atatgaaata ttatttcttc tattggaaga ataatttccc ttattagaat tttttatttt 660
atctttatta tttgagaaag attttcctct catcgaaaaa gaatttctgc tttaaattta 720
aattcatcaa ttgaatgact acaaaatctt ccaccgactg atcat 765
<210>4
<211>766
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>4
cccaggattt ggactaattt cccatattat tagccaagaa agaggaaaaa aagaagcttt 60
tggagttcta ggtataattt atgcaataac agcgattggt cttctaggtt ttgtagtttg 120
agctcatcat atatttacag taggaataga tgttgatacc cgagcctact tcacctcagc 180
cactataatt attgctgtac ctacaggaat taaaattttt agttgactag ctactttcca 240
tggagcacaa atttctttta atccttcaag actttgatct ttaggattta tttttctttt 300
cactttagga ggtctaactg gagttattct cgctaattct tctattgata ttattttaca 360
tgacacttat tatgtagttg ctcattttca ttatgtactc tcaataggag ctgtatttgc 420
tattatcgca ggaattgtac aatgatttcc attatttaca ggtttaacat taaataaaaa 480
atacttaaaa attcactttt ttactatatt tgctggagta aatttaacat tctttccgca 540
acactttcta ggtttaagag gaatacctcg acgttactca gactaccctg atgcttatct 600
aatatgaaat attatttctt ctattggaag aataatttcc cttattagaa ttttttattt 660
tatctttatt atttgagaaa gattttcctc tcatcgaaaa agaatttctg ctttaaattt 720
aaattcatca attgaatgac tacaaaatct tccaccgact gatcat 766
<210>5
<211>766
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>5
cccaggattt gggttaattt cccatattat cagccaagaa agaggaaaaa aagaagcttt 60
tggagtccta ggtataattt atgcaataac agcaattgga cttctaggtt ttgtagtttg 120
agctcaccat atatttacgg taggaataga tgttgatacc cgagcctact ttacctcagc 180
cactataatt attgctgtac ccacaggaat taaaatcttt agttgattag ctactttcca 240
cggagcacaa atttctttta atccctcaag actttgatct ttagggttta ttttcctctt 300
cactttgggg ggtttaactg gagttatcct tgctaattct tctattgaca ttattttaca 360
tgatacttat tatgtagttg ctcacttcca ctatgtactc tctataggag ctgtttttgc 420
tattattgca ggaattgtcc aatgatttcc gctatttaca ggtttaacat taaataaaaa 480
atatttaaaa attcactttt tcactatatt tacaggagta aatttaacat tcttccctca 540
gcactttcta ggtttaagag gcataccccg acgttattca gattaccctg atgcctatct 600
tttatgaaat attatttctt ctattggaag aataatttca cttattagaa ttttttattt 660
tatctttatt atttgagaaa gattttcttc tcaccgaaaa agaatctctg ctttaaacct 720
aaattcatca attgaatgac tacaaaatct tcctccaact gatcat 766
<210>6
<211>766
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>6
cccaggattt gggttaattt cccatattat cagccaagaa agaggaaaaa aagaagcttt 60
tggagtccta ggtataattt atgcaataac agcaattgga cttctaggtt ttgtagtttg 120
agctcaccat atatttacgg taggaataga tgttgatacc cgagcctact ttacctcagc 180
cactataatt attgctgtac ccacaggaat taaaatcttt agttgattag ctactttcca 240
cggagcacaa atttctttta atccctcaag actttgatct ttagggttta ttttcctctt 300
cactttgggg ggtttaactg gagttatcct tgctaattct tctattgaca ttattttaca 360
tgatacttat tatgtagttg ctcacttcca ctatgtactc tctataggag ctgtttttgc 420
tattattgca ggaattgtcc aatgatttcc gctatttaca ggtttaacat taaataaaaa 480
atatttaaaa attcactttt tcactatatt tacaggagta aatttaacat tcttccctca 540
gcactttcta ggtttaagag gcataccccg acgttattca gattaccctg atgcctatct 600
tttatgaaat attatttctt ctattggaag aataatttca cttattagaa ttttttattt 660
tatctttatt atttgagaaa gattttcttc tcaccgaaaa agaatctctg ctttaaacct 720
aaattcatca attgaatgac tacaaaatct tcctccaact gatcat 766
<210>7
<211>872
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>7
atagctttcc cacgacttaa caacataaga ttttgacttc ttccaccttc tttaatattc 60
ttaatcataa gaagaattgt agataaaggg gcaggaacag gatgaactgt gtacccccca 120
ctgtcagcta atattgctca tgaaggaaca tcagtagatc tagctatttt tagtttacac 180
atatcaggaa tttcctcaat tttaggagcc attaatttta tttctacaat tattaatata 240
cacccaaaag gagttgctcc agaacaacta accctattta cctgagcagt aaaaattaca 300
gcaatccttc ttcttctttc cctgcctgta cttgcaggag caattactat actacttaca 360
gatcgtaata ttaatacctc tttttttgac cctgctggag gtggagaccc tattttatac 420
caacatttat tttgattctt tggacaccca gaagtgtata ttttaattct cccaggattt 480
ggactaattt cccatattat tagccaagaa agaggaaaaa aagaagcttt tggagttcta 540
ggtataattt atgcaataac agcaattggg cttctaggtt ttgtagtttg agctcatcat 600
atatttacag taggaataga tgttgatacc cgagcctact tcacctcagc cactataatt660
attgctgtac ctacaggaat taaaattttt agttgactag ctactttcca tggagcacaa 720
atttctttta atccttcaag actttgatct ttaggattta tttttctttt cactttagga 780
ggtctaactg gagttattct cgctaattct tctattgata ttattttaca tgacacttat 840
tacgtagttg ctcatttcca ctatgttcta tc 872
<210>8
<211>766
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>8
cccaggattt ggattaatct cccacattat cagtcaagaa agaggaaaaa aagaagcttt 60
tggagtccta ggtataattt atgcaataac agcaattgga cttctaggtt ttgtagtttg 120
agctcaccat atatttacag taggaataga tgtagatacc cgagcctact ttacctcagc 180
cactataatt attgcagttc ccacaggaat taaaatcttt agttgattag ctactttcca 240
tggagcacaa atttcttcta acccttcaag actttgatct ttaggattta ttttcctctt 300
cactttaggg ggtctaactg gagttatcct tgctaattct tctattgata ttattttaca 360
tgatacttat tatgtagttg ctcatttcca ctatgtacta tctataggag ctgtttttgc 420
tattattgca ggaattgtcc aatgattccc actatttaca ggtttaacat taaataaaaa 480
atatttaaaa attcactttt tcactatatt tacaggagta aatttaactt tcttccctca 540
acacttccta ggtttaaggg gaatacctcg acgttattca gactatcctg acgcctatat 600
tttatgaaat attatttctt ctattgggag aataatttca cttattagaattttttattt 660
tatctttatt atttgagaaa gattttcttc tcaccgaaaa agaatctctg ctttaaatct 720
aaattcatca attgaatggc tacaaaatct acctccaact aatcat 766
<210>9
<211>766
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>9
tcctggattt ggtttaattt cccatattat tagacaagaa agaggtaaaa aagaagcttt 60
tggagtatta ggaataatct atgcaataac agctattgga ctattaggat ttgtagtctg 120
agcccatcat atatttacag ttggaataga cgtagacact cgagcctatt ttacttcagc 180
tactataatt attgcagtac ctacaggtat taaaattttt agatgattag ctacttttca 240
tggtgcacaa atttctttta atccctcaag attatgatcc cttggcttta tctttctttt 300
cacaatagga ggattaactg gagtaatttt agctaactct tctattgata ttattcttca 360
tgatacttac tatgtagtag ctcacttcca ttatgttctt tctatagggg ccgtttttgc 420
tattattgca ggaattgttc aatgattccc tttatttacc ggtcttacac tcaataaaaa 480
attcttaaaa attcattttt ttactatatt tacaggagta aatttaacct ttttccctca 540
acatttctta ggattaagag gtatacctcg acgttactca gactatcctg acgcatatct 600
aatatgaaac attatttcat caattgggag aataatctcc ctcattagaa ttttttattt 660
tatctttatt atttgagaaa gattttcttc acatcgacaa agaatttttg ctttaaactt 720
aaactcttca ttagaatgat tacaaagtct acctcctaca gatcat 766
<210>10
<211>755
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>10
cccaggattt ggactaattt cccatattat tagccaagaa agaggaaaaa aagaagcttt 60
tggagttcta ggtataattt atgcaataac agcaattggg cttctaggtt ttgtagtttg 120
agctcatcat atatttacag taggaataga tgttgatacc cgagcctact tcacctcagc 180
cactataatt attgctgtac ctacaggaat taaaattttt agttgactag ctactttcca 240
tggagcacaa atttctttta atccttcaag actttgatct ttaggattta tttttctttt 300
cactttagga ggtctaactg gagttattct cgctaattct tctattgata ttattttaca 360
tgacacttat tatgtagttg ctcattttca ttatgtactc tcaataggag ctgtttttgc 420
tattatcgca ggaattgtac aatgatttcc attatttaca ggtttaacat taaataaaaa 480
atacttaaaa attcactttt ttactatatt tgctggagta aatttaacat tctttccgca 540
acactttcta ggtttaagag ggatacctcg acgttactca gactaccctg atgcttatct 600
aatatgaaat attatttctt ctattggaag aataatttcc cttattagaa ttttttattt 660
tatctttatt atttgagaaa gattttcctc tcatcgaaaa agaatttctg ctttaaattt 720
aaattcatca attgaatgac tacaaaatgt tccac 755
<210>11
<211>766
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>11
cccaggattt ggactaattt cccatattat tagccaagaa agaggaaaaa aagaagcttt 60
tggagttcta ggtataattt atgcaataac agcaattggg cttctaggtt ttgtagtttg 120
agctcatcat atatttacag taggaataga tgttgatacc cgagcctact tcacctcagc 180
cactataatt attgctgtac ctacaggaat taaaattttt agttgactag ctactttcca 240
tggagcacaa atttctttta atccttcaag actttgatct ttaggattta tttttctttt 300
cactttagga ggtctaactg gagttattct cgctaattct tctattgata ttattttaca 360
tgacacttat tatgtagttg ctcattttca ttatgtactc tcaataggag ctgtttttgc 420
tattatcgca ggaattgtac aatgatttcc attatttaca ggtttaacat taaataaaaa 480
atacttaaaa attcactttt ttactatatt tgctggagta aatttaacat tctttccgca 540
acactttcta ggtttaagag ggatacctcg acgttactca gactaccctg atgcttatct 600
aatatgaaat attatttctt ctattggaag aataatttcc cttattagaa ttttttattt 660
tatctttatt atttgagaaa gattttcctc tcatcgaaaa agaatttctg ctttaaattt 720
aaattcatca attgaatggc tacaaaatct tccaccgact gatcat 766
Claims (10)
1. A specific primer is characterized by comprising a primer J2246 and a primer N2723,
the sequence of the primer J2246 is shown as SEQ ID NO. 1;
the sequence of the primer N2723 is shown as SEQ ID NO. 2.
2. The use of the specific primer of claim 1 for identifying insects of the genus ips.
3. A kit for identifying an insect of the genus ips, the kit comprising a DNA barcode sequence and the specific primers of claim 1.
4.The kit of claim 3, wherein the kit further comprises DNA standards, PremixTaq, ddH2O。
5. The kit of claim 3, wherein the ips insect is an ips insect carried by a Picea plant from Canada.
6. The kit of claim 3, wherein the ips insects include Douglas fir ips, Peuglas fir ips, and Douglas fir ips.
7. The kit of claim 3, wherein the DNA barcode sequence comprises the Dolichos denticulata barcode sequence, the bark beetle barcode sequence, and the Dolichos denticulata barcode sequence; the ips typographus bar code sequence is shown in SEQ ID NO.3-SEQ ID NO. 4; the ips typographus bar code sequence is shown in SEQ ID NO.5 to SEQ ID NO. 8; the sequence of the ips typographus is shown as SEQ ID NO. 9; the cissus macrantha barcode sequence is shown in SEQ ID NO.10 to SEQ ID NO. 11.
8. Use of the kit of any one of claims 3 to 7 for identifying insects of the genus ips.
9. A method for identifying ips insects based on the kit of any one of claims 3 to 7, comprising the steps of:
1) extracting DNA of a species to be detected;
2) carrying out PCR amplification on the DNA of the species to be detected by using the specific primer;
3) and after sequencing the PCR amplification product, comparing a sequence result obtained by sequencing with the DNA barcode sequence to identify whether the species to be detected is the ips insect.
10. The method for identifying ips insects as claimed in claim 9, wherein in step 3), the sequence result obtained by sequencing is compared with the DNA barcode sequence with a similarity of 100%, and the species to be tested is determined to be ips insects;
if the comparison similarity between the sequence result obtained by sequencing and the DNA barcode sequence is less than 100%, translating the sequence result of the species to be detected into amino acid according to invertebrate codons, calculating the genetic distance with the amino acid corresponding to the DNA barcode sequence by using a P-distance model, wherein the genetic distance is less than or equal to 2%, and then the species to be detected is one of ips typographus, ips meretrix and ips maximus; and if the genetic distance is more than 2%, the species to be detected is not in the range of ips typographus, ips meretrix, ips magnus.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010735848.4A CN111748638A (en) | 2020-07-28 | 2020-07-28 | Specific primer, kit and method for identifying ips insects based on DNA bar code |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010735848.4A CN111748638A (en) | 2020-07-28 | 2020-07-28 | Specific primer, kit and method for identifying ips insects based on DNA bar code |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN111748638A true CN111748638A (en) | 2020-10-09 |
Family
ID=72711986
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010735848.4A Pending CN111748638A (en) | 2020-07-28 | 2020-07-28 | Specific primer, kit and method for identifying ips insects based on DNA bar code |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111748638A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113604586A (en) * | 2021-09-03 | 2021-11-05 | 上海市园林科学规划研究院 | Specific primer of bark beetle of black branch |
| CN115786531A (en) * | 2022-09-08 | 2023-03-14 | 浙江省检验检疫科学技术研究院 | Method for identifying lycoris radiate mealybugs and application |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103146811A (en) * | 2012-12-28 | 2013-06-12 | 北京林业大学 | Method to detect dendroctonus valens and detection kit |
| CN107164500A (en) * | 2017-06-14 | 2017-09-15 | 北京林业大学 | Detect primer, kit and the detection method of Douglas fir large small moth |
| CN110804664A (en) * | 2019-11-26 | 2020-02-18 | 拱北海关技术中心 | Primer pair and kit for identifying bark beetles in Meidiao and application of primer pair and kit |
| CN111187844A (en) * | 2020-01-16 | 2020-05-22 | 南京海关动植物与食品检测中心 | Gene bar code detection kit and detection method for ips insect |
-
2020
- 2020-07-28 CN CN202010735848.4A patent/CN111748638A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103146811A (en) * | 2012-12-28 | 2013-06-12 | 北京林业大学 | Method to detect dendroctonus valens and detection kit |
| CN107164500A (en) * | 2017-06-14 | 2017-09-15 | 北京林业大学 | Detect primer, kit and the detection method of Douglas fir large small moth |
| CN110804664A (en) * | 2019-11-26 | 2020-02-18 | 拱北海关技术中心 | Primer pair and kit for identifying bark beetles in Meidiao and application of primer pair and kit |
| CN111187844A (en) * | 2020-01-16 | 2020-05-22 | 南京海关动植物与食品检测中心 | Gene bar code detection kit and detection method for ips insect |
Non-Patent Citations (4)
| Title |
|---|
| 常虹: "口岸截获小蠹科昆虫DNA条形码技术研究", 《中国优秀硕士学位论文全文数据库（农业科技辑）》, no. 3, 15 March 2014 (2014-03-15), pages 23 - 29 * |
| 常虹等: "基于线粒体COI基因的齿小蠹属昆虫DNA条形码研究", 《昆虫学报》 * |
| 常虹等: "基于线粒体COI基因的齿小蠹属昆虫DNA条形码研究", 《昆虫学报》, vol. 55, no. 9, 20 September 2012 (2012-09-20), pages 1075 - 1081 * |
| 杨清玲等: "《分子生物学实验指导》", 中国科学技术大学出版社, pages: 186 - 195 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113604586A (en) * | 2021-09-03 | 2021-11-05 | 上海市园林科学规划研究院 | Specific primer of bark beetle of black branch |
| CN115786531A (en) * | 2022-09-08 | 2023-03-14 | 浙江省检验检疫科学技术研究院 | Method for identifying lycoris radiate mealybugs and application |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111748638A (en) | Specific primer, kit and method for identifying ips insects based on DNA bar code | |
| CN111733283A (en) | Triple fluorescence PCR detection kit for infectious spleen and kidney necrosis virus, largemouth black bass virus and mandarin fish rhabdovirus | |
| CN112359125A (en) | Method for rapidly detecting cryptococcus gatherensis | |
| CN105624290A (en) | Application of Aspergillus fumigatus annexin anxC4 gene (anxC4 gene) | |
| CN103820580B (en) | Porcine circovirus 2 type LAMP diagnostic kit | |
| CN113151542B (en) | Development method and application of Huashansong genome SNP | |
| JP5522820B2 (en) | Method for detecting pathogens of strawberry important diseases and primers for detection | |
| CN111187844B (en) | Gene bar code detection kit and detection method for bark beetle insects | |
| CN101748206B (en) | Detection and identification kit of composite PCR of wheat stripe rust fungus and leaf rust fungus | |
| CN111690759B (en) | Specific primer, kit and method for detecting RPA of citrus canker pathogen | |
| CN113265452A (en) | Bioinformatics pathogen detection method based on Nanopore metagenome RNA-seq | |
| CN109097454B (en) | Detection method for residual quantity of HEK293gDNA | |
| CN108384881B (en) | Fluorescent quantitative PCR detection method of cercospora fuscogilva | |
| CN105695626A (en) | RT-PCR (Reverse Transcription-Polymerase Chain Reaction) primer and method for detecting pepper vein yellows virus (PeVYV) | |
| CN112522434B (en) | Primer group and kit for simultaneously detecting multiple pathogenic fungi | |
| CN104975077A (en) | Pig-sourced eperythrozoon fluorogenic quantitative PCR detection kit and application thereof | |
| CN104946657B (en) | Reference gene, its screening technique and the application of the stable expression of white backed planthopper different larval instar | |
| Li et al. | Oligonucleotide array and VITEK matrix-assisted laser desorption ionization-time of flight mass spectrometry in species identification of blood yeast isolates | |
| CN103882150A (en) | Primer, probe and real-time fluorescent PCR (polymerase chain reaction) method for detecting TTSuV II (torque teno sus virus II) | |
| RU2642273C1 (en) | Method of differentiating yersinia pestis strains on basic and nonbasic subtypes by pcr method in real time mode | |
| CN114958875B (en) | Screening and application of reference gene metG of citrus yellow-long pathogen | |
| CN104651507B (en) | For distinguishing specific primer and the method for bollworm and oriental tobacco budworm | |
| CN114107539B (en) | LAMP primer composition for detecting pythium delicicola and application thereof | |
| CN116970735B (en) | Rapid detection method and kit for maize rust recombinant polymerase | |
| CN110578018B (en) | LAMP (loop-mediated isothermal amplification) method for rapidly detecting nuclear polyhedrosis virus |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |