CN112322745B - Specific primer pair, kit and method for rapidly identifying pumpkin fruit fly - Google Patents
Specific primer pair, kit and method for rapidly identifying pumpkin fruit fly Download PDFInfo
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- 238000000034 method Methods 0.000 title description 10
- 241001116648 Zeugodacus depressus Species 0.000 title description 6
- 241000255588 Tephritidae Species 0.000 claims abstract description 31
- 240000001980 Cucurbita pepo Species 0.000 claims abstract description 24
- 235000000832 Ayote Nutrition 0.000 claims abstract description 23
- 235000009854 Cucurbita moschata Nutrition 0.000 claims abstract description 23
- 235000009804 Cucurbita pepo subsp pepo Nutrition 0.000 claims abstract description 23
- 235000015136 pumpkin Nutrition 0.000 claims abstract description 23
- 241001124181 Bactrocera dorsalis Species 0.000 claims description 7
- 150000007523 nucleic acids Chemical group 0.000 claims 3
- 241000894007 species Species 0.000 abstract description 23
- 238000012408 PCR amplification Methods 0.000 abstract description 10
- 238000013461 design Methods 0.000 abstract description 5
- 241000255925 Diptera Species 0.000 abstract description 2
- 108020004414 DNA Proteins 0.000 description 10
- 150000001875 compounds Chemical class 0.000 description 6
- 238000001514 detection method Methods 0.000 description 6
- 241000255581 Drosophila <fruit fly, genus> Species 0.000 description 4
- 241000238631 Hexapoda Species 0.000 description 4
- 238000001962 electrophoresis Methods 0.000 description 4
- 230000000877 morphologic effect Effects 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 3
- 241001136527 Anastrepha suspensa Species 0.000 description 2
- 241000218495 Bactrocera correcta Species 0.000 description 2
- 241000255579 Ceratitis capitata Species 0.000 description 2
- 238000007400 DNA extraction Methods 0.000 description 2
- 101000797990 Homo sapiens Putative activator of 90 kDa heat shock protein ATPase homolog 2 Proteins 0.000 description 2
- 102100032319 Putative activator of 90 kDa heat shock protein ATPase homolog 2 Human genes 0.000 description 2
- 241000157279 Rhagoletis cerasi Species 0.000 description 2
- 241001136529 Zeugodacus cucurbitae Species 0.000 description 2
- 238000000246 agarose gel electrophoresis Methods 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 238000000137 annealing Methods 0.000 description 2
- 238000004925 denaturation Methods 0.000 description 2
- 230000036425 denaturation Effects 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 238000012257 pre-denaturation Methods 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 241001136525 Anastrepha ludens Species 0.000 description 1
- 241001124183 Bactrocera <genus> Species 0.000 description 1
- 241001490361 Bactrocera minax Species 0.000 description 1
- 241001612102 Carpomya incompleta Species 0.000 description 1
- 241000663354 Carpomya vesuviana Species 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 230000004544 DNA amplification Effects 0.000 description 1
- 241000157278 Dacus <genus> Species 0.000 description 1
- 241000163301 Dacus punctatifrons Species 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 108020005196 Mitochondrial DNA Proteins 0.000 description 1
- 241000607479 Yersinia pestis Species 0.000 description 1
- 101150116184 abi gene Proteins 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 238000012268 genome sequencing Methods 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
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- 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
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- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
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Abstract
The invention discloses a specific sequence for rapidly identifying fruit fly of pumpkin, wherein the specific sequence is shown in SEQ ID NO: 1. The invention discovers species-specific sequences of the pumpkin fly by utilizing genome data of 11 quarantine flies, designs primers by software, detects specific strips only on the pumpkin fly after PCR amplification, and finally verifies that the species-specific sequences of the pumpkin fly and the primers thereof can effectively distinguish the pumpkin fly, thereby providing theoretical guidance significance for high-efficiency and rapid identification of the pumpkin fly.
Description
Technical Field
The invention relates to the field of molecular biology, in particular to a specific primer pair, a kit and a method for rapidly identifying fruit fly of pumpkin.
Background
Fruit fly is a phytophagous insect, and larvae are all predatory, and most of the insect pests are fruit, vegetable and flower crops, so that agricultural economy in China is seriously affected. Traditional insect quarantine identification is mainly morphological identification, but has certain limitation in port quarantine for samples with small individuals and similar morphologies and difficult morphological identification. On the one hand, for hidden seeds and compound seeds with similar morphology, the morphology difference is very small, and an identification expert of a special group with long-term identification experience is required to give accurate judgment; on the other hand, for samples with fuzzy morphological characteristics captured by quarantine, such as larvae and damaged samples or non-adult insects, the samples are difficult to identify effectively by morphological methods. In addition, the fruit fly has a large number of compound species, the identification of the compound species is ten times difficult, and no classification search table capable of covering all the compound species of the fruit fly exists at present.
With the rapid development of modern molecular biology technology and bioinformatics, more and more DNA-based identification systems are being applied to species identification and identification. At present, mitochondrial genes are most commonly used in fruit fly insects, and are characterized in that sequences at two ends of the genes are relatively conserved, and a third base of a codon can be freely mutated, so that the difference between different species is large, and the genes are commonly used for distinguishing similar species, but the difference between hidden species and compound species is small, and the hidden species and the compound species cannot be effectively distinguished, so that missed detection and false detection occur in the epidemic detection process, and international disputes are caused.
The trade traffic of China and the surrounding important countries and regions is greatly increased, potential invasive organisms are continuously introduced into the living beings to burst, and new invasive organisms are scattered to harm the living beings wantonly, so that the situation is severe. There is a need to develop new technologies, new products and new methods for precise identification and rapid detection of invading organisms, and create new lines of biosafety for the state gate. Therefore, a more efficient molecular identification means is needed to rapidly identify the fruit fly insects, and a rapid detection technology system of the fruit fly insects is improved to help the port to form relatively perfect.
Disclosure of Invention
The invention aims to provide a specific primer pair, a kit and a method for rapidly identifying fruit fly of pumpkin, so as to overcome the defects in the prior art.
In order to solve the technical problems, the technical scheme of the invention is as follows:
a specific sequence for rapid identification of a pumpkin fly, said specific sequence being as set forth in SEQ ID NO: 1.
In a preferred embodiment of the present invention, the method further comprises a specific primer pair obtained according to a specific sequence, wherein the specific primer pair is a primer pair Hch1, and the sequence of the primer pair Hch1 is shown in SEQ ID NO: 2-3.
In a preferred embodiment of the invention, a kit for rapid identification of fruit fly from pumpkin, comprising the specific sequence of claim 1 and the specific primer sequence of claim 2 is further included.
In a preferred embodiment of the invention, the use of the specific primers described above for identifying Drosophila species is further included.
In a preferred embodiment of the invention, the kit further comprises the use of the kit in identifying a Drosophila species.
In a preferred embodiment of the present invention, there is further provided a method for designing primer identification based on a specific sequence for rapid identification of fruit fly from pumpkin, comprising the steps of:
(1) According to the identification of the species-specific sequence of the fruit fly, designing a primer by using software, and comparing in the genome of the sequencing fruit fly, and screening out the specific primer of the fruit fly;
(2) Performing PCR amplification by using the Bactrocera's genome DNA as a template;
(3) The PCR products were checked by agarose gel electrophoresis for the presence of bands of the expected size.
In a preferred embodiment of the present invention, further comprising, in the step (1), designing the primer, the amplification product is within 1000 bp.
In a preferred embodiment of the present invention, further comprising, in step (2), extracting DNA using a QIAGEN genomic DNA extraction kit using 11 quarantine fruit fly adults.
In a preferred embodiment of the present invention, further comprising, in the step (2), 20. Mu.L of a PCR amplification system comprising 10. Mu.L of 2 XPromix Ex Taq (Takara), 1. Mu.L of a DNA template, 1. Mu.L of each of 10. Mu.M concentration of the upstream and downstream primers, and the balance of sterilized distilled water.
In a preferred embodiment of the present invention, the PCR amplification conditions further comprise: pre-denaturation at 95℃for 3min, denaturation at 94℃for 45s, annealing at 61℃for 45s, extension at 72℃for 45s, and extension at 72℃for 5min after 35 cycles.
The invention has the beneficial effects that:
(1) The electrophoresis result shows that the specific band only exists in the corresponding species and does not exist in other species, which indicates that the found specific sequence of the pumpkin fruit fly species can be used for designing primers to distinguish the pumpkin fruit fly and can be used for rapidly identifying the pumpkin fruit fly.
(2) The invention discovers species-specific sequences of the pumpkin fly by utilizing genome data of 11 quarantine flies, designs primers by software, detects specific strips only on the pumpkin fly after PCR amplification, and finally verifies that the species-specific sequences of the pumpkin fly and the primers thereof can effectively distinguish the pumpkin fly, thereby providing theoretical guidance significance for high-efficiency and rapid identification of the pumpkin fly.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described below, and it is apparent that the drawings in the following description are only some embodiments described in the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 shows the effect of PCR amplification electrophoresis of specific primers of Bactrocera dorsalis;
Detailed Description
Technical aspects of embodiments of the present invention will be clearly and fully described in the following description of the embodiments of the present invention with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some, but not all embodiments of the present invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the present invention without making any inventive effort, are intended to fall within the scope of the present invention.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced otherwise than as described herein, and therefore the scope of the present invention is not limited to the specific embodiments disclosed below.
Example 1
The invention uses the low abundance genome sequencing data of 11 important cross-border fruit flies (Mexico fruit fly (Anastrepha ludens), caribbean fruit fly (Anastrepha suspensa), guava fruit fly (Bactrocera correcta), orange fruit fly (Bactrocera dorsalis), bactrocera minax, melon fruit fly (Bactrocera cucurbitae), jujube fruit fly (Carpomya vesuviana), mediterranean fruit fly (Ceratitis capitata), pumpkin fruit fly (Dacus cinnatidus), black spine fruit fly (Dacus punctatifrons) and European cherry fruit fly (Rhagoletis cerasi)), and identifies and discovers species specific sequences as shown in SEQ ID NO: 1.
Example 2
The present invention uses the specific sequence in example 1 to design specific primers, and the primers used for identifying the specific sequence of the species of the fruit fly of pumpkin are shown in Table 1.
TABLE 1 species-specific sequences and primer sequences of Drosophila parvifolia
The experimental method comprises the following steps:
(1) The Primer is designed by utilizing software Primer Premier 5.0, the size of an amplified product is set within 1000bp, and the designed Primer is screened among the fruit fly genomes through NCBI Primer-blast tool to obtain a specific Primer.
(2) 11 quarantine fruit fly adults were collected, DNA was extracted using a QIAGEN genomic DNA extraction kit, and the concentration was determined by an ultraviolet spectrophotometer.
(3) PCR amplification was performed using 11 quarantine fruit fly DNAs as templates and ExTaq enzyme (Takara).
Wherein, the PCR amplification system is 20 mu L, which comprises 10 mu L of 2 XPremix Ex Taq (Takara), 1 mu L of DNA template, 1 mu L of upstream and downstream primers with 10 mu M concentration and the balance of sterilized distilled water;
PCR amplification conditions: pre-denaturation at 95℃for 3min, denaturation at 94℃for 45s, annealing at 61℃for 45s, extension at 72℃for 45s, and extension at 72℃for 5min after 35 cycles.
The amplification reaction of the invention is carried out in Veriti TM The sample was subjected to a 96-well American ABI gene amplification apparatus.
And (3) electrophoresis detection: the PCR products were detected by 3% agarose gel electrophoresis separation.
As shown in FIG. 1, the electrophoresis results showed that the specific band was only present in the Bactrocera dorsalis species and not present in the remaining species, indicating that the found Bactrocera dorsalis species specific sequence can be used to design primers to discriminate Bactrocera dorsalis.
The invention discovers species specific sequences of the fruit flies with 11 quarantine fruit flies by utilizing genome data of the fruit flies, designs primers by software, and detects specific strips only on the fruit flies with pumpkin after PCR amplification. The experimental result proves that the species-specific sequence and the primer thereof of the fruit fly with pumpkin can effectively distinguish the fruit fly with pumpkin, and have theoretical guiding significance for high-efficiency and rapid identification of the fruit fly with pumpkin.
Furthermore, it should be understood that although the present disclosure describes embodiments, not every embodiment is provided with a single embodiment, and that this description is provided for clarity only, and that the embodiments of the disclosure may be suitably combined to form other embodiments that will be understood by those skilled in the art.
Sequence listing
<110> Shanghai customs animals and plants and food inspection and quarantine technology center
ZHEJIANG University
Guangzhou Customs Technical Center
<120> a specific primer pair, kit and method for rapid identification of Drosophila parvifolia
<130> 1
<141> 2020-08-28
<160> 3
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tttctataag caaataaatg ttatatttta tagtattgtt cccaaactat caatttcagt 180
aatactatac caaaaccaat atctaataga ataacatgta tgatttagaa agtactgaaa 240
agaaaaaatt attagaacta ttaatagatt actttaaaaa tatttcgtat attttcagtt 300
gaccaagcaa aattaaaatt cgcgtttctt taaatggata aaatctgtac ttcgcgtctt 360
agtatttgtt tattgaatta aatagaaatg atgaaactta atgtaaaatg tcgatcttct 420
ttatacttag ctatgacatc tgttacccat tatccaaaac cataatgtat ataaaatcta 480
tttctcttgc ttcttcaata agatctatta aaatatctca aaattatcac tttccaaata 540
aaaattatca ggacagtcct catctttata tgtaatgtgc actgatgtag cacttctaaa 600
ataatataaa taaagctgaa acaacctcgc aatacaatat agcaatactt atgaatgtgt 660
acactcattc gaccctcctt gtctaccaag gggcaatgtg aaatatttgc ttttgtactt 720
taacggctat cgaaatcaat tgaagctccg tgatttaaat gtttatattt ttaagagcta 780
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Claims (5)
1. A specific nucleic acid fragment for rapidly identifying a pumpkin fly, wherein the specific nucleic acid fragment has a sequence as set forth in SEQ ID NO: 1.
2. A specific primer pair for identifying fruit fly of pumpkin, which is characterized in that the sequence of the specific primer pair is shown as SEQ ID NO: 2-3.
3. A kit for rapid identification of bactrocera dorsalis, comprising the specific nucleic acid fragment of claim 1 and the specific primer pair of claim 2.
4. Use of the specific primer of claim 2 for identifying a pumpkin drosophila species.
5. Use of the kit of claim 3 for identifying a bactrocera dorsalis species.
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| CN202010888861.3A CN112322745B (en) | 2020-08-28 | 2020-08-28 | Specific primer pair, kit and method for rapidly identifying pumpkin fruit fly |
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106367506A (en) * | 2016-09-09 | 2017-02-01 | 中国检验检疫科学研究院 | Quarantine fruit fly multi-target rapid identification method |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106367506A (en) * | 2016-09-09 | 2017-02-01 | 中国检验检疫科学研究院 | Quarantine fruit fly multi-target rapid identification method |
Non-Patent Citations (1)
| Title |
|---|
| G EHAN H AJI M ARONSY，S HAMAL A BDULLAH A L -M UFFTI.MOLECULAR IDENTIFICATION OF CUCURBIT FLY Dacus ciliatus (DIPTERA: TEPHRITIDAE) INFEST CUCURBITACEAE FAMILY BASED ON MITOCHONDRIAL GENE IN KURDISTAN REGION- IRAQ.《Journal of University of Duhok》.2019,第22卷(第2期),第229-238页. * |
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