CN111893194A - Specific primer pair, kit and method for rapidly identifying Lecanicillium involving Hibiscus sordida - Google Patents
Specific primer pair, kit and method for rapidly identifying Lecanicillium involving Hibiscus sordida Download PDFInfo
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- 241001121967 Lecanicillium Species 0.000 title claims abstract description 30
- 241000218033 Hibiscus Species 0.000 title claims abstract description 17
- 235000005206 Hibiscus Nutrition 0.000 title claims abstract description 17
- 235000007185 Hibiscus lunariifolius Nutrition 0.000 title claims abstract description 17
- 238000000034 method Methods 0.000 title claims description 18
- 241001492664 Solenopsis <angiosperm> Species 0.000 claims abstract description 25
- 241000894007 species Species 0.000 claims abstract description 23
- 238000012408 PCR amplification Methods 0.000 claims abstract description 13
- 241001465977 Coccoidea Species 0.000 claims description 24
- 230000003321 amplification Effects 0.000 claims description 4
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 4
- 238000007400 DNA extraction Methods 0.000 claims description 3
- 238000000246 agarose gel electrophoresis Methods 0.000 claims description 3
- 238000000137 annealing Methods 0.000 claims description 3
- 238000004925 denaturation Methods 0.000 claims description 3
- 230000036425 denaturation Effects 0.000 claims description 3
- 239000012153 distilled water Substances 0.000 claims description 3
- 238000012257 pre-denaturation Methods 0.000 claims description 3
- 238000011144 upstream manufacturing Methods 0.000 claims description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical group O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- 244000048199 Hibiscus mutabilis Species 0.000 claims 1
- 235000003973 Hibiscus mutabilis Nutrition 0.000 claims 1
- 238000002474 experimental method Methods 0.000 abstract description 2
- 108020004414 DNA Proteins 0.000 description 10
- 240000000249 Morus alba Species 0.000 description 5
- 241000722234 Pseudococcus Species 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 238000001962 electrophoresis Methods 0.000 description 4
- 241000196324 Embryophyta Species 0.000 description 3
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- 241000238631 Hexapoda Species 0.000 description 2
- 108020005196 Mitochondrial DNA Proteins 0.000 description 2
- 241000607479 Yersinia pestis Species 0.000 description 2
- XLOMVQKBTHCTTD-UHFFFAOYSA-N Zinc monoxide Chemical compound [Zn]=O XLOMVQKBTHCTTD-UHFFFAOYSA-N 0.000 description 2
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- 235000014692 zinc oxide Nutrition 0.000 description 2
- 239000011787 zinc oxide Substances 0.000 description 2
- 244000099147 Ananas comosus Species 0.000 description 1
- 235000007119 Ananas comosus Nutrition 0.000 description 1
- 241001285221 Breviceps Species 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 241001266001 Cordyceps confragosa Species 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 241000259121 Ferrisia virgata Species 0.000 description 1
- 241000319062 Lycoris radiata Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000122904 Mucuna Species 0.000 description 1
- 241000691880 Planococcus citri Species 0.000 description 1
- 241000125161 Planococcus lilacinus Species 0.000 description 1
- 241000631435 Planococcus minor Species 0.000 description 1
- 241000596535 Pseudococcus comstocki Species 0.000 description 1
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- 235000013601 eggs Nutrition 0.000 description 1
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- 238000012268 genome sequencing Methods 0.000 description 1
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Abstract
The invention discloses a specific sequence for rapidly identifying a Hibiscus solenopsis Lecanicillium, which is shown as SEQ ID NO: 1 is shown. The invention discovers the species specificity sequence of the Hibiscus solenopsis Lecanicillium by utilizing the genome data of 11 quarantine Lecanicillium, and a primer is designed through software, and a specificity strip is only detected in the Hibiscus solenopsis Lecanicola after PCR amplification, and finally, a molecular biology experiment verifies that the species specificity sequence of the Hibiscus solenopsis Lecanicola and the primer thereof can effectively distinguish the Hibiscus solenopsis Lecanicola, and the invention has theoretical guiding significance for the high-efficiency rapid identification of the Hibiscus solenopsis Lecanicola.
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 quickly identifying Lecanicillium solenopsis.
Background
The mealybugs are tiny and special insects, and damage various crops, forest trees and ornamental plants mainly by sucking juice of young parts of the plants. The pest is a highly alert pest in China because of the very strong adaptability and reproductive capacity and the easy outbreak and disaster formation in the agriculture and forestry areas. In recent years, the invasion of the mealybugs continuously occurs on the global scale, and the mealybugs become one of the international risk quarantine objects.
On one hand, the hidden seeds and the compound seeds with similar forms are often ignored by form identification, and accurate judgment can be given only by an identification expert of a special group with long-term identification experience; on the other hand, the coccid captured in port field quarantine is often eggs, nymphs, male pupae or residues, and is difficult to identify effectively by a morphological method. In addition, the mealybugs belong to tiny and highly specialized insects, the identification of the mealybugs is mainly distinguished according to the morphological characteristics of female adults at present, the identification can be carried out only by preparing slide specimens, and the preparation requirements of the slide specimens are higher, and the process is complicated, so the morphological identification of the mealybugs is more difficult. In view of the fact that the traditional taxonomy method is difficult to meet the needs of the contemporary identification work, the research of a novel rapid identification technology applied to the classification research of the mealybugs is urgently needed.
With the rapid development of modern molecular biology technology and bioinformatics, more and more DNA-based identification systems are applied to species identification and identification. At present, mitochondrial genes are most commonly used in mealybug insects, and are characterized in that sequences at two ends of the genes are relatively conserved, the third base of a codon can be freely varied and has larger difference among different species, so that the mitochondrial genes are commonly used for distinguishing similar species, but the interspecific difference between a hidden species and a compound species is small and cannot be effectively distinguished, so that missed detection and false detection occur in the quarantine process, and international disputes are caused. Therefore, a more efficient molecular identification means is needed to quickly identify the mealybug insects, and a more complete quick detection technical system of the mealybug insects is formed at the port.
Disclosure of Invention
The invention aims to provide a specific sequence of the species of the Lecanicillium Funarii, a specific primer obtained based on the specific sequence and a method for identifying the species of the Lecanicillium Funarii through the specific primer, 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 rapidly identifying the Lecanicillium involving Hibiscus, wherein the specific sequence is shown as SEQ ID NO: 1 is shown.
In a preferred embodiment of the present invention, the sequence of the specific primer pair is as shown in SEQ id no: 2-3.
In a preferred embodiment of the present invention, the kit further comprises the specific sequence of claim 1 and the specific primer pair sequence of claim 2.
In a preferred embodiment of the invention, the application of the specific primer in identifying the species Leptococcus solonii is further included.
In a preferred embodiment of the invention, the kit is further used for identifying the species Leptococcus solonii.
In a preferred embodiment of the present invention, the method further comprises a method for designing primer identification based on identification of specific sequences of Lecanicillium solenopsis, comprising the following steps:
(1) designing primers by using software according to species specific sequences, and comparing in the genome of sequenced mealybugs to screen out specific primers of the Lecanicillium solenopsis;
(2) performing PCR amplification by using the genomic DNA of the mealybugs as a template;
(3) the PCR product was checked for the presence of bands of the expected size by agarose gel electrophoresis.
In a preferred embodiment of the present invention, the method further comprises, in step (1), designing primers such that the amplification product is within 1000 bp.
In a preferred embodiment of the present invention, the method further comprises, in the step (2), extracting DNA using QIAGEN genomic DNA extraction kit from 11 kinds of adult quarantine mealybugs.
In a preferred embodiment of the present invention, the PCR amplification system in step (2) is 20. mu.L, which includes 2 XPrimx Ex Taq (Takara) 10. mu.L, DNA template 1. mu.L, 10. mu.M concentration of upstream and downstream primers 1. mu.L, and the balance of sterilized distilled water.
In a preferred embodiment of the present invention, the PCR amplification conditions are further as follows: pre-denaturation at 94 ℃ for 4min, denaturation at 94 ℃ for 1min, annealing at 50 ℃ for 1min, extension at 72 ℃ for 90s, and extension at 72 ℃ for 4min after 40 cycles.
The invention has the beneficial effects that:
(1) the electrophoresis result of the invention shows that the specific band only exists in the species of the Lecanicillium Funarii, but does not exist in other species, which shows that the discovered species specific sequence of the Lecanicillium Funarii can be used for designing a primer to distinguish the Lecanicillium Funarii.
(2) The invention utilizes the genome data of 11 quarantine mealybugs to discover the species specificity sequence of the Chinese white mulberry mealybugs, and the primers are designed through software, and the specific bands are only detected in the Chinese white mulberry mealybugs after PCR amplification.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments described in the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 shows the effect of PCR amplification electrophoresis of specific primers of Lecanicillium solenopsis;
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection 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 in other ways than those specifically described herein, and therefore the scope of the present invention is not limited by the specific embodiments disclosed below.
Example 1
The invention utilizes the low abundance genome sequencing data of 11 important trans-border mealybugs of 6 genera (Lycoris radiata (Phenococcus solani), Hibiscus solenopsis (Phenococcus solenopsis), Pseudobulbus calomycus (Planococcus minor), Pseudobulbus pellucida (Planococcus lilacinus), Pseudobulbus neobrevicella (Dysmicoccus neobrevicella), Pseudobulbus pineapple (Dysmicoccus breviceps), double-stripe mealyrus (Ferrisia virgata), Pseudobulbus conraddea (Pseudococcus comstocki), Mucuna quincunea (Paracoccaceae), Pseudobulbus pseudolaris (Pseudococcus conatatus), and Pseudobulbus pseudobulbus pseudolaris (Pseudococcus citri), sequence specificity identification (Ci) such as species specificity identification of SEQ ID: 1 is shown.
Example 2
The invention utilizes the specific sequence in example 1 to design specific primers, and the primers for identifying the species specific sequence of the Lecanicillium solenopsis are shown in Table 1.
TABLE 1 species-specific sequences and primer sequences of Hibiscus solenopsis Lecanicillium
The experimental method comprises the following steps:
(1) the software Primer Premier 5.0 is used for designing primers, the size of an amplification product is set within 1000bp, and the designed primers are screened among the mealybug genomes through an NCBI Primer-blast tool to obtain specific primers.
(2) 11 quarantine mealybug adults are respectively collected, DNA is extracted by using QIAGEN genome DNA extraction kit, and the concentration is determined by an ultraviolet spectrophotometer.
(3) PCR amplification reaction was performed using ExTaq enzyme (Takara) using 11 DNA of quarantine Lecanicillium lecanii as a template.
The PCR amplification system was 20. mu.L, which included 2 XPromix Ex Taq (Takara) 10. mu.L, DNA template 1. mu.L, 10. mu.M concentration of upstream and downstream primers 1. mu.L each, and the balance of sterilized distilled water.
PCR amplification conditions: pre-denaturation at 94 ℃ for 4min, denaturation at 94 ℃ for 1min, annealing at 50 ℃ for 1min, extension at 72 ℃ for 90s, and extension at 72 ℃ for 4min after 40 cycles.
Amplification reaction in VeritiTM96-well American ABI Gene Amplifier.
And (3) electrophoresis detection: the PCR product was separated and detected by 2.5% agarose gel electrophoresis.
As shown in FIG. 1, the electrophoresis results show that the specific band is only present in the species Lecanicillium involving the mulberry and is absent in the other species, which indicates that the specific sequence of Lecanicillium involving the mulberry can be used to design primers to distinguish Lecanicillium involving the mulberry.
The invention utilizes the genome data of 11 quarantine mealybugs to find the species specific sequence of the Hibiscus solenopsis Leyanus, and primers are designed through software, and a specific band is only detected in the Hibiscus solenopsis Leyanus after PCR amplification. The experimental results prove that the species specificity sequence of the pseudococcus solenopsis and the primers thereof can effectively distinguish the pseudococcus solenopsis, and have theoretical guiding significance for the high-efficiency and rapid identification of the pseudococcus solenopsis.
Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.
Sequence listing
<110> Shanghai customs animal and plant and food inspection and quarantine technology center
ZHEJIANG University
Guangzhou Customs Technical Center
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Claims (10)
1. A specific sequence for rapidly identifying the Lecanicillium involving Hibiscus, which is characterized in that the specific sequence is shown as SEQ ID NO: 1 is shown.
2. A specific primer pair obtained according to the specific sequence of claim 1, wherein the sequence of the specific primer pair is as shown in SEQ ID NO: 2-3.
3. A kit for rapidly identifying the mealybugs of hibiscus sinensis, which comprises the specific sequence of claim 1 and the specific primer pair sequence of claim 2.
4. The use of the specific primer of claim 2 for identifying the species Lecanicillium solenopsis.
5. Use of the kit of claim 3 for identifying the species Lecanicillium involving Hibiscus sordida.
6. A method for designing primer identification based on rapid identification of specific sequences of Lecanicillium solenopsis is characterized by comprising the following steps:
(1) designing primers by using software according to species specific sequences, and comparing in the genome of sequenced mealybugs to screen out specific primers of the Lecanicillium solenopsis;
(2) performing PCR amplification by using the genomic DNA of the mealybugs as a template;
(3) the PCR product was checked for the presence of bands of the expected size by agarose gel electrophoresis.
7. The method for designing primer identification based on rapid identification of specific sequence of Lecanicillium solenopsis according to claim 6, wherein in step (1), the primer is designed such that the amplification product is within 1000 bp.
8. The method for designing primer identification based on rapid identification of specific sequence of Lecanicillium involving Hibiscus sordida according to claim 6, wherein in step (2), DNA is extracted from 11 adult quarantine Lecanicillium involving QIAGEN genomic DNA extraction kit.
9. The method for designing primer identification based on rapid identification of specific sequence of Lecanicillium solenopsis according to claim 6, wherein in step (2), the PCR amplification system is 20 μ L, which comprises 2 XPromix Ex Taq (Takara)10 μ L, DNA template 1 μ L, upstream and downstream primers with 10 μ M concentration 1 μ L each, and the balance is distilled water.
10. The method for designing primer identification based on rapid identification of specific sequence of Lecanicillium solenopsis according to claim 6, wherein the PCR amplification conditions are as follows: pre-denaturation at 94 ℃ for 4min, denaturation at 94 ℃ for 1min, annealing at 50 ℃ for 1min, extension at 72 ℃ for 90s, and extension at 72 ℃ for 4min after 40 cycles.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114736907A (en) * | 2022-04-14 | 2022-07-12 | 浙江大学 | Waxy synthesis gene PsFAR of Hibiscus solenopsis Lecanicillium and application thereof |
| CN117051123A (en) * | 2023-09-06 | 2023-11-14 | 广州海关技术中心 | Papaya gecko population traceability SNP molecular marker and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103131700A (en) * | 2013-02-25 | 2013-06-05 | 中国农业科学院植物保护研究所 | Pair of phenacoccus solenopsis specific SS-COI primers, and rapid PCR detection method and kit |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103131700A (en) * | 2013-02-25 | 2013-06-05 | 中国农业科学院植物保护研究所 | Pair of phenacoccus solenopsis specific SS-COI primers, and rapid PCR detection method and kit |
Non-Patent Citations (3)
| Title |
|---|
| LING MA, ET AL: "Development of novel microsatellites for population genetic analysis of Phenacoccus solenopsis Tinsley (Hemipeta: Pseudoccoccidae) based on genomic analysis", INTERNATIONL JOURNAL OF BIOLOGICAL MACROMOLECULES, vol. 121, pages 1135 - 1144 * |
| 田虎;李小凤;万方浩;张桂芬;张金良;: "利用种特异性COI引物(SS-COI)鉴别扶桑绵粉蚧", 昆虫学报, vol. 56, no. 06, pages 689 - 696 * |
| 罗梅，等: "基于转录组数据高通量发掘扶桑绵粉蚧微卫星引物", 昆虫学报, vol. 57, no. 04, pages 395 - 400 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114736907A (en) * | 2022-04-14 | 2022-07-12 | 浙江大学 | Waxy synthesis gene PsFAR of Hibiscus solenopsis Lecanicillium and application thereof |
| CN114736907B (en) * | 2022-04-14 | 2023-07-11 | 浙江大学 | Wax synthesis gene PsFAR of Hibiscus rosa-sinensis mealy bugs and application thereof |
| CN117051123A (en) * | 2023-09-06 | 2023-11-14 | 广州海关技术中心 | Papaya gecko population traceability SNP molecular marker and application thereof |
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