CN111235286A - Spodoptera frugiperda specific SS-CO I primer and application thereof - Google Patents
Spodoptera frugiperda specific SS-CO I primer and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of agricultural biology, and particularly relates to a Spodoptera frugiperda specific SS-COI primer and application thereof. The specific primer only has amplification capacity on spodoptera frugiperda, the size of an amplification product is 323bp, and the specific primer also has good detection effects on single eggs and adult residues.
Description
Technical Field
The invention belongs to the technical field of agricultural biology, and particularly relates to a Spodoptera frugiperda specific SS-COI primer and application thereof.
Background
Spodoptera frugiperda JE Smith, also known as fall armyworm, pseudoarmyworm, alfalfa worm and spodoptera exigua belong to Lepidoptera, Spodoptera and Spodoptera, are native to America, and are migratory agricultural pests. The host range is wide, and the plant is 350 plants of 76 families, especially plants of Gramineae, Compositae and Leguminosae. The larvae eat the leaves and eat the tender cores, the stalks and the reproductive organs, so that serious damage is caused, and the larvae are strong in diffusivity and very easy to outbreak and cause disasters. Therefore, the enhancement of the detection and monitoring of spodoptera frugiperda is a necessary premise for guaranteeing the agricultural safety production.
Spodoptera frugiperda larvae are boring insects and are often hidden, and are difficult to discover early particularly in the early stage of occurrence, the traditional morphological identification method is difficult and low in accuracy, and a set of rapid and accurate molecular detection method needs to be established for strengthening quarantine and monitoring. At present, the international method for identifying Spodoptera frugiperda mainly comprises 1) morphological characteristic identification; 2) PCR techniques, and the like. However, at present, no standard detection method for Spodoptera frugiperda exists in China.
COI and RFLP techniques have been used to identify Spodoptera frugiperda and its related species. The mtDNA COI technical detection is to use a universal primer to amplify target DNA in vitro under the catalysis of DNA polymerase, then recover, purify and sequence a PCR product, and judge a detection result according to sequence comparison and phylogenetic tree construction. The RFLP technology detection is that restriction enzyme is utilized to cut DNA, gel electrophoresis separation is carried out on the cut DNA, then the separated DNA fragments are transferred to a filter membrane, hybridization is carried out by utilizing a radioactive labeled probe, and the detection result is judged according to the specific DNA fragments displayed by the hybridization.
The target species specificity SS-COI PCR detection technology is a specific sequence amplification region marker developed on the basis of mtDNA COI technology, a detection result is judged according to the existence and the size of an expected DNA band by using a specific primer, sequencing and sequence comparison are not needed, enzyme digestion and hybridization are not needed, and the method has the advantages of high sensitivity, strong specificity, quickness, simplicity, convenience, good reproducibility, strong stability and the like.
The design of specific primers depends on the sequence information available in the database. Currently, there are few known COI sequences in databases for feeding noctuidae pests, of which the number of related sibling closely related species is only 7. Meanwhile, analysis shows that the homology of the base sequences among the different species is high, and a specific segment targeting Spodoptera frugiperda is difficult to find.
Disclosure of Invention
The invention aims to provide a pair of Spodoptera frugiperda specific SS-COI primers.
Still another object of the present invention is to provide a specific detection method for Spodoptera frugiperda.
Still another object of the present invention is to provide a spodoptera frugiperda detection kit.
The sequence of the Spodoptera frugiperda specific SS-COI primer according to the specific embodiment of the invention is as follows:
SFZYF3：5’-TTAGATTTCGATCAGTAAGTAAT-3’；
SFZYR3：5’-TTTAACTTTATTAATTTCTAGTAGC-3’。
the spodoptera frugiperda specific SS-COI primer is used for amplifying to obtain a spodoptera frugiperda specific gene fragment, and the nucleotide sequence of the spodoptera frugiperda specific gene fragment is as follows:
according to the specific detection method of spodoptera frugiperda, the method comprises the step of performing PCR amplification by using the spodoptera frugiperda specific SS-COI primer.
According to the specific detection kit for spodoptera frugiperda, which is provided by the embodiment of the invention, the kit comprises the spodoptera frugiperda specific SS-COI primer.
The invention has the beneficial effects that:
(1) the primers SFZYF3 and SFZYR3 designed according to the Spodoptera frugiperda species specificity SS-COI marker can amplify a 323bp fragment when PCR (polymerase chain reaction) is used for rapidly detecting Spodoptera frugiperda, so that not only can single adult and 2-6 instar larvae of Spodoptera frugiperda be detected, but also single eggs and single newly hatched larvae can be accurately detected;
(2) the method is simple, convenient and quick to operate, the PCR technology is adopted, the whole process can be completed within 5 hours, and the operation process is simple, quick and efficient;
(3) the primer designed by the invention can specifically detect Spodoptera frugiperda, and has no amplification product in related species and moths.
Therefore, the method has strong practicability and can meet the requirements of plant quarantine and pest monitoring/detection and prevention.
Drawings
FIG. 1 shows the PCR amplification results of specific primers SFZYF3/SFZYR3 on Spodoptera frugiperda and its kindred species, genus moths, wherein M is molecular weight standard Trans2KTM1 Spodoptera frugiperda JE Smith, 2 Spodoptera exigua (H ü bner), 3 Spodoptera litura (Fabricius), 4 Athetis lepigone (Moschler), 5 Scotoptera litura rotorvata rofibr, 6 Agrotis lutescens segetum (Denis et Schifferm ü r), 7 Helicoverpa armigera (H ü bner), 8 Mythimna setaria segata (Walker), 9 Dichrocispora segertilis Guenee, Cn 10 Ostrinia ostrinialis (Ostrinia furalis) Gutiena, 11 rice leaf borer (Localciphila ostrinia), 12 Lobelita (Localciphila ostrinia) Guenee;
FIG. 2 shows the PCR amplification results of specific primers SFZYF3/SFZYR3 on Spodoptera frugiperda in different insect states and different larval instar stages, wherein M is molecular weight standard Trans2KTM(ii) a 1: egg (single grain); 2:1 instar larvae (single head); larvae at 3:2 instar (1/2 body length); 4:3 instar larvae (1/4 body length); 5:4 instar larvae (abdominal 1/4); 6:5 instar larvae (abdominal 1/8); 7:6 instar larvae (abdominal 1/8); pupae (female, abdomen 1/4); pupae (male, abdomen 1/4); adult (female, abdomen 1/4); adult (male, abdomen 1/4); 12:negative control (ultrapure water);
FIG. 3 shows the detection result of specific primer SFZYF3/SFZYR3 on Spodoptera frugiperda imago residues, wherein M is molecular weight standard Trans2KTM(ii) a 1, antennae (1 root); 2, lower lip beard; 3, beak; 4, front wings (1); 5, back wings (1); 6, head part; 7: breast (1/4); 8: abdomen (1/4); 9, forefoot (1 strip); 10, midfoot (1/2); 11: hind feet (1/2); negative control (ultrapure water);
FIG. 4 shows the determination of the minimum detection threshold for Spodoptera frugiperda using the specific primer SFZYF3/SFZYR3, M: molecular weight Standard Trans2KTM(ii) a 1-16, 1265X 10 respectively3,126.5×103,12.65×103,6.33×103,3.16×103,1.58×103790.63,395.31,197.66,98.83,49.41,24.71,12.35,6.18,3.09,1.54 pg/. mu.L, 17: negative control (ultrapure water).
Detailed Description
Example 1 preparation of Spodoptera frugiperda-specific primers
A Spodoptera frugiperda mitochondrion mtDNA COI genome sequence is taken as a target sequence, and Spodoptera frugiperda specific primers (SFZYF3/SFZYR3) are designed, wherein the sequence of the primers is as follows:
Primer SFZYF3：5’-TTAGATTTCGATCAGTAAGTAAT-3’；
Primer SFZYR3：5’-TTTAACTTTATTAATTTCTAGTAGC-3’。
and (3) performing temperature gradient PCR with the annealing temperature of 46-53 ℃ by using Spodoptera frugiperda DNA as a template to determine the optimal amplification condition. For the primer pair SFZYF3/SFZYR3, 323bp single bands can be obtained at the annealing temperatures of 52 ℃ and 53 ℃, and the amplified bands are clearer and brighter at the annealing temperature of 53 ℃, so that the annealing temperature of 53 ℃ is selected.
Example 2 specific amplification Effect of primers on Spodoptera frugiperda
The primers SFZYF3/SFZYR3 are used for carrying out SS-COI PCR amplification by taking Spodoptera frugiperda DNA as a template and 11 other common species of moths such as beet armyworm, prodenia litura, Athetis lepigone, Spodoptera nutans, cutworms, cotton bollworms, oriental armyworms, pillworm, Asian corn borer, Cnaphalocrocis medinalis guenee, meadow moth and the like as controls.
Collecting moth larvae (2-instar or 5-instar), identifying and confirming via morphological characteristics and DNA bar codes, extracting DNA, and storing at-20 deg.C.
mu.L of the PCR product was electrophoretically separated on a 1.0% (w/v) agarose gel containing Gold view, and the results were determined on the basis of the size of the amplified product on a gel imaging system.
As shown in FIG. 1, 323bp of the target band was amplified in Spodoptera frugiperda in lane 1, and no amplification products were obtained in all of the other 11 kindred and species of moths, and therefore, the SS-COIPCR amplification primers derived from Spodoptera frugiperda mitochondrial DNA selected by the present invention had high specificity.
Example 3 amplification Effect of primers SFZYF3/SFZYR3 on Spodoptera frugiperda in different insect stages and larval instar
PCR amplification is carried out by using primers SFZYF3/SFZYR3 and Spodoptera frugiperda DNA of different insect states and different larval instars as templates.
Taking single-head/single-grain Spodoptera frugiperda (wherein 1/2 body length is taken for 2-year larvae, 1/4 body length is taken for 3-year larvae, belly 1/4 is taken for 4-year larvae, belly 1/8 is taken for 5-6-year larvae, and belly 1/4 is taken for pupae and adults) at different development stages (eggs, 1-6-year larvae, female pupae and male pupae, and female adult and male adult), extracting DNA, and storing at-20 ℃ for later use.
The results are shown in FIG. 2, lanes 1-11 all show 323bp target bands, which shows that the target genes can be successfully amplified by single eggs of Spodoptera frugiperda, single-head newly hatched larvae, 2-instar larvae, 3-instar larvae, 4-instar larvae, 5-instar larvae, 6-instar larvae, pupae (female and male), and female adults and male adults of Spodoptera frugiperda.
Example 4 amplification Effect of primers SFZYF3/SFZYR3 on Spodoptera frugiperda adult residues
PCR amplification was performed using primers SFZYF3/SFZYR3 and DNA from different residues of Spodoptera frugiperda adults as templates.
The DNA of the residues was obtained from different adult residues (male, including antennal/1 root, lower labial whisker, beak, antelope/1, hind fin/1, head, breast (1/4), abdomen (1/4), forefoot/1, midfoot (1/2), hind paw (1/2), respectively.
As shown in FIG. 3, 323bp target bands are amplified on lanes corresponding to an antennal, a lower labial whisker, a beak, an antennal, a hind fin, a head, a chest, an abdomen, an anterior foot, a middle foot and a hind foot, so that the specific primer SFZYF3/SFZYR3 has high detection accuracy on adult residues.
Example 5 determination of threshold value of detection of Spodoptera frugiperda by primers SFZYF3/SFZYR3
Performing PCR amplification by using DNA of Spodoptera frugiperda with different dilution multiples as a template, and diluting the obtained DNA of the original template of Spodoptera frugiperda to the concentration of 1.54 pg/mu L by 10-fold and 2-fold descending gradient, wherein the concentration is about equivalent to 1/120,1/1200,1/12000,1/24000,1/48000,1/96000,1/192000,1/384000,1/768000,1/1536000,1/3072000,1/6144000,1/12288000,1/24576000,1/49152000,1/98304000 and 1/196608000 female adults; 2 mu L of the obtained product is taken as a template for PCR amplification and is directly added into a PCR reaction system.
As shown in FIG. 4, a 323bp band was amplified in 24.71 pg/. mu.L corresponding to lane 13, and therefore the lowest template DNA concentration detected by the present invention was 24.71 pg/. mu.L, which is equivalent to 1/12288000 adult females, and the sensitivity was higher.
Example 6 screening comparison of different primers
The results of comparing 3 pairs of primers provided by software design with the primers SFZYF3/SFZYR3 of the present invention are shown in Table 1:
TABLE 1 screening and evaluation of Spodoptera frugiperda-specific SS-COI primers
The result shows that the 3 pairs of primers provided by the software design have low specificity, the sequence consistency of the specific segment and the target species Spodoptera frugiperda is 97.6-100%, the consistency with other related species is less than 96.0-96.3% according to different primer pairs, but the overlapping phenomenon is serious, the band is not single, and the specific segment cannot be amplified. Compared with the primer provided by software design, the primer provided by the invention has strong specificity, the homology of the amplified specific segment and the COI gene sequence of the target species Spodoptera frugiperda reaches 97.80-100%, and the homology with other related species is less than 96.2%.
Sequence listing
<110> institute of plant protection of Chinese academy of agricultural sciences
<120> Spodoptera frugiperda specificity SS-CO I primer and application thereof
<160>3
<170>SIPOSequenceListing 1.0
<210>1
<211>23
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>1
ttagatttcg atcagtaagt aat 23
<210>2
<211>25
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>2
tttaacttta ttaatttcta gtagc 25
<210>3
<211>323
<212>DNA
<213> Spodoptera frugiperda JE Smith
<400>3
ttagatttcg atcagtaagt aatatagtaa tagctccagc taaaacaggt aaagataata 60
ataataaaaa tgcggtaata cctacagctc aaataaataa aggtatttga tcaaatgata 120
aattatttaa tcgtatatta ataatagtgg taataaagtt aatagctcct aaaatagatg 180
aaattccagc taaatgaagt gagaaaatag ctaaatctac tgaactacca ccatgagcaa 240
tattagagga gagggggggg taaactgttc atccagttcc tgctccattt tctacaatgc 300
tactagaaat taataaagtt aaa 323
Claims (4)
1. The Spodoptera frugiperda specific SS-COI primer pair is characterized by comprising primers with the sequences as shown in the specification:
primer SFZYF 3: 5'-TTAGATTTCGATCAGTAAGTAAT-3', respectively;
primer SFZYR 3: 5'-TTTAACTTTATTAATTTCTAGTAGC-3' are provided.
2. A method for the specific detection of Spodoptera frugiperda, comprising the step of PCR amplification using the Spodoptera frugiperda-specific SS-COI primer according to claim 1.
3. A spodoptera frugiperda-specific detection kit, characterized in that the kit comprises spodoptera frugiperda-specific SS-COI primers according to claim 1.
4. Use of Spodoptera frugiperda-specific SS-COI primers as claimed in claim 1.
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Cited By (6)
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| CN111832441A (en) * | 2020-06-28 | 2020-10-27 | 广东省农业科学院植物保护研究所 | Spodoptera frugiperda prevention and control system and method |
| CN112143817A (en) * | 2020-09-25 | 2020-12-29 | 中国农业科学院植物保护研究所 | SCAR primer for Nanmei tomato leaf miner and application thereof |
| CN113308552A (en) * | 2021-06-10 | 2021-08-27 | 四川大学 | Spodoptera frugiperda species specific primer pair, kit and identification method |
| CN113981111A (en) * | 2021-12-24 | 2022-01-28 | 北京林业大学 | Specific primer and detection method for identifying Asian gypsy moth |
| CN114075594A (en) * | 2020-08-12 | 2022-02-22 | 四川大学华西医院 | Method for detecting DNA content of Sf9 cell |
| CN117089632A (en) * | 2023-10-19 | 2023-11-21 | 中国热带农业科学院三亚研究院 | Sequence combination for rapidly detecting spodoptera frugiperda based on CRISPR/Cas12a-RPA and application thereof |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111832441A (en) * | 2020-06-28 | 2020-10-27 | 广东省农业科学院植物保护研究所 | Spodoptera frugiperda prevention and control system and method |
| CN114075594A (en) * | 2020-08-12 | 2022-02-22 | 四川大学华西医院 | Method for detecting DNA content of Sf9 cell |
| CN114075594B (en) * | 2020-08-12 | 2023-11-14 | 四川大学华西医院 | Method for detecting DNA content of Sf9 cells |
| CN112143817A (en) * | 2020-09-25 | 2020-12-29 | 中国农业科学院植物保护研究所 | SCAR primer for Nanmei tomato leaf miner and application thereof |
| CN112143817B (en) * | 2020-09-25 | 2021-12-24 | 中国农业科学院植物保护研究所 | SCAR primer for Nanmei tomato leaf miner and application thereof |
| CN113308552A (en) * | 2021-06-10 | 2021-08-27 | 四川大学 | Spodoptera frugiperda species specific primer pair, kit and identification method |
| CN113981111A (en) * | 2021-12-24 | 2022-01-28 | 北京林业大学 | Specific primer and detection method for identifying Asian gypsy moth |
| CN117089632A (en) * | 2023-10-19 | 2023-11-21 | 中国热带农业科学院三亚研究院 | Sequence combination for rapidly detecting spodoptera frugiperda based on CRISPR/Cas12a-RPA and application thereof |
| CN117089632B (en) * | 2023-10-19 | 2024-05-14 | 中国热带农业科学院三亚研究院 | Sequence combination for rapidly detecting spodoptera frugiperda based on CRISPR/Cas12a-RPA and application thereof |
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