CN112143817B - SCAR primer for Nanmei tomato leaf miner and application thereof - Google Patents
SCAR primer for Nanmei tomato leaf miner 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 south American tomato leaf miner specific SCAR primer and application thereof. The specific primer of the invention has amplification capacity only for south American tomato leaf miner, and the size of an amplification product is 397 bp. The kit has good detection effect on single eggs and adult residues, improves the detection accuracy of the south American tomato leaf miner, saves the detection time, and is suitable for popularization in entry plant quarantine, domestic plant quarantine and field control in the form of a kit.
Description
Technical Field
The invention belongs to the technical field of agricultural biology, and particularly relates to a south American tomato leaf miner specific SCAR primer and application thereof.
Background
South America tomato leaf miner Tuta absoluta (Meyrinck), namely tomato leaf miner, tomato leaf miner and tomato wheat moth, the common name of the South American tomato leaf miner is selected in English, belongs to Lepidoptera (Lepidotera) Merlongensis (Gelechiideae), is originally produced in Peru in South America, and is a worldwide quarantine invasive pest. The Nanmei tomato leaf miner host has a wide range, is harmful to vegetables (such as tomatoes, eggplants, pimientos, beans, spinach and the like), fruits (such as cherry tomatoes/cherry tomatoes, physalis pubescens, ginseng fruits and the like), food crops (such as potatoes), sugar crops (such as beets), tobacco and weed plants (such as black nightshade, Convolvulus arvensis and the like), is rapid in diffusion, has extremely high yield reduction rate when serious occurrence occurs, and even destroys seeds for reseeding, and stopping production. At present, although the south American tomato leaf miner only occurs in local areas of China, serious harm is caused to tomatoes in protected areas and open areas, and therefore, the strengthening of detection and monitoring of the south American tomato leaf miner is the primary premise for guaranteeing the healthy development of vegetable and fruit industries.
The south American tomato leaf miner is a boring insect, is usually hidden, is difficult to discover early particularly in the early stage of occurrence, the traditional morphological identification method is difficult to realize 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 identification method for the south American tomato leaf miner mainly comprises the following steps: 1) identifying morphological characteristics; 2) PCR techniques, and the like. However, at present, no standard detection method exists for south American tomato leaf miner in China.
RAPD and ITS, COI technology (and SS-COI technology) have been used to identify Nanmei tomato leaf miner and ITS kindred species. The RAPD technology uses genome DNA as a template, uses a single artificially synthesized random polymorphic nucleotide sequence as a primer, performs PCR amplification under the catalysis of DNA polymerase, and judges a detection result according to polymorphism on an ultraviolet fluoroscope after agarose or polyacrylamide electrophoretic separation and ethidium bromide staining of an amplification product. The rDNA ITS technology and mtDNA COI technology are used for detecting, a universal primer is utilized, target DNA is amplified in vitro under the catalysis of DNA polymerase, then PCR products are recovered, purified and sequenced, and the detection result is judged according to sequence comparison and phylogenetic tree construction. The target species specificity SCAR-PCR detection technology is a specific sequence amplification area marker developed on the basis of RAPD technology, utilizes specific primers, judges a detection result according to the existence and the size of an expected DNA band, does not need sequencing and sequence comparison, and has the advantages of high sensitivity, strong specificity, quickness, simplicity, convenience, good reproducibility, strong stability and the like.
Disclosure of Invention
The invention aims to provide a pair of specific SCAR primers for Nanmei tomato leaf miner.
The invention further aims to provide a specific detection method for the plutella xylostella of the south American tomato.
The invention further aims to provide a kit for detecting the leaf miner of the south American tomato.
According to the specific SCAR primer of the Nanmei tomato leaf miner, the primer sequence is as follows:
TAZBF1：5′-ACGAAAACACACAGCGACAT-3′；
TAZBR1：5′-AACACACCTGCGAGAGAA-3′。
the specific gene fragment 397bp of the south American tomato leaf miner is obtained by amplifying the specific SCAR primer of the south American tomato leaf miner, and the nucleotide sequence is as follows:
acgaaaacac acagcgacat taattggcat agccattatt tcaagacttt atactatgca 60
aaaatatgtt tgcttcaacg ttcaatatcc caaatgtggg tcatagatca gagggaacag 120
tttcaaatgg gaatcgttct ccgcgcgctt aattgctgcc tctccggtgc agcgccgaga 180
tggatgcccg caaatggctt tttaatcttt cgatcgagag ttgagtctca cctcattgct 240
tctgcggaag tttatttatc tttatagatg ggaaatcgtc agtttatccc gtgggagcaa 300
tcagcgctgg tgtcggcgtg acccagatcg gccgaaactc atgcacgtcg tttagcagag 360
tccgcggtag tgtcgtgcat tctctcgcag gtgtgtt 397
according to the specific detection method of the south American tomato leaf miner, the method comprises the step of carrying out PCR amplification by using the south American tomato leaf miner specific SCAR primer.
According to the specific detection kit for the south American tomato leaf miner, the kit comprises the south American tomato leaf miner specific SCAR primer.
The invention has the beneficial effects that:
(1) the primers TAZBF1 and TAZBR1 designed according to the species-specific SCAR marker of the Nanmei tomato leaf miner can amplify a 397bp fragment when PCR is used for rapidly detecting the Nanmei tomato leaf miner, so that single-head adults and 2-4 instar larvae of the Nanmei tomato leaf miner can be detected, and single-egg and single-head primarily hatched larvae as well as adult residues (including feet, wings, tentacles, beaks and the like) 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 the leafminer of the south American tomato, and has no amplification product in related species, leafminer/fruit-eating pests.
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 TAZBF1/TAZBR1 against Nanmei tomato leaf miner and related species, leaf eating/leaf miner/fruit eating pests, wherein M is molecular weight standard Trans2 KTM; 1,26, south American tomato leaf miner Tuta absoluta (Meyrinck); 2, tuber moth Phthyimaea operculella (Zeller); 3, sweet potato moth Brachymia macrocopa (Meyrinck); 4, the pink bollworm Pectinophora gossypiella (Saunders); 5, the wheat moth Sitotroga cerealella (Olivier); 6 Liriomyza sativae (Blancard); 7, Liriomyza trifolii (Burgess); 8, Liriomyza huidobrensis Blanchard; 9, Liriomyza chinensis (Kato); 10, pea color fly Phytomyza horticola Gourean; plutella xylostella (L.); spodoptera frugiperda (J.E. Smith); 13,14 negative control (ultrapure water); 15 Spodoptera exigua (Hubner); spodoptera litura (Fabricius); 17 Helicoverpa armigera (Hubner); 18, Anta trifolii (Hufnagel) of Spodoptera nutans; mythimna separata (Walker); 20 Argyrogramma agnata (Staudinger); 21, Conogethes punctiferalis (Guen bee); asiatic corn borer Ostrinia furnacalis (Guenee); 23 Indian meal moth Plodia interpunctella (Hubener) 24 Grapholita molesta Busck; 25 Cydia pomonella L.;
FIG. 2 shows the PCR amplification results of primers TAZBF1/TAZBR1 for different insect states, different larval instar stages and different adult sexes of Nanmei tomato leaf miner, wherein M is molecular weight standard Trans2KTM(ii) a 1: egg (single grain); 2:1 instar larvae (single head); 3:2 instar larvae (single head); 4:3 instar larvae (abdominal 1/2); 5:4 instar larvae (abdominal 1/3); pupa (abdomen 1/2); adult (female, abdominal 1/4); adult (male, ventral 1/4); negative control (ultrapure water);
FIG. 3 shows the results of the detection of adult tomato leaf miner residues in south America by primers TAZBF1/TAZBR1, wherein M is molecular weight standard Trans2KTM(ii) a 1, antennae (1 root); 2, lower lip beard (1 root); 3, beak (1); 4, forefoot (1 strip); 5, middle feet (1 strip); 6, hind feet (1 strip); 7, front wings (1); 8, back wings (1); 9, head (excluding tentacle, lower lip beard and beak); chest (except feet and wings, 1/2); 11, abdomen (1/2); negative control (ultrapure water);
FIG. 4 shows the determination of the minimum detection threshold for the tomato leaf miner south America with the specific primers TAZBF1/TAZBR1,
m molecular weight Standard Trans2KTM(ii) a 1-13, respectively 138.4 +/-10.7 multiplied by 103,69.2×103,34.6×103,17.3×103,8.65×103,4.33×103,2.16×103,1.08×103540.63,270.31,135.16,67.58 pg/. mu.L, 13 negative control (ultrapure water).
Detailed Description
Example 1 preparation of specific primers for Nanmei tomato leaf miner
A specific segment in a genomic DNA gene sequence of the south American tomato leaf miner is taken as a target sequence, and a south American tomato leaf miner specific primer (TAZBF1/TAZBR1) is designed, wherein the sequence of the primer is as follows:
TAZBF1：5′-ACGAAAACACACAGCGACAT-3′；
TAZBR1：5′-AACACACCTGCGAGAGAA-3′。
the invention takes south American tomato leaf miner DNA as a template, the dosage of the DNA template is 0.5-2.0 muL (0.5,0.6,0.8,0.9,1.0,2.0 muL), the dosage of an upstream primer and a downstream primer is 0.2-0.5 muL (0.2,0.3, 0.35, 0.4,0.5 muL), the dosage of Taq DNA polymerase is 0.15-0.25 muL (0.15,0.20,0.25 muL), so as to determine an optimal amplification system; meanwhile, temperature gradient PCR with 27-38 cycles (27,28,29,30,35,38 cycles), denaturation time 30-60s (30,60s), annealing time 30-40s (30,45,60s), extension time 30-60s (30,60s), and annealing temperature 52-62 deg.C (52,54,56,58,60,62 deg.C) was performed to determine the optimal amplification conditions. For the primer pair TAZBF1/TAZBR1, a single band of 397bp can be obtained under each DNA template, and the band obtained by amplification is the clearest and bright when the amount of the DNA template is 0.8 mu L; a single band of 397bp can be obtained under the dosage of each primer, and the band obtained by amplification is the clearest and brightest when the upstream primer and the downstream primer are 0.3 mu L, so that the dosage of the selected primer is 0.3 mu L; a single band of 397bp can be obtained under the condition of using the Taq DNA polymerase, and the band obtained by amplification is the clearest and brightest when the using amount of the polymerase is 0.2 mu L, so that the using amount of the DNA polymerase is 0.2 mu L. A single band of 397bp can be obtained under each cycle, and the band obtained by amplification is the clearest and brightest when the cycle is 29, so that the cycle is 29; a single band of 397bp can be obtained in all denaturation time, annealing time and extension time, and the amplified band is the clearest and brightest when the denaturation time is 30s and the annealing time and the extension time are 30s, so that the denaturation time, the annealing time and the extension time are all 30 s; a single band of 397bp can be obtained at each annealing temperature, and the band obtained by amplification is the clearest and brighter when the annealing temperature is 60 ℃, so that the annealing temperature is selected to be 60 ℃.
Example 2 specific amplification Effect of primers TAZBF1/TAZBR1 on tomato leaf miner
The primer TAZBF1/TAZBR1 is used for carrying out SCAR PCR amplification by using DNA of Nanmei tomato leaf miners as templates and 22 other common species of leaf miners/fruit borers such as potato tuber moth, sweet potato wheat moth, cotton bollworm, wheat moth, American leaf miners, south America leaf miners, onion leaf miners, pea color miners, plutella xylostella, spodoptera frugiperda, beet armyworm, prodenia litura, cotton bollworms, helicoverpa virescens, armyworm, silverworm, dichocrocis punctifera, Asian corn borers, Indian meal moth, oriental fruit borers, codling moth and the like as controls.
Collecting leaf-hiding/leaf-eating/fruit-eating insect larvae (2-3 years old) in the field, identifying and confirming through morphological characteristics and DNA bar codes, extracting DNA, and storing at-20 ℃ for later use.
mu.L of the PCR product was electrophoretically separated on a 1.5% (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, the target band of 397bp was amplified in the tomato leaf miner of lanes 1 and 26, and no amplification products were obtained in all 22 other related species of leaf miner/leaf eating/fruit eating insects, therefore, the SCAR PCR amplification primers from the genomic DNA of tomato leaf miner of the invention have strong specificity.
Example 3 amplification Effect of primers TAZBF1/TAZBR1 on different insect status, sex and larval age of Nanmei tomato leaf miner
PCR amplification is carried out by using primers TAZBF1 and TAZBR1 and using Nanmei tomato leaf miner DNAs with different insect states, different sexes and different larva instars as templates.
Taking single-head/single-grain tomato leaf miner of different development stages (eggs, 1-4 instar larvae and pupae) and adults of different sexes (female adults and male adults), wherein 3 instar larvae take abdomen 1/2, 4 instar larvae take abdomen 1/3, pupae take abdomen 1/2, adult take abdomen 1/4, extracting DNA, and storing at-20 ℃ for later use.
The results are shown in FIG. 2, lanes 1-8 all show target bands of 397bp, which indicates that the target genes can be successfully amplified by single eggs and single-head primarily hatched larvae of the Spodoptera frugiperda, and female adults and male adults of 2-instar larvae, 3-instar larvae, 4-instar larvae, pupae and Spodoptera frugiperda, so that the specific primers disclosed by the invention are high in accuracy.
Example 4 amplification Effect of primers TAZBF1/TAZBR1 on adult Nanmei tomato leaf miner residues
PCR amplification is carried out by using primers TAZBF1 and TAZBR1 and DNA of different residues of the adult tomato leaf miner in south America as a template.
The DNA of the various adult residues (female, including single antennal, lower labial whisker and beak, single forefoot, midfoot and hind paw, single anterior wing and posterior wing, and head (excluding antennal, lower labial whisker and beak), thorax (3 vs thoracic feet and 2 vs wings, 1/2), and abdomen 1/2) were obtained separately.
As a result, as shown in FIG. 3, 397bp bands were amplified in lanes corresponding to the antennal, lower labial whisker, beak, forefoot, midfoot, hind paw, anterior wing, posterior wing, head, chest and abdomen, and thus the detection accuracy of the adult insect residues by the specific primers TAZBF1 and TAZBR1 of the present invention was high.
Example 5 determination of the threshold of detection of primers TAZBF1/TAZBR1 against Nanmey tomato leaf miner
Carrying out PCR amplification by taking the DNA of the south American tomato leaf miner with different dilution multiples as a template, diluting the obtained original template solution by a 2-fold descending gradient to the concentration of 67.58 pg/mu L, which is equivalent to 1/100, 1/200, 1/400, 1/800, 1/1600, 1/3200, 1/6400, 1/12800,1/25600, 1/51200, 1/102400 and 1/204800 head female adults; taking 0.8 mu L as a template for PCR amplification, and directly adding the template into a PCR reaction system.
As shown in FIG. 4, 1.08 ng/. mu.L corresponding to lane 8 amplified a target band of 397bp, so that the lowest template DNA concentration detected by the method of the invention was 1.08 ng/. mu.L, which is equivalent to 1/12800 adult female adults, and the sensitivity was high.
Example 6 screening comparison of different primers
The results of comparing 3 primers provided by the software design with the primers TAZBF1/TAZBR1 of the present invention are shown in Table 1:
TABLE 1 screening and evaluation of Nanmei tomato leaf miner specific SCAR primers
The result shows that the specificity of the 2 pairs of primers provided by the software design is not strong, and the primers cannot be matched with the sequence of the target species of the Nanmei tomato leaf miner. 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 DNA gene sequence of the target species of the south American tomato leaf miner reaches 99.24-99.75%, and the homologous sequences of other related species are not searched.
Sequence listing
<110> institute of plant protection of Chinese academy of agricultural sciences
SCAR primer for <120> Nanmei tomato leaf miner and application thereof
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
<210> 2
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
<210> 3
<211> 397
<212> DNA
<213> Nanmei tomato leaf miner (Tuta absoluta)
<400> 3
acgaaaacac acagcgacat taattggcat agccattatt tcaagacttt atactatgca 60
aaaatatgtt tgcttcaacg ttcaatatcc caaatgtggg tcatagatca gagggaacag 120
tttcaaatgg gaatcgttct ccgcgcgctt aattgctgcc tctccggtgc agcgccgaga 180
tggatgcccg caaatggctt tttaatcttt cgatcgagag ttgagtctca cctcattgct 240
tctgcggaag tttatttatc tttatagatg ggaaatcgtc agtttatccc gtgggagcaa 300
tcagcgctgg tgtcggcgtg acccagatcg gccgaaactc atgcacgtcg tttagcagag 360
tccgcggtag tgtcgtgcat tctctcgcag gtgtgtt 397
<210> 4
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
<210> 5
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
<210> 6
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
<210> 7
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
Claims (9)
1. The specific SCAR primer pair for the south American tomato leaf miner is characterized by comprising primers with the following sequences:
primer TAZBF 1: 5'-ACGAAAACACACAGCGACAT-3';
primer TAZBR 1: 5'-AACACACCTGCGAGAGAA-3'.
2. The method for detecting the specificity of the south American tomato leaf miner is characterized by comprising the step of carrying out PCR amplification by using the south American tomato leaf miner specificity SCAR primer pair disclosed by claim 1, and if an amplification product obtains a single band of 397bp, judging that a sample to be detected contains the south American tomato leaf miner.
3. The specific detection method of the Nanmei tomato leaf miner according to claim 2, wherein the amount of the primer is 0.3 μ L.
4. The method for detecting the specificity of the tomato leaf miner in south America according to claim 2, wherein the dosage of the DNA polymerase is 0.2 μ L.
5. The method for specifically detecting the leafminer of the south American tomato as claimed in claim 2, wherein the number of cycles is 29.
6. The method for detecting the specificity of the Nanmei tomato leaf miner according to claim 2, wherein the denaturation time, annealing time and extension time are all 30 s.
7. The method for detecting the specificity of the tomato leaf miner in south america as claimed in claim 2 wherein the annealing temperature is 60 ℃.
8. The kit for detecting the specificity of the Nanmei tomato leaf miner is characterized by comprising the specific SCAR primer pair of the Nanmei tomato leaf miner, which is disclosed by claim 1.
9. The application of the south American tomato leaf miner specific SCAR primer pair in detecting or identifying the south American tomato leaf miner in claim 1 is characterized in that the south American tomato leaf miner specific SCAR primer pair in claim 1 is used for carrying out PCR amplification on a sample to be detected, and if an amplification product obtains a single band of 397bp, the sample to be detected is judged to contain the south American tomato leaf miner.
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