CN109706252B - One-step dual-specificity SS-COI primer combination of Frankliniella occidentalis strain and application thereof - Google Patents
One-step dual-specificity SS-COI primer combination of Frankliniella occidentalis strain and application thereof Download PDFInfo
- Publication number
- CN109706252B CN109706252B CN201910187153.4A CN201910187153A CN109706252B CN 109706252 B CN109706252 B CN 109706252B CN 201910187153 A CN201910187153 A CN 201910187153A CN 109706252 B CN109706252 B CN 109706252B
- Authority
- CN
- China
- Prior art keywords
- strain
- greenhouse
- lupin
- frankliniella occidentalis
- primer
- 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.)
- Active
Links
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention belongs to the technical field of agricultural biology, and particularly relates to a one-step dual-specificity SS-COI primer combination of a Frankliniella occidentalis strain and application thereof. The one-step dual-specificity SS-COI primer combination of the Frankliniella occidentalis strain comprises a universal upstream primer TFZ6 of a greenhouse strain and a lupin strain, a specific downstream primer GRZ32 of the greenhouse strain and a specific downstream primer LRZ12 of the lupin strain. The one-step dual-specificity PCR primer combination amplification product of the invention has the sizes of 362bp and 541bp respectively, has good detection effect on single eggs and newly hatched larvae, improves the detection accuracy of the Frankliniella occidentalis greenhouse strain and lupin strain, 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 one-step dual-specificity SS-COI primer combination of a Frankliniella occidentalis strain and application thereof.
Background
Frankliniella occidentalis pergand, also known as alfalfa thrips, belonging to the genus Thysanoptera (Thysanoptera) thrips (Thripidae) Frankliniella, which is native to the Western North American, besides direct damage, can transmit a variety of crop viruses, such as tomato Betula virus, causing viral epidemics. The western flower thrips feeding sex is not only a worldwide quarantine pest but also an important foreign and internal quarantine object in China, wherein the types of host plants are known to be more than 500 in more than 60 families. There are two lines of frankliniella occidentalis, the greenhouse line (GR) and the lupine Line (LR), of which the greenhouse line has strong resistance to drugs. At present, greenhouse strains and lupin strains of Frankliniella occidentalis invade China and pose great threats to agriculture and forestry production. Therefore, the enhancement of the monitoring of the frankliniella occidentalis strain, particularly the resistant strain (i.e., the greenhouse strain), is an essential prerequisite for the health development of the vegetable industry, the fruit industry, and the flower industry.
The Frankliniella occidentalis is a microminiature insect, and the two strains are extremely similar in body type, body color, external morphological characteristics and the like, so that 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 enhancing quarantine and monitoring supervision. In the prior art, the PCR technology is mainly adopted to identify the Frankliniella occidentalis strain.
COI and COII and 28SD2 marking techniques have been used to identify Frankliniella occidentalis/lines and related species. The rDNA28S technical detection is to design a strain specific primer according to a D2 region sequence, judge a detection result according to the existence and the size of an expected DNA band, and do not need sequencing and sequence comparison, but the stability is poor and the specificity is not strong. The mtDNA COI and mtDNACEI technical detection is that a universal primer is utilized to amplify target DNA 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. Although there are currently about 130 known COI sequences of thrips in the database, among which there are few known COI sequences for thrips, only 4 related species, and the homology of the base sequence between the Frankliniella occidentalis greenhouse line and the lupin line is relatively high, it is difficult to find specific segments that target the Frankliniella occidentalis greenhouse line and the lupin line, respectively.
Disclosure of Invention
The invention aims to provide a one-step dual-specificity SS-COI primer combination of a Frankliniella occidentalis greenhouse strain and a lupin strain.
The invention further aims to provide application of the SS-COI primer combination with one-step dual specificity for the Frankliniella occidentalis greenhouse strain and the lupin strain.
Still another objective of the present invention is to provide a specific one-step double PCR detection method for Frankliniella occidentalis greenhouse strain and lupin strain.
Still another object of the present invention is to provide a one-step double detection kit for a Frankliniella occidentalis greenhouse strain and a lupin strain.
Still another object of the present invention is to provide a specific SS-COI primer combination for a Frankliniella occidentalis greenhouse strain.
The invention further aims to provide a PCR detection method for the Frankliniella occidentalis greenhouse strain.
The invention further aims to provide a specific detection kit for the Frankliniella occidentalis greenhouse strain.
Still another object of the present invention is to provide a specific SS-COI primer combination for lupin strain of Frankliniella occidentalis.
The invention further aims to provide a PCR detection method of the lupin strain of frankliniella occidentalis.
Still another object of the present invention is to provide a specific detection kit for lupin strain of frankliniella occidentalis.
The one-step dual-specificity SS-COI primer combination of the Frankliniella occidentalis greenhouse strain and the lupin strain comprises the following sequences:
universal upstream primers TFZ6 for greenhouse and lupin lines:
5’-CGACTTAATAACATAAGATTTT-3’；
greenhouse strain specific downstream primer GRZ 32: 5'-GATGTATCTAAGTCTCGGTCT-3', respectively;
lupin strain specific downstream primer LRZ 12: 5'-GCATAGCATAGATTAGTCCC-3' are provided.
The specific gene segments of the Frankliniella occidentalis strain are obtained by amplifying the specific one-step double SS-COI combined primers of the Frankliniella occidentalis strain, and the nucleotide sequences are respectively as follows:
greenhouse strain:
lupin strain:
the invention relates to a specific SS-COI primer combination of a Frankliniella occidentalis greenhouse strain, which comprises the following components:
universal upstream primer TFZ 6: 5'-CGACTTAATAACATAAGATTTT-3', respectively;
greenhouse strain specific downstream primer GRZ 32: 5'-GATGTATCTAAGTCTCGGTCT-3' are provided.
The specific SS-COI primer combination of the lupin strain of frankliniella occidentalis comprises the following components:
universal upstream primer TFZ 6: 5'-CGACTTAATAACATAAGATTTT-3', respectively;
lupin strain specific downstream primer LRZ 12: 5'-GCATAGCATAGATTAGTCCC-3' are provided.
According to the specific one-step double detection method of the Frankliniella occidentalis greenhouse line and the lupin line, the method comprises the step of carrying out PCR amplification by using the SS-COI combined primer specific to the Frankliniella occidentalis greenhouse line and the lupin line.
According to the one-step double PCR detection method of the Frankliniella occidentalis strain, the molar ratios of the universal upstream primer TFZ6 of the greenhouse strain and the lupin strain, the specific downstream primer GRZ32 of the greenhouse strain and the specific downstream primer LRZ12 of the lupin strain are 1.0, 0.2 and 0.8 respectively.
According to the one-step double PCR detection method of the Frankliniella occidentalis strain, the annealing temperature is 44 ℃.
According to the dual specificity detection kit for the Frankliniella occidentalis greenhouse strain and the lupin strain, the kit comprises the dual specificity SS-COI combined primer for the Frankliniella occidentalis greenhouse strain and the lupin strain.
According to the PCR detection method of the Frankliniella occidentalis greenhouse strain, the method comprises the step of carrying out PCR amplification on the Frankliniella occidentalis greenhouse strain by using the specific SS-COI primer combination of the Frankliniella occidentalis greenhouse strain.
According to the detection kit of the Frankliniella occidentalis greenhouse strain, the detection kit comprises the specific SS-COI primer combination of the Frankliniella occidentalis greenhouse strain.
According to the PCR detection method of the Frankliniella occidentalis strain, the method comprises the step of carrying out PCR amplification on the Frankliniella occidentalis strain by using the specific SS-COI primer combination of the Frankliniella occidentalis strain.
According to the detection kit of the lupin western flower thrips strain provided by the embodiment of the invention, the detection kit comprises the specific SS-COI primer combination of the lupin western flower thrips strain.
The invention has the beneficial effects that:
(1) according to the invention, the combined primer TFZ6/GRZ32/LRZ12 designed according to the dual specificity SS-COI marker of the Frankliniella occidentalis greenhouse strain and the lupin strain can respectively amplify 362bp and 541bp fragments when the Frankliniella occidentalis greenhouse strain and the lupin strain are rapidly detected by PCR; the method can be used for detecting single-head adults, 2-instar larvae, prepupa and pupae of a Frankliniella occidentalis greenhouse strain and a lupin strain, and can also be used for accurately detecting single-egg and single-head newly hatched larvae, so that the detection accuracy is high;
(2) by adopting the detection method, two fragments of 362bp and 541bp are amplified, the DNA fragments have different sizes, base sequence determination and comparison analysis are not needed, one-step dual specificity detection is completed, the whole process can be completed within 5 hours, and the operation process is simple, rapid and efficient;
(3) the dual-specificity primer combination can specifically detect the western flower thrips greenhouse strain and the lupin strain, and has no amplification product in the kindred and thrips, thereby having stronger specificity.
(4) The lowest template DNA concentration of the greenhouse strain which can be detected by the primer combination is 35.90 pg/mu L, which is equivalent to 1/10240 of head female imagoes; the lowest template DNA concentration detectable for the lupin line was 146.95 pg/. mu.L, corresponding to 1/2560 female adults. Therefore, the sensitivity is stronger.
Drawings
FIG. 1 shows the PCR amplification results of the combined primers TFZ6/GRZ32/LRZ12 on Frankliniella occidentalis greenhouse strain and lupin strain and their kindred species, thrips, wherein M is molecular weight standard Trans2KTM(ii) a 1 greenhouse strain Frankliniella occidentalis (Pergande) greenhouse race; 2, lupin line Frankliniella occidentalis (Pergande) lupine race; 3 Frankliniella intransa (Trybom); 4, Frankliniella graminicola Tenuicornis (Uzel); 5, Thrips palmi thunbergii Hawaiiensis (Morgan); 6, thistle threads major Uzel; thrips palmi Kamy; 8, Thrips tabaci thunb (Lindeman); 9, Megallurothrips usitatus (Bagnall) of common Cirsium japonicum; 10, Myrterothrips glabries (Okamoto); 11, Susseicothrips meliloti Han, a species of sweet clover; 12, thrips palmekiola serrata (Kobus); rice simple pipe thrips Haplothrips acutus (Fabricius); 14, root of Ficus auriculata, Mesothrips jordani Zimmermann; 15, Gynaikothrips ficorum (Marchal); 16, the lateral striated thrips fasciatus (L.); negative control (ultrapure water);
FIG. 2 shows the PCR amplification results of the combined primers TFZ6/GRZ32/LRZ12 on different insect states and different larval stages of Frankliniella occidentalis greenhouse line and lupin greenhouse line, wherein M is molecular weight standard Trans2KTM(ii) a Western thrips greenhouse lines: 1: egg (single grain); 2:1 instar larvae (single head); 3, 2 instar larvae; 4, prepupa; pupa; 6, adult male;7, female imagoes; western thrips lupine line: egg (single grain); 9:1 instar larvae (single head); 10:2 instar larva; 11, prepupa; 12, pupa; 13, adult male; 14, female imagoes; negative control (ultrapure water);
FIG. 3 shows the results of the determination of the lowest detection threshold of the Frankliniella occidentalis greenhouse line and lupin line with the combined primers TFZ6/GRZ32/LRZ12, M: molecular weight Standard Trans2KTMthe greenhouse strains 1-13 are respectively 18.38 plus or minus 5.40 × 103,9.19×103,4.60×103,2.30×103,1.15×103574.38,287.19,143.59,71.80,35.90,17.95,8.97,4.49 pg/mu L, lupin strain 15-27, 18.81 plus or minus 5.71 × 103,9.41×103,4.70×103,2.35×103,1.18×103587.81,293.91,146.95,73.48,36.74,18.37,9.18,4.59pg/μ L; 14,28 negative control (ultrapure water).
Detailed Description
EXAMPLE 1 preparation of specific composite primers
Specific segments in mitochondrial mtDNACEI gene sequences of a Frankliniella occidentalis greenhouse strain and a lupin strain are respectively taken as target sequences, strain specific combined primers (TFZ6/GRZ32/LRZ12) are designed, and the method specifically comprises the following steps:
universal upstream primers TFZ6 for greenhouse and lupin lines:
5’-CGACTTAATAACATAAGATTTT-3’；
greenhouse strain specific downstream primer GRZ 32: 5'-GATGTATCTAAGTCTCGGTCT-3', respectively;
lupin strain specific downstream primer LRZ 12: 5'-GCATAGCATAGATTAGTCCC-3' are provided.
PCR is carried out with the Frankliniella occidentalis greenhouse strain and lupin strain DNAs as templates, respectively, at primer combination ratios TFZ6/GRZ32/LRZ12 of 1.0/0.0/1.0, 1.0/0.1/0.9, 1.0/0.2/0.8, 1.0/0.3/0.7, 1.0/0.4/0.6, 1.0/0.5/0.5, 1.0/0.6/0.4, 1.0/0.7/0.3, 1.0/0.8/0.2, 1.0/0.9/0.1, 1.0/1.0/0. mu.L to determine the optimal primer ratio and reaction system. When greenhouse strain DNA is used as a template, 362bp single bands can be obtained under the primer combination of TFZ6/GRZ32/LRZ12 in each proportion, and the bands obtained when the primer combination proportion is 1.0/0.2/0.8 are the clearest and brightest; when lupin strain DNA is used as a template, 541bp single bands can be obtained under the condition of TFZ6/GRZ32/LRZ12 primer combination in each proportion, and the bands obtained when the primer combination proportion is 1.0/0.2/0.8 are the clearest and brightest. Therefore, the ratio of the selected primer combination is 1.0/0.2/0.8.
And (3) carrying out temperature gradient PCR with the annealing temperature of 36-54 ℃ by taking the DNA of the Frankliniella occidentalis greenhouse strain and the lupin strain as templates to determine the optimal amplification condition. When greenhouse strain DNA is used as a template, a single band of 362bp can be obtained at each annealing temperature for the primer combination ratio of TFZ6/GRZ32/LRZ12 of 1.0/0.2/0.8, and the band obtained by amplification is the clearest and brightest when the annealing temperature is 44 ℃; when lupin strain DNA is used as a template, a single 541bp band can be obtained at the annealing temperature of 36-46 ℃ in the combination ratio of TFZ6/GRZ32/LRZ12 primers of 1.0/0.2/0.8, and the amplified band is the clearest and brightest at the annealing temperature of 44 ℃. Thus, an annealing temperature of 44 ℃ was selected.
Example 2 verification of the specificity of composite primers
The method is characterized in that a combined primer TFZ6/GRZ32/LRZ12 is utilized, western flower thrips greenhouse strain and lupin strain DNA are used as templates, 14 other common thrips such as thrips loti, thrips graminifolia, thrips palmi, thrips tabaci, common thrips, thrips microphylla, thrips meliloti, thrips choisy, thrips ventricosi, thrips oryzae, thrips ficus indica, thrips banianus, thrips gynandrae, thrips striatus and the like are used as controls, and one-step double SS-COI PCR amplification is carried out.
And identifying and confirming the adult thrips insects collected in the field through morphological characteristics and DNA bar codes, extracting DNA, and storing at the temperature of 20 ℃ below zero for later use.
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, 362bp of target band is amplified in the Frankliniella occidentalis greenhouse line in lane 1, 541bp of target band is amplified in the Frankliniella occidentalis lupin line in lane 2, and other 14 kindred and species of thistle have no amplification product, so that the specificity of the one-step double SS-COI PCR amplification combined primer from the Frankliniella occidentalis greenhouse line and lupin mitochondrial DNA screened by the invention is strong.
Example 3 verification of the accuracy of composite primers
PCR amplification is carried out by using combined primers TFZ6/GRZ32/LRZ12 and taking DNA of Frankliniella occidentalis greenhouse strains and lupin strains with different insect states and different larval instars as templates.
Taking eggs, 1-2 instar larvae, prepupa, pupae, single-headed/single-grain Frankliniella occidentalis greenhouse strains and lupin strains at different development stages of male imago and female imago, extracting DNA, and storing at-20 deg.C for use.
The results are shown in FIG. 2, lanes 1-7 all show the target band of 362bp greenhouse strain, lanes 8-14 all show the target band of 541bp lupin strain, which indicates that the target gene can be successfully amplified by greenhouse strain of Frankliniella occidentalis, single egg of lupin strain, single-head first-hatched larva, 2-instar larva, pre-pupa, male imago and female imago. Therefore, the one-step dual specificity composite primer has high accuracy.
Example 4 verification of sensitivity of composite primers
And carrying out PCR amplification by taking the DNAs of the Frankliniella occidentalis greenhouse strain and the lupin strain with different dilution times as templates. Diluting the obtained primary template solution with 2-fold decreasing gradient to obtain the concentrations of 4.49 pg/muL and 4.59 pg/muL of a greenhouse strain and a lupin strain, which are respectively equal to 1/20,1/40,1/80,1/160,1/320,1/640,1/1280,1/2560,1/5120,1/10240,1/20480,1/40960 and 1/81920 female imagoes; taking 1 mu L as a template for PCR amplification, and directly adding the template into a PCR reaction system.
The result is shown in FIG. 3, 35.90 pg/. mu.L corresponding to lane 10 can amplify the target band of the 362bp greenhouse strain; 146.95 pg/. mu.L in lane 8 amplified a band of 541bp lupin strain. Therefore, the lowest template DNA concentration of the greenhouse strain detected by the primer combination is 35.90 pg/mu L, which is equivalent to 1/10240 of female adults, and the sensitivity is higher; the lowest template DNA concentration of the lupin strain which can be detected is 146.95 pg/mu L, which is equivalent to 1/2560 female imagoes, and has higher sensitivity.
Example 5 screening Effect of different primers on greenhouse lines and lupin lines
The results of comparing the 22 primer pairs (11 double combinations) provided by the software design with the primer TFZ6/GRZ32 and TFZ6/LRZ12 combinations of the present invention are shown in tables 1,2 and 3:
TABLE 1 primer screening results of Frankliniella occidentalis greenhouse lines/GR
TABLE 2 screening results of lupin line/LR primer of Frankliniella occidentalis
TABLE 3 evaluation results of Frankliniella occidentalis greenhouse lines/GR and lupin lines/LR primers
The result shows that 22 pairs of 11 double combinations provided by the software design have weak specificity, the sizes of the amplified DNA fragments are completely the same, and the base sequence determination and the comparison analysis are needed to determine the strain type; or PCR amplification cannot be carried out in the same reaction system, and one-step double PCR targeting specific segments of the greenhouse strain and the lupin strain respectively cannot be carried out. Compared with a primer combination provided by software design, the optimally designed combined primer has strong specificity, the homology of the amplified greenhouse strain specific section and the COI gene sequence of the target species Frankliniella occidentalis greenhouse strain reaches 96.95-100%, the homology with the lupin strain is 94.20-96.41%, and the homology with other kindred species is less than 87%; the homology of the specific segment of the amplified lupin strain and the COI gene sequence of the target species of the lupin strain of frankliniella occidentalis reaches 98.34-100%, the homology with the greenhouse strain is 92.74-96.49%, and the homology with other kindred species is less than 84%. And the greenhouse strain and the lupin strain can be simultaneously detected in the same PCR reaction system, and the strain type can be judged according to the existence and the size of a specific strip without base sequence determination and comparative analysis.
Sequence listing
<110> institute of plant protection of Chinese academy of agricultural sciences
<120> one-step dual-specificity SS-COI primer combination of Frankliniella occidentalis strain and application thereof
<160>3
<170>SIPOSequenceListing 1.0
<210>1
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>1
<210>2
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>2
gatgtatcta agtctcggtc t 21
<210>3
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>3
Claims (3)
1. Use of a one-step dual-specific SS-COI primer combination of a frankliniella occidentalis strain for identifying a frankliniella occidentalis strain in a greenhouse and a lupin strain, wherein the primer combination comprises the following primers:
universal upstream primers TFZ6 for greenhouse and lupin lines:
5’-CGACTTAATAACATAAGATTTT-3’；
greenhouse strain specific downstream primer GRZ 32: 5'-GATGTATCTAAGTCTCGGTCT-3', respectively;
lupin strain specific downstream primer LRZ 12: 5'-GCATAGCATAGATTAGTCCC-3' are provided.
2. The use of claim 1, wherein the molar ratio of the universal upstream primer TFZ6 for the greenhouse line and lupin line, the GRZ32 for the greenhouse line specific downstream primer GRZ 12 for the lupin line specific downstream primer LRZ12 is 1.0, 0.2, 0.8, respectively.
3. The use according to claim 1, wherein the primer anneals at a temperature of 44 ℃.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910187153.4A CN109706252B (en) | 2019-03-13 | 2019-03-13 | One-step dual-specificity SS-COI primer combination of Frankliniella occidentalis strain and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910187153.4A CN109706252B (en) | 2019-03-13 | 2019-03-13 | One-step dual-specificity SS-COI primer combination of Frankliniella occidentalis strain and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109706252A CN109706252A (en) | 2019-05-03 |
| CN109706252B true CN109706252B (en) | 2020-09-18 |
Family
ID=66265801
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910187153.4A Active CN109706252B (en) | 2019-03-13 | 2019-03-13 | One-step dual-specificity SS-COI primer combination of Frankliniella occidentalis strain and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109706252B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112522419B (en) * | 2020-10-06 | 2022-09-02 | 中国农业科学院植物保护研究所 | Double PCR detection method for parthenogenetic female strain and amphoteric strain of Brachymyza virgata |
| CN114317768B (en) * | 2021-12-21 | 2023-04-07 | 广西壮族自治区农业科学院 | Dual PCR detection primer and method for identifying frankliniella occidentalis and frankliniella occidentalis |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101838644A (en) * | 2010-01-22 | 2010-09-22 | 中国农业科学院植物保护研究所 | SCAR primer, detection method and kit for Frankliniella occidentalis specificity |
| CN101818146B (en) * | 2010-01-22 | 2011-11-23 | 中国农业科学院植物保护研究所 | Specific COI primer, detection method and reagent kit of Frankliniella occidentalis |
-
2019
- 2019-03-13 CN CN201910187153.4A patent/CN109706252B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101838644A (en) * | 2010-01-22 | 2010-09-22 | 中国农业科学院植物保护研究所 | SCAR primer, detection method and kit for Frankliniella occidentalis specificity |
| CN101818146B (en) * | 2010-01-22 | 2011-11-23 | 中国农业科学院植物保护研究所 | Specific COI primer, detection method and reagent kit of Frankliniella occidentalis |
Non-Patent Citations (2)
| Title |
|---|
| Sudden Widespread Distribution of Frankliniella occidentalis (Thysanoptera: Thripidae) in Shandong Province, China;Hui-Sheng Duan等;《Florida Entomologist》;20130930;第96卷(第3期);第933-940页 * |
| 入侵种西花蓟马与本地近缘种花蓟马的双基因鉴定;田虎等;《中国生物防治学报》;20171031;第33卷(第5期);摘要、第1.3部分 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109706252A (en) | 2019-05-03 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108707687B (en) | PCR detection and identification method capable of distinguishing blast bacteria and rice blast bacteria | |
| CN106987653B (en) | LAMP (loop-mediated isothermal amplification) detection primer for potato late blight bacteria and visual detection method thereof | |
| CN111235286A (en) | Spodoptera frugiperda specific SS-CO I primer and application thereof | |
| Walsh et al. | Rapid detection of Grapevine leafroll-associated virus type 3 using a reverse transcription loop-mediated amplification method | |
| CN109706252B (en) | One-step dual-specificity SS-COI primer combination of Frankliniella occidentalis strain and application thereof | |
| Opiyo et al. | Development of a multiplex PCR technique for simultaneous detection of Sweet potato feathery mottle virus and Sweet potato chlorotic stunt virus | |
| CN104745698A (en) | Ring mediated isothermal amplification primer composite for Didymella bryoniae | |
| Topkaya et al. | Presence of cucurbit viruses in Ankara and Antalya province and molecular characterization of coat protein gene of zucchini yellow mosaic virus Turkish isolates | |
| Przybylska et al. | PCR-RFLP method to distinguish Frankliniella occidentalis, Frankliniella intonsa, Frankliniella pallida and Frankliniella tenuicornis | |
| CN108949916B (en) | Rape black shank bacterium specific sequence, LAMP detection primer and application | |
| CN102277444A (en) | Method for quickly distinguishing grape varieties by random amplified polymorphic deoxyribonucleic acid (RAPD) | |
| Mantip et al. | Comparison of nucleotide sequences of recent and previous lineages of peste-des-petits-ruminants viruses of sheep and goats in Nigeria | |
| CN110257528B (en) | Codling moth specific SS-COII primer and application thereof | |
| KR102238486B1 (en) | Primer sets for the detection of Phytophthora species and use thereof | |
| Schlenzig et al. | Thuja occidentalis: a new host for Phytophthora lateralis | |
| CN104673890B (en) | Cotton black aphid PCR quick determination methods based on specific SS COI primers | |
| CN104498593A (en) | Primer pair and kit for identification or assisted identification of stored bean weevils | |
| KR101662312B1 (en) | Primer set for reverse transcription loop-medicated isothermal amplification(RT-LAMP) for rapid detection of Korean field rabies virus, and uses there of | |
| CN103773860A (en) | Harmonia axyridis specific COI primer of harmonia axyridis, kit containing primer and detection method for harmonia axyridis | |
| CN103451298B (en) | Kit for detecting physiological races of brussels sprouts wilt pathogens I and II and detection method thereof | |
| CN106978505B (en) | Primer pair for detecting pedunculosis solani and application thereof | |
| CN109371173A (en) | A method of based on RPA detection tomato chlorisis virus | |
| CN107663547B (en) | PCR (polymerase chain reaction) rapid detection method for frankliniella occidentalis based on specific SS-COI (sequence specific-polymerase chain reaction) primer | |
| CN104250665B (en) | No. 5 microspecies RTQ-PCR detection primers of Ralstonia solanacearum and method | |
| CN114317768B (en) | Dual PCR detection primer and method for identifying frankliniella occidentalis and frankliniella occidentalis |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |