CN113584208B - Method for detecting Diadorthe novem by using PCR primer - Google Patents
Method for detecting Diadorthe novem by using PCR primer Download PDFInfo
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- 238000000034 method Methods 0.000 title claims description 16
- 238000001514 detection method Methods 0.000 claims abstract description 20
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- 238000003199 nucleic acid amplification method Methods 0.000 claims description 3
- 238000012408 PCR amplification Methods 0.000 claims description 2
- 238000000246 agarose gel electrophoresis Methods 0.000 claims description 2
- 230000003321 amplification Effects 0.000 claims description 2
- 238000003556 assay Methods 0.000 claims 1
- 238000005516 engineering process Methods 0.000 abstract description 2
- 244000000004 fungal plant pathogen Species 0.000 abstract description 2
- 238000011895 specific detection Methods 0.000 abstract description 2
- 244000068988 Glycine max Species 0.000 description 9
- 235000010469 Glycine max Nutrition 0.000 description 9
- 241000196324 Embryophyta Species 0.000 description 8
- 241000235349 Ascomycota Species 0.000 description 6
- 241000866066 Diaporthe caulivora Species 0.000 description 5
- 241000802132 Diaporthe aspalathi Species 0.000 description 4
- 241001235320 Diaporthe conorum Species 0.000 description 4
- 241000533786 Diaporthe longicolla Species 0.000 description 4
- 241001508801 Diaporthe phaseolorum Species 0.000 description 4
- 241000233866 Fungi Species 0.000 description 4
- 239000013642 negative control Substances 0.000 description 4
- 244000052769 pathogen Species 0.000 description 4
- 230000001717 pathogenic effect Effects 0.000 description 4
- 241000983238 Diaporthe novem Species 0.000 description 3
- 241001373666 Diaporthe sp. Species 0.000 description 3
- 244000046052 Phaseolus vulgaris Species 0.000 description 3
- 235000010627 Phaseolus vulgaris Nutrition 0.000 description 3
- 208000025865 Ulcer Diseases 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 231100000397 ulcer Toxicity 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241001512584 Diaporthe vaccinii Species 0.000 description 2
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- 235000003222 Helianthus annuus Nutrition 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 235000013399 edible fruits Nutrition 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 235000012424 soybean oil Nutrition 0.000 description 2
- 239000003549 soybean oil Substances 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 244000298697 Actinidia deliciosa Species 0.000 description 1
- 235000009436 Actinidia deliciosa Nutrition 0.000 description 1
- 241000167854 Bourreria succulenta Species 0.000 description 1
- 244000089742 Citrus aurantifolia Species 0.000 description 1
- 235000008733 Citrus aurantifolia Nutrition 0.000 description 1
- 244000175448 Citrus madurensis Species 0.000 description 1
- 208000011231 Crohn disease Diseases 0.000 description 1
- 241001508802 Diaporthe Species 0.000 description 1
- 235000017317 Fortunella Nutrition 0.000 description 1
- 244000267823 Hydrangea macrophylla Species 0.000 description 1
- 235000014486 Hydrangea macrophylla Nutrition 0.000 description 1
- 241000228456 Leptosphaeria Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 108700001094 Plant Genes Proteins 0.000 description 1
- 241001107098 Rubiaceae Species 0.000 description 1
- 239000005708 Sodium hypochlorite Substances 0.000 description 1
- 244000269722 Thea sinensis Species 0.000 description 1
- 235000006468 Thea sinensis Nutrition 0.000 description 1
- 235000011941 Tilia x europaea Nutrition 0.000 description 1
- 235000003095 Vaccinium corymbosum Nutrition 0.000 description 1
- 235000017537 Vaccinium myrtillus Nutrition 0.000 description 1
- 244000077233 Vaccinium uliginosum Species 0.000 description 1
- 235000009754 Vitis X bourquina Nutrition 0.000 description 1
- 235000012333 Vitis X labruscana Nutrition 0.000 description 1
- 240000006365 Vitis vinifera Species 0.000 description 1
- 235000014787 Vitis vinifera Nutrition 0.000 description 1
- 241000607479 Yersinia pestis Species 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 235000020279 black tea Nutrition 0.000 description 1
- 235000021014 blueberries Nutrition 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 235000019693 cherries Nutrition 0.000 description 1
- 238000012272 crop production Methods 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000003337 fertilizer Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
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- 239000001963 growth medium Substances 0.000 description 1
- 239000004571 lime Substances 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 239000003921 oil Substances 0.000 description 1
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- 238000002360 preparation method Methods 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
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- 230000001954 sterilising effect Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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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
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
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- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
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Abstract
The present invention relates to the detection of plants and plant products using PCR technologyDiaporthe novemBelongs to the field of plant pathogenic fungi detection. The invention designs and synthesizesD.novemFor specific primers for (A) toD.novemSpecific detection of genomic DNA. The invention establishes a rapid, simple, convenient, high-specificity, accurate and reliableD.novemThe whole detection is completed within one working day.
Description
Technical Field
The present invention relates to the detection of plants and plant products using PCR technologyDiaporthe novem J.M. Santos, Vrandečić & A.J.L. Phillips, sp. nov.Belongs to the field of plant pathogenic fungi detection.
Background
Diaporthe novemBelonging to the fungus kingdom (Fungi), ascomycota (Ascomycota), ascomycetes (Ascomycetes), ascomycetes (Diadorthales), ascomycetes (Diadorthaceae), ascomycetesDiaporthe) The asexual stage isPhomopsissp.9. It is used for curing northern stem ulcer of soybeanBacteria of ulcer diseaseD. caulivoraLeptosphaeria sojae (Fr.) KummerD. aspalathiAnd soybean phomopsisD. longicollaIs similar to the species, wherein the northern stem canker germ and southern stem canker germ are listed in the entry plant quarantine pest directory of China.
D. novemOriginally formally named by Jorge Manuel Santos et al in 2011, the prior art shows that the pathogen is distributed in areas such as chile, crohn's disease, italy, portugal, south africa, australia, etc. Serious economic losses are caused by the damage to important commercial crops such as soybean, sunflower, kiwi fruit, grape, kumquat, lime and the like, and serious economic losses are caused by the decay of seeds and fruits, and the method has separation reports on hydrangea, south african black tea and Rubiaceae Lavine, but has not been reported in China.
As an important grain and oil serving as crops and feed raw materials, the soybean has great demands in China, is one of the most important imported agricultural products in China, and the total imported soybean quantity in 2019 China reaches 8851 ten thousand tons. China's port multiple detectionD. novemSince the pathogen is not in the quarantine directory, its hazard is often easily ignored. The germ causes soybean stem ulcers and seed rot, resulting in reduced soybean yield and quality. The soybean oil produced by the damaged seeds and other products such as feed, fertilizer, bean cake and the like obtained after the soybean oil is squeezed are low in quality and are not suitable for being used as the feed. In addition, the germ host is wide, once the soybeans enter China along with the entering environment, immeasurable losses are caused to the soybeans and other crop production and agricultural ecology in China.
In view of the followingD. novemThe risk and the severity of the pathogen hazard transmitted into China, in order to protect the production safety of agriculture and forestry in China, maintain the national interests, further standardize the quarantine of the important diseases, improve the accuracy and the verifying and placing speed of the epidemic situation detection of the port, prevent the pathogen from being transmitted into China, and establish a set of rapid, accurate and complete detection and prevention methodsD. novemThe detection method of (2) is urgent.
Disclosure of Invention
The invention collects 8 fungi (see table 1) in the mesothelium, and establishes a fast, simple, convenient, high-specificity, accurate and reliable mesotheliumThe method for detecting the fungus molecules of the genus Fabricius can be used for detectingD. novemAnd (5) accurately identifying. The method is rapid in detection, reliable in method, and capable of being effectively popularized and applied in port detection, and the whole process is completed within one working day.
The specific technical scheme is as follows:
the invention aims to provide a detection methodD. novemA method of PCR of genomic DNA comprising the steps of:
(1) Extracting plant genome DNA;
（2）D. novemthe sequence of the specific primer is as follows:
DnoF1：CTCACACTGGCTGGATTATG
DnoR1：AGCCGCTTATCTCCTATGC
the primer is used forD. novemGenomic DNA specific detection, amplified fragment of about 329bp.
Compared with the prior art, the invention has the beneficial effects that: the invention establishes the rapid, simple, convenient, strong, accurate and reliableD. novemMolecular detection method, capable ofD. novemIs different from other kinds of seat shells. The method is rapid in detection, reliable in method, and capable of being effectively popularized and applied in port detection, and the whole process is completed within one working day.
The invention is described in further detail below with reference to the drawings and the detailed description.
Drawings
FIG. 1D. novemAmplification of genomic DNA specific primers
Lanes 1-9 in the figure are respectivelyD. novem, D. longicolla , D. caulivora, D. aspalathi, Diaporthe sp., D. eres , D.phaseolorum, D. vacciniiAnd a negative control, lane M is a 2000 bp DNA marker.
Detailed Description
Example 1: extraction of genomic DNA from plant material
This experimentD. novemThe strain is derived from Tianjin customs animal-plant feeding center plant detection laboratory. A total of 8 strains, and the related information is shown in Table 1.
TABLE 1 test Material code, latin name, chinese name and year of collection
| Sequence number | Latin name | Chinese name | Year of collection |
| 1 | Diaporthe novem | - | 2019 |
| 2 | Diaporthe longicolla | Seed rot of soybean | 2019 |
| 3 | Diaporthe caulivora | North stem canker of soybean | 2019 |
| 4 | Diaporthe aspalathi | Leptosphaeria sojae (Fr.) Kummer | 2019 |
| 5 | Diaporthesp. | Sunflower chamber seat shell | 2020 |
| 6 | Diaporthe eres | Seat shell between cherry | 2020 |
| 7 | Diaporthephaseolorum | Bean compartment base shell | 2020 |
| 8 | Diaporthe vaccinii | Blueberry fruit rot germ | 2020 |
Selecting shrunken disease beans with disease spots, sterilizing with 1% sodium hypochlorite for 5 min, washing with sterilized water for 3 times, culturing at 25deg.C for 24 h, freezing at-20deg.C for 24 h, and culturing on PDA culture medium at 22deg.C under 12 h alternatively. After 5d, the culture results were observed. Separating and purifying suspicious colony with PDA, collecting proper amount of mycelium blocks, grinding with liquid nitrogen, extracting DNA with QIAGEN Dneasy plant mini kit plant gene extraction kit, and dissolving DNA in 100μL1 xTE buffer, and storing at-20deg.C.
Example 2: design of specific primers
Artificial synthesisD. novemThe primer sequences are as follows:
DnoF1：CTCACACTGGCTGGATTATG
DnoR1：AGCCGCTTATCTCCTATGC
example 3:D. novemPCR amplification of specific primers
1. Preparation of the reaction Mixed solution
The configuration of the reaction system is shown in Table 2.
TABLE 2D. novemSpecific amplification system
| Reagent(s) | Sample addition amount/. Mu.l |
| 2× Premix | 12.5 |
| ddH 2 O | 10.0 |
| DnoF1 | 0.5 |
| DnoR1 | 0.5 |
| Template | 1.5 |
| Total | 25.0 |
2. PCR reaction procedure
Pre-denaturation: 94 ℃ for 3min
Denaturation: 94 ℃ and 30s
Annealing: 60 ℃ and 20s
Extension: 72 ℃ and 20s
Cycle number: 35
Extension: 72 ℃ for 6min
2.3 Analysis of results
The genomic DNA of 8 experimental materials was amplified with the specific primers DnoF1/DnoR1, and sterile water was added as a template as a negative control. The amplified product is subjected to 1.5% agarose gel electrophoresis, the specific size band of the target can be observed by EB staining, the amplified fragment of the universal primer is about 329bp, onlyD. novemA specific size of the band of interest is shown (see lane 1 in the figure),D. longicolla , D. caulivora, D. aspalathi, Diaporthe sp., D. eres , D.phaseolorum, D. vaccinii and negative control without corresponding product (see lanes 2-9, lanes 1-9, respectivelyD. novem, D. longicolla, D. caulivora, D. aspalathi, Diaporthe sp., D. eres, D.phaseolorum, D. vacciniiAnd negative control), lane M is a 2000 bp DNA marker.
Sequence listing
<110> Tianjin customs animals and plants and food detection center
<120> a method for detecting Diadorthe novem using PCR primer
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> Diaporthe novem
<400> 1
ctcacactgg ctggattatg 20
<210> 2
<211> 19
<212> DNA
<213> Diaporthe novem
<400> 2
agccgcttat ctcctatgc 19
Claims (3)
1. PCR primer detectionDiaporthe novemIs characterized by thatD. novem The sequences of the PCR specific primer pairs are as follows:
DnoF1：CTCACACTGGCTGGATTATG
DnoR1：AGCCGCTTATCTCCTATGC
the amplified fragment is 329bp forD. novemGenomic DNA specific amplification detection.
2. A method for detecting by PCR primer as set forth in claim 1D. novemIs characterized in that it comprises the following steps:
(1) Extracting plant genome DNA;
(2) Establishment ofD. novemA PCR amplification system of genome DNA, which is amplified according to a reaction program;
(3) Agarose gel electrophoresis detection of amplified products.
3. The assay as claimed in claim 1D. novemSpecific PCR primer pair or detection according to claim 2D. novemMethod for detecting specificallyD. novemApplication to the above.
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|---|---|---|---|---|
| CN102094086A (en) * | 2010-12-01 | 2011-06-15 | 天津出入境检验检疫局动植物与食品检测中心 | Method for detecting Solanum rostratum Dunal by PCR (Polymerase Chain Reaction) primer |
| CN102373233A (en) * | 2010-08-23 | 2012-03-14 | 深圳出入境检验检疫局动植物检验检疫技术中心 | Positive standard molecule for detecting diaporthe phaseolorum (Cooke etEll) Sacc. var. caulivora Athow et Caldwell (DPC) and diaporthe phaseolorum (Cooke etEll) Sacc. var. meridionalis F.A.Fernandex (DPM) as well as preparation method and detection method thereof |
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2021
- 2021-08-06 CN CN202110899407.2A patent/CN113584208B/en active Active
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