CN114480722A - 一种叶斑菌病原菌lamp检测引物、试剂盒及lamp快速检测方法 - Google Patents
一种叶斑菌病原菌lamp检测引物、试剂盒及lamp快速检测方法 Download PDFInfo
- Publication number
- CN114480722A CN114480722A CN202210272056.7A CN202210272056A CN114480722A CN 114480722 A CN114480722 A CN 114480722A CN 202210272056 A CN202210272056 A CN 202210272056A CN 114480722 A CN114480722 A CN 114480722A
- Authority
- CN
- China
- Prior art keywords
- pathogenic bacteria
- lamp
- primer
- leaf spot
- reaction
- 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.)
- Pending
Links
- 238000001514 detection method Methods 0.000 title claims abstract description 70
- 244000052616 bacterial pathogen Species 0.000 title claims abstract description 67
- 238000007397 LAMP assay Methods 0.000 title description 2
- 241000519856 Phyllosticta Species 0.000 title description 2
- 238000006243 chemical reaction Methods 0.000 claims description 43
- 201000010099 disease Diseases 0.000 claims description 26
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 26
- 108020004414 DNA Proteins 0.000 claims description 20
- 241000196324 Embryophyta Species 0.000 claims description 16
- 239000000047 product Substances 0.000 claims description 13
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 13
- 239000007795 chemical reaction product Substances 0.000 claims description 12
- 238000000246 agarose gel electrophoresis Methods 0.000 claims description 11
- 239000001963 growth medium Substances 0.000 claims description 8
- 238000000034 method Methods 0.000 claims description 8
- 239000008223 sterile water Substances 0.000 claims description 8
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 7
- 230000003321 amplification Effects 0.000 claims description 7
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 7
- 210000001519 tissue Anatomy 0.000 claims description 7
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 claims description 6
- 238000011901 isothermal amplification Methods 0.000 claims description 5
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 5
- 238000004458 analytical method Methods 0.000 claims description 4
- 238000002955 isolation Methods 0.000 claims description 4
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 claims description 3
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 claims description 3
- 241000233866 Fungi Species 0.000 claims description 3
- 108010068204 Peptide Elongation Factors Proteins 0.000 claims description 3
- 101100213970 Schizosaccharomyces pombe (strain 972 / ATCC 24843) ypt3 gene Proteins 0.000 claims description 3
- 238000000137 annealing Methods 0.000 claims description 3
- NNBFNNNWANBMTI-UHFFFAOYSA-M brilliant green Chemical compound OS([O-])(=O)=O.C1=CC(N(CC)CC)=CC=C1C(C=1C=CC=CC=1)=C1C=CC(=[N+](CC)CC)C=C1 NNBFNNNWANBMTI-UHFFFAOYSA-M 0.000 claims description 3
- 238000004925 denaturation Methods 0.000 claims description 3
- 230000036425 denaturation Effects 0.000 claims description 3
- 230000035484 reaction time Effects 0.000 claims description 3
- 210000003705 ribosome Anatomy 0.000 claims description 3
- 238000004659 sterilization and disinfection Methods 0.000 claims description 3
- 229960005322 streptomycin Drugs 0.000 claims description 3
- 238000013519 translation Methods 0.000 claims description 3
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 3
- 239000012498 ultrapure water Substances 0.000 claims description 3
- 238000011144 upstream manufacturing Methods 0.000 claims description 3
- 108020000949 Fungal DNA Proteins 0.000 claims description 2
- 241000589636 Xanthomonas campestris Species 0.000 claims description 2
- 101150087698 alpha gene Proteins 0.000 claims description 2
- 238000012257 pre-denaturation Methods 0.000 claims description 2
- 238000010008 shearing Methods 0.000 claims description 2
- 241000275067 Phyllotreta Species 0.000 claims 1
- 244000000005 bacterial plant pathogen Species 0.000 abstract description 2
- 241000756137 Hemerocallis Species 0.000 description 25
- 102000053602 DNA Human genes 0.000 description 18
- 208000024891 symptom Diseases 0.000 description 9
- 238000012360 testing method Methods 0.000 description 9
- 238000011081 inoculation Methods 0.000 description 7
- 244000052769 pathogen Species 0.000 description 7
- 230000001717 pathogenic effect Effects 0.000 description 7
- 238000005516 engineering process Methods 0.000 description 6
- 230000003902 lesion Effects 0.000 description 6
- CGNLCCVKSWNSDG-UHFFFAOYSA-N SYBR Green I Chemical compound CN(C)CCCN(CCC)C1=CC(C=C2N(C3=CC=CC=C3S2)C)=C2C=CC=CC2=[N+]1C1=CC=CC=C1 CGNLCCVKSWNSDG-UHFFFAOYSA-N 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- 239000002609 medium Substances 0.000 description 5
- 230000000877 morphologic effect Effects 0.000 description 5
- 229920001817 Agar Polymers 0.000 description 4
- 239000008272 agar Substances 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 230000035945 sensitivity Effects 0.000 description 4
- 238000000926 separation method Methods 0.000 description 4
- 235000013311 vegetables Nutrition 0.000 description 4
- UHPMCKVQTMMPCG-UHFFFAOYSA-N 5,8-dihydroxy-2-methoxy-6-methyl-7-(2-oxopropyl)naphthalene-1,4-dione Chemical compound CC1=C(CC(C)=O)C(O)=C2C(=O)C(OC)=CC(=O)C2=C1O UHPMCKVQTMMPCG-UHFFFAOYSA-N 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 241000223218 Fusarium Species 0.000 description 3
- 241000813090 Rhizoctonia solani Species 0.000 description 3
- 206010039509 Scab Diseases 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 238000001962 electrophoresis Methods 0.000 description 3
- 238000001502 gel electrophoresis Methods 0.000 description 3
- 239000003550 marker Substances 0.000 description 3
- 239000013642 negative control Substances 0.000 description 3
- 230000007918 pathogenicity Effects 0.000 description 3
- IAKHMKGGTNLKSZ-INIZCTEOSA-N (S)-colchicine Chemical compound C1([C@@H](NC(C)=O)CC2)=CC(=O)C(OC)=CC=C1C1=C2C=C(OC)C(OC)=C1OC IAKHMKGGTNLKSZ-INIZCTEOSA-N 0.000 description 2
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 2
- 241001529387 Colletotrichum gloeosporioides Species 0.000 description 2
- 240000006497 Dianthus caryophyllus Species 0.000 description 2
- 235000009355 Dianthus caryophyllus Nutrition 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- 244000172419 Hemerocallis flava Species 0.000 description 2
- 235000017209 Hemerocallis flava Nutrition 0.000 description 2
- 244000061456 Solanum tuberosum Species 0.000 description 2
- 235000002595 Solanum tuberosum Nutrition 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 125000001475 halogen functional group Chemical group 0.000 description 2
- 238000011392 neighbor-joining method Methods 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 239000001100 (2S)-5,7-dihydroxy-2-(3-hydroxy-4-methoxyphenyl)chroman-4-one Substances 0.000 description 1
- 241000605447 Anemarrhena Species 0.000 description 1
- 208000019901 Anxiety disease Diseases 0.000 description 1
- 241000193738 Bacillus anthracis Species 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- JMGZEFIQIZZSBH-UHFFFAOYSA-N Bioquercetin Natural products CC1OC(OCC(O)C2OC(OC3=C(Oc4cc(O)cc(O)c4C3=O)c5ccc(O)c(O)c5)C(O)C2O)C(O)C(O)C1O JMGZEFIQIZZSBH-UHFFFAOYSA-N 0.000 description 1
- 241000207199 Citrus Species 0.000 description 1
- 241001480643 Colletotrichum sp. Species 0.000 description 1
- 108050009160 DNA polymerase 1 Proteins 0.000 description 1
- 241001245666 Didymella macrostoma Species 0.000 description 1
- 241001492222 Epicoccum Species 0.000 description 1
- 241001506775 Epicoccum nigrum Species 0.000 description 1
- 241001022589 Fusarium andiyazi Species 0.000 description 1
- 241001149959 Fusarium sp. Species 0.000 description 1
- 241000461774 Gloeosporium Species 0.000 description 1
- 241001053920 Gloeosporium sp. Species 0.000 description 1
- 241000827798 Hemerocallis citrina Species 0.000 description 1
- 240000009206 Hemerocallis fulva Species 0.000 description 1
- 235000002941 Hemerocallis fulva Nutrition 0.000 description 1
- QUQPHWDTPGMPEX-UHFFFAOYSA-N Hesperidine Natural products C1=C(O)C(OC)=CC=C1C1OC2=CC(OC3C(C(O)C(O)C(COC4C(C(O)C(O)C(C)O4)O)O3)O)=CC(O)=C2C(=O)C1 QUQPHWDTPGMPEX-UHFFFAOYSA-N 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 241000234280 Liliaceae Species 0.000 description 1
- 241001547796 Macrophoma Species 0.000 description 1
- 206010030113 Oedema Diseases 0.000 description 1
- 102000002508 Peptide Elongation Factors Human genes 0.000 description 1
- 241001503951 Phoma Species 0.000 description 1
- 241000437063 Phyllotreta striolata Species 0.000 description 1
- 208000004880 Polyuria Diseases 0.000 description 1
- 239000005708 Sodium hypochlorite Substances 0.000 description 1
- 235000011684 Sorghum saccharatum Nutrition 0.000 description 1
- 244000062793 Sorghum vulgare Species 0.000 description 1
- 241000082085 Verticillium <Phyllachorales> Species 0.000 description 1
- 244000085595 Zizania latifolia Species 0.000 description 1
- 235000004259 Zizania latifolia Nutrition 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 229930191283 anemarrhena Natural products 0.000 description 1
- 230000036506 anxiety Effects 0.000 description 1
- QUQPHWDTPGMPEX-UTWYECKDSA-N aurantiamarin Natural products COc1ccc(cc1O)[C@H]1CC(=O)c2c(O)cc(O[C@@H]3O[C@H](CO[C@@H]4O[C@@H](C)[C@H](O)[C@@H](O)[C@H]4O)[C@@H](O)[C@H](O)[C@H]3O)cc2O1 QUQPHWDTPGMPEX-UTWYECKDSA-N 0.000 description 1
- 210000000270 basal cell Anatomy 0.000 description 1
- 230000003796 beauty Effects 0.000 description 1
- 238000004820 blood count Methods 0.000 description 1
- 230000001914 calming effect Effects 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 235000020971 citrus fruits Nutrition 0.000 description 1
- APSNPMVGBGZYAJ-GLOOOPAXSA-N clematine Natural products COc1cc(ccc1O)[C@@H]2CC(=O)c3c(O)cc(O[C@@H]4O[C@H](CO[C@H]5O[C@@H](C)[C@H](O)[C@@H](O)[C@H]5O)[C@@H](O)[C@H](O)[C@H]4O)cc3O2 APSNPMVGBGZYAJ-GLOOOPAXSA-N 0.000 description 1
- 229960001338 colchicine Drugs 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 239000008121 dextrose Substances 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 230000003467 diminishing effect Effects 0.000 description 1
- 230000035619 diuresis Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- IVTMALDHFAHOGL-UHFFFAOYSA-N eriodictyol 7-O-rutinoside Natural products OC1C(O)C(O)C(C)OC1OCC1C(O)C(O)C(O)C(OC=2C=C3C(C(C(O)=C(O3)C=3C=C(O)C(O)=CC=3)=O)=C(O)C=2)O1 IVTMALDHFAHOGL-UHFFFAOYSA-N 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000004438 eyesight Effects 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 244000053095 fungal pathogen Species 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 229940025878 hesperidin Drugs 0.000 description 1
- VUYDGVRIQRPHFX-UHFFFAOYSA-N hesperidin Natural products COc1cc(ccc1O)C2CC(=O)c3c(O)cc(OC4OC(COC5OC(O)C(O)C(O)C5O)C(O)C(O)C4O)cc3O2 VUYDGVRIQRPHFX-UHFFFAOYSA-N 0.000 description 1
- QUQPHWDTPGMPEX-QJBIFVCTSA-N hesperidin Chemical compound C1=C(O)C(OC)=CC=C1[C@H]1OC2=CC(O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@@H](CO[C@H]4[C@@H]([C@H](O)[C@@H](O)[C@H](C)O4)O)O3)O)=CC(O)=C2C(=O)C1 QUQPHWDTPGMPEX-QJBIFVCTSA-N 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 229910017053 inorganic salt Inorganic materials 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000001338 necrotic effect Effects 0.000 description 1
- ARGKVCXINMKCAZ-UHFFFAOYSA-N neohesperidine Natural products C1=C(O)C(OC)=CC=C1C1OC2=CC(OC3C(C(O)C(O)C(CO)O3)OC3C(C(O)C(O)C(C)O3)O)=CC(O)=C2C(=O)C1 ARGKVCXINMKCAZ-UHFFFAOYSA-N 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 238000013081 phylogenetic analysis Methods 0.000 description 1
- 239000001965 potato dextrose agar Substances 0.000 description 1
- 235000012015 potatoes Nutrition 0.000 description 1
- 238000004382 potting Methods 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- FDRQPMVGJOQVTL-UHFFFAOYSA-N quercetin rutinoside Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC=2C(C3=C(O)C=C(O)C=C3OC=2C=2C=C(O)C(O)=CC=2)=O)O1 FDRQPMVGJOQVTL-UHFFFAOYSA-N 0.000 description 1
- JQXXHWHPUNPDRT-WLSIYKJHSA-N rifampicin Chemical compound O([C@](C1=O)(C)O/C=C/[C@@H]([C@H]([C@@H](OC(C)=O)[C@H](C)[C@H](O)[C@H](C)[C@@H](O)[C@@H](C)\C=C\C=C(C)/C(=O)NC=2C(O)=C3C([O-])=C4C)C)OC)C4=C1C3=C(O)C=2\C=N\N1CC[NH+](C)CC1 JQXXHWHPUNPDRT-WLSIYKJHSA-N 0.000 description 1
- IKGXIBQEEMLURG-BKUODXTLSA-N rutin Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](C)O[C@@H]1OC[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](OC=2C(C3=C(O)C=C(O)C=C3OC=2C=2C=C(O)C(O)=CC=2)=O)O1 IKGXIBQEEMLURG-BKUODXTLSA-N 0.000 description 1
- ALABRVAAKCSLSC-UHFFFAOYSA-N rutin Natural products CC1OC(OCC2OC(O)C(O)C(O)C2O)C(O)C(O)C1OC3=C(Oc4cc(O)cc(O)c4C3=O)c5ccc(O)c(O)c5 ALABRVAAKCSLSC-UHFFFAOYSA-N 0.000 description 1
- 235000005493 rutin Nutrition 0.000 description 1
- 229960004555 rutoside Drugs 0.000 description 1
- 238000007790 scraping Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 description 1
- 235000000346 sugar Nutrition 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 238000003239 susceptibility assay Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Analytical Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Immunology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Botany (AREA)
- Mycology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明提供一种叶斑病病原菌的LAMP检测引物,涉及植物病原菌检测的技术领域,所述引物包括正向外引物F3、反向外引物B3、正向内引物FIP和反向内引物BIP,所述引物系列如下:F3:5’‑ACCAGTGCGGTGGTATCG‑3’;B3:5’‑CAAGAGCGCCTGTCACTC‑3’;FIP:5’‑GGATCGATGGGAAGAAGGGCGCAAG CGAACCATCGAGAAGT‑3’;BIP:5’‑CATACGACGACTCGACAAGCGCCCACCACCCAAAAAAACGAC‑3’。
Description
技术领域
本发明涉及植物病原菌检测的技术领域,具体而言,涉及一种叶斑病病原菌的LAMP检测引物、试剂盒及LAMP快速检测方法。
背景技术
黄花菜(Hemerocallis citrina Baroni),又称为金针菜、安神菜、忘忧草,学名为萱草,属百合科多年生宿根草本植物。其食用部分为含苞末放的花蕾,营养丰富,含多种糖类、蛋白质、维生素、无机盐及多种人体必需的氨基酸,是我国传统特色蔬菜之一。该植物广泛种植于中国、朝鲜韩国和日本等地,具有观赏和蔬菜价值。此外,黄花菜的花蕾合成代谢物含有丰富的芦丁、橙皮苷、秋水仙碱等,对治疗焦虑、浮肿、抑郁等症状有效,具有抗菌消炎、清热利湿、明目安神、美容养颜等功效,可作为中药材和现代药用植物。随着黄花菜栽培的兴起和特色蔬菜产业的发展,种植面积逐渐扩大,其病害发生情况也日趋严重,对产业造成了巨大的经济损失。
黄花菜上常发生多种叶部及花薹病害,包括叶枯病、叶斑病、褐斑病、茎枯病、黄叶病、炭疽病、红腐病等,其病原多为炭疽菌(Colletotrichum sp.)、镰刀菌(Fusarium sp.)、轮斑病菌(Pestallozzia sp.)、大茎点霉(Macrophoma sp.)、茎点霉(Phoma sp.)和盘长孢菌(Gloeosporium sp.)等。
目前对病原菌的检测常见采用PCR技术,但是该技术检测的效率低,不利于对植物的防护。
发明内容
本发明的目的在于提供一种叶斑病病原菌的LAMP检测引物、试剂盒及LAMP快速检测方法,用以实现快速对叶斑病病原菌的检测,提高检测效率。
本发明通过以下技术方案实现:所述引物包括正向外引物F3、反向外引物B3、正向内引物FIP和反向内引物BIP,所述引物系列如下:
F3:5’-ACCAGTGCGGTGGTATCG-3’;
B3:5’-CAAGAGCGCCTGTCACTC-3’;
FIP: 5’-GGATCGATGGGAAGAAGGGCGCAAGCGAACCATCGAGAAGT-3’;
BIP: 5’-CATACGACGACTCGACAAGCGCCCACCACCCAAAAAAACGAC-3’。
提供一种叶斑病病原菌的LAMP检测试剂盒,包括上述引物。
为了更好的实现本发明,进一步的,所述试剂盒还包括dNTPs、8U/μL Bst DNA聚合酶、10×isothermal amplificationbuffer和MgSO4。
提供一种叶斑病病原菌的LAMP检测反应体系,反应体系包括:10 uM的FIP和BIP引物各4μL,10uM F3和B3引物0.5μL,10mM dNTPs 2.5μL,8U/μLBst DNA聚合酶1μL,50mMMgSO44μL,10×isothermal amplificationbuffer 2.5μL,用灭菌超纯水补平至25μL。
提供一种叶斑病病原菌的LAMP检测方法,包括以下步骤:
S1:获取叶斑菌病原菌的EF-1α基因保守区利用Primer Explore V5 在线设计引物;
S2:制备25μL的检测反应体系;
S3:将待测样品总DNA于S2所述的检测反应体系中进行LAMP扩增,反应结束后,取反应产物;
S4:将反应产物用琼脂糖凝胶电泳检测扩增结果,或/和用SYBR Green I染料观察LAMP反应液变化;
S5:LAMP反应产物在琼脂糖凝胶电泳出现连续呈弥散状条带为阳性,存在叶斑病病原菌;和/或反应液呈现翠绿色为阳性,存在叶斑病病原菌,淡橙色为阴性,不存在叶斑菌病原菌。
为了更好的实现本发明,进一步的,所述检测反应体系中的反应条件为:温度为60℃、反应时间为60min。
为了更好的实现本发明,进一步的,所述S1中获取叶斑病病原菌的操作步骤如下:
步骤一:剪取罹病叶片病健交界部分的组织进行消毒,再用无菌水漂洗,用无菌滤纸吸干水分后将其放置于含有链霉素的PDA平板上,26℃黑暗培养;
步骤二:将长出的菌落转接至PDA培养基,通过单孢分离进行纯化后用PDA斜面保存于4℃备用,得到分离纯化的菌株。
为了更好的实现本发明,进一步的,还包括对叶斑病病原菌的鉴定,包括以下步骤:
Sa:将分离纯化的菌株接种与PDA培养基中,26℃黑暗培养7天后刮取菌丝利用CTAB法提取真菌DNA;
Sb:通过引物ITS4/ITS5和EF1-728F/EF1-986R扩增核糖体转录间区和翻译延长因子基因部分序列;
Sc:构建PCR反应体系,该PCR反应体系为2×Es Taq Master Mix 25 μL、总DNA模板2μL、10μM上下游引物各2μL、ddH2O 19μL,扩展反应取反应产物;
Sd:反应产物经琼脂糖凝胶电泳检测后,进行序列测定,获序列在 NCBI数据库进行BLASTN比对分析,利用MEGA6软件中邻接法构建系统发育树,采用自举法进行1000次重复检验。
为了更好的实现本发明,进一步的,PCR反应条件94℃预变性5min; 94℃变性30s,56℃退火30s,72℃延伸1min,共35个循环;最后72℃延伸10min,4℃保存。
一种试剂盒在植物叶斑病病原菌鉴定中的应用,所述植物为黄花菜。
本发明的有益效果是:
本发明基于叶斑病病原菌的EF-1α序列设计了一组可以特异性检测叶斑病病原菌的LAMP检测引物,该叶斑病病原菌为F.armeniacum,通过该检测引物能够快速检测出病原菌,提高检测的工作效率。
本发明通过设置LAMP检测引物,并通过LAMP检测引物和LAMP 检测技术的结合快速检测叶斑病病原菌,LAMP检测技术的检测特异性强、检测时间短、灵敏度高、对仪器设备的要求低,结果可视化易判断。
本发明所提供的LAMP检测相比常规的PCR检测的灵敏度高10倍,能够满足染病植物病原体的检测。
附图说明
为了更清楚地说明本发明的技术方案,下面将对本发明中所需要使用的附图作简单地介绍,应当理解,以下附图仅示出了本发明的某些实施例,因此不应被看作是对范围的限定,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他相关的附图。
图1为本发明提供的LAMP反应特异性检测的实验图;
图2为本发明提供的LAMP反应灵敏度检测图;
图3为本发明提供的实际样品叶斑病病原菌检测图;
图4为本发明提供的叶斑病致病菌测试图一;
图5为本发明提供的叶斑病致病菌测试图二;
图6为本发明提供的叶斑病病原菌的形态图;
图7为本发明提供的系统发育树。
具体实施方式
下面将结合本发明中的附图,对本发明中的技术方案进行描述。
实施例:
本发明提供一种叶斑病病原菌的LAMP检测引物:
基于叶斑病病原菌EF-1α序列基因保守区利用Primer Explore V5在线设计引物,引物包括2条外引物F3与B3以及2条内引物FIP与BIP。所用引物由生工生物工程(上海)股份有限公司完成,引物序列如下:
F3:5’-ACCAGTGCGGTGGTATCG-3’;
B3:5’-CAAGAGCGCCTGTCACTC-3’;
FIP:
5’-GGATCGATGGGAAGAAGGGCGCAAGCGAACCATCGAGAAGT-3’;
BIP: 5’-CATACGACGACTCGACAAGCGCCCACCACCCAAAAAAACGAC-3’。
构建LAMP检测反应体系,LAMP检测反应体系包括10uM的FIP 和BIP引物各4μL,10uM F3和B3引物各0.5μL,10mM dNTPs 2.5μL, 8U/μL Bst DNA聚合酶1μL,50mM MgSO44μL,10×isothermal amplificationbuffer 2.5μL,模板DNA 1μL,用灭菌超纯水补平至25μL。在LAMP检测反应过程中,反应条件为:温度60℃,反应时间60min。
反应结束后,取3μL的反应产物用1%的琼脂糖凝胶电泳检测扩增结果,另外还加入1μL的SYBR Green I染料观察LAMP反应液是否发生颜色变化。
反应结果:
电泳观察中LAMP反应产物在1%琼脂糖凝胶电泳出现连续呈弥散状条带为阳性,存在叶斑病病原菌,观察反应液呈现翠绿色为阳性,存在叶斑病病原菌,淡橙色为阴性。
实验例-LAMP检测引物特异性检测;
LAMP检测引物特异性检测:将黄花菜叶斑病病原菌、茭白鞘腐病原菌(Fusariumandiyazi)、柑橘炭疽病菌(Colletotrichum gloeosporioides)、立枯丝核菌(Rhizoctoniasolani)、高粱附球菌(Epicoccum nigrum)按实施例 LAMP检测方法进行检测,通过加入SYBR Green I染料染色和1%琼脂糖凝胶电泳观察结果,来判断LAMP引物特异性,检测结果如图1所示,图中所显示的a为:SYBR Green I检测LAMP产物,b为凝胶电泳检测LAMP产物,M为DL2000 marker;另外数字1~3为黄花菜叶斑病原菌; 4为茭白鞘腐病菌;5为胶孢炭疽菌;6为立枯丝核菌;7为高粱附球菌; 8为无菌水阴性对照。
测试结果为:黄花菜叶斑病病原菌DNA为模板的LAMP产物电泳条带呈弥散状,颜色变为绿色,说明发生了阳性反应,存在叶斑菌病原菌,而以其他病原真菌DNA为模板的LAMP检测产物电泳无条带,颜色仍为橙色,表明其为阴性反应,从而表明该LAMP检测体系可以特异性地检测出黄花菜叶斑病病原菌。
实验例-LAMP及常规PCR检测的灵敏度检测
将1000ng/μL的标准的黄花菜叶斑病病原菌的DNA按照10倍浓度梯度稀释至1fg/μL,分别作为反应模板,用常规PCR检测体系及LAMP 体系进行检测,根据扩增产物的颜色变化和琼脂糖凝胶电泳观察结果显示,如图2所示,图中a为SYBR Green I检测LAMP产物,b为凝胶电泳检测LAMP产物,c为凝胶电泳检测PCR产物,M为DL 2000marker;其中1~7为1000ng/μL、100ng/μL、10ng/μL、1ng/μL、100pg/μL、10pg/μL、 1pg/μL浓度梯度的黄花菜叶斑病菌DNA。
从LAMP反应体系的最低检测限度为100pg/μL,而普通PCR反应的最低检测限度为1ng/μL,说明该LAMP检测方法的灵敏度是常规PCR 检测方法的10倍,如图中所示。
实验例-实际样品中病原菌的检测
操作方法如下:
将人工接种黄花菜叶斑病病原菌的黄花菜叶片在接种4d后提取总DNA,将其作为模板进行LAMP扩增,结果显示,未接种叶斑病病原菌的黄花菜叶片没有检测到黄花菜叶斑病菌,而接种后的植株上都检测到了病原菌,如图3的a所述,图中3的a为人工接种黄花菜叶片LAMP检测;M为DL 2000marker;另外1为阳性黄花菜叶斑病菌株;2和3为接种了黄花菜叶斑病病原菌的叶片;4和5为未接种病原菌的健康植株;6 为无菌水阴性对照。
另外对2株采集于浙江丽水市缙云县显示黄花菜叶斑病症状的叶片样品进行了组织分离,通过菌株形态学和分子生物学等的鉴定,确定2 株样品都分离得到了黄花菜叶斑病菌,而另外取2株健康植株叶片未分离到病原菌。同时用相同样品进行LAMP检测,结果显示这2份病样均产生了阳性反应,与病原菌分离结果一致,而黄花菜健康组织中没有发生阳性反应。这说明可以用该LAMP技术进行黄花菜叶斑病田间样品的快速诊断,如图3的b所示,图中b为田间采样发病植株叶片LAMP检测; 1~3为田间发病植株叶片;4和5为田间健康植株叶片;6为无菌水阴性对照。
本发明还提供获取叶斑病病原菌的操作以及对叶斑病病原菌鉴定,分别是形态学鉴定和分子生物学鉴定,具体操作如下:
采集明显有叶斑病症状的叶片,摘取典型症状的叶片进行观察,记录发病症状。采用组织分离法分离病原菌,剪取罹病叶片病健交界部分大小为5×5mm的组织置于75%酒精中消毒30s,2%次氯酸钠消毒1min,再用无菌水漂洗3次,用无菌滤纸吸干水分后将其放置于含有链霉素(50 μg/ml)的PDA平板上,26℃黑暗培养。将长出的菌落转接至新的PDA 培养基,通过单孢分离进行纯化后用PDA斜面保存于4℃备用。
培养基:马铃薯葡萄糖琼脂(potato dextrose agar,PDA)培养基:葡萄糖20g、土豆200g、琼脂20g、蒸馏水1000mL;不加琼脂为马铃薯葡萄糖(potato dextrose,PD)液体培养基;康乃馨琼脂(carnation leaf-piece agar,CLA)培养基:康乃馨叶片若干、琼脂粉20g、蒸馏水1 L。
对黄花菜叶斑病病原菌的致病性测定:
采用孢子液活体接种进行柯赫氏法则验证。选取代表性供试菌株在 PD培养基中26℃,180r/min震荡培养5d,用无菌纱布过滤后,收集孢子液。通过血球计数板计算,用无菌水配制浓度为1×106个/mL的分生孢子悬浮液,选取健康的40cm高的盆栽黄花菜植株进行致病力测定,将植株叶片用70%乙醇擦拭表面消毒后,用移液枪吸取孢子液滴于叶片上,每片叶片滴3~4个点,每个点10μL。用无菌水接种作为对照。将所有接种体置于25℃恒温、相对湿度80%~90%条件下培养,逐日观察发病情况。叶片发病后,对发病叶片再次进行病原菌分离鉴定。接种实验重复2次,每个处理3个盆栽重复。
致病性的测试结果:
接种4d后观察到叶片发病,黄花菜叶斑病主要为害叶片,在叶面或叶缘出现。最初为水浸状淡黄色斑点,后扩大成梭形病斑,边缘褐色,中央灰色,具有黄色晕圈,如图4所示。病害发生后期,病斑扩大,引起穿孔、叶片破裂。病斑也可扩展至茎部,在茎秆上也出现褐色、梭形病斑,后扩大环绕茎部,引起茎秆枯萎。通常,在田间高湿度条件下,侵染的叶片和茎秆病斑处可见白色或红色霉层。对采集的罹病叶片和茎秆进行组织分离,从20份病害样品中共分离纯化获得20株菌株。
如图5所示,图5中a为盆栽活体接种6d后发病症状;b为接种叶片典型病斑;CK为对照,a中具体表现为水浸状到黄褐色的坏死梭形斑,并具有明显黄色晕圈,后期病斑扩展引起穿孔和叶片枯萎。该接种症状与田间自然发病症状相似,而对照接种叶片不表现出症状。从接种病斑处可镜检到病原菌分生孢子,且可再次从病斑处分离出病原菌,经形态学和分子鉴定为与接种所用一致的病原菌为F.armeniacum,由此说明F.armeniacum 为黄花菜叶斑病的病原菌。
另外还对病原菌进行形态学鉴定:
将分离纯化的菌株接种于PDA平板上,将菌株接种于CLA平板上 26℃黑暗培养,待其产生孢子后在光学显微镜下观察大小分生孢子和厚垣孢子形态特征,肾形或卵圆形,壁较厚,具有0~1个隔膜,大小为7.7~14.1 ×2.7~5.0μm。在CLA培养基上培养7d后或者在发病植株病斑处,易产生大型分生孢子,镰刀状,薄壁,有一个长而尖的顶端细胞和一个有凹口的基底细胞,3~5个隔膜,大小为20.5~38.5×2.8~5.1μm。厚垣孢子近圆形,壁光滑,常串生如图6-c~f,依据该病原菌的形态学特征可将该病原菌初步鉴定为Fusariumarmeniacum。如图6所示,其中a为PDA平板上菌落正面;b为PDA平板上菌落背面;c为大型分生孢子梗;d为大型分生孢子;e为小型分生孢子:f为厚垣孢子。
除此持外,还有对病原菌分子生物学鉴定,具体操作如下:
将分离纯化的菌株接种于PDA培养基中,26℃黑暗培养7d后刮取菌丝利用CTAB法提取真菌DNA(Lee and Taylor,1990)。采用通用引物 ITS4/ITS5(White et al.,1990)和EF1-728F/EF1-986R(Carbone et al.,1999)扩增核糖体转录间区(ITS)和翻译延长因子(EF-1α)基因部分序列。
PCR反应体系为50μL:2×Es Taq MasterMix 25μL、总DNA模板2 μL、10μM上下游引物各2μL、ddH2O 19μL。
PCR反应程序为:94℃预变性5min;94℃变性30s,56℃退火30s, 72℃延伸1min,共35个循环;最后72℃延伸10min,4℃保存。
扩增产物经1%琼脂糖凝胶电泳检测后,委托上海生物工程有限公司进行序列测定。所获序列在NCBI数据库进行BLASTN比对分析。利用 MEGA 6软件中邻接法(neighbor-joining,NJ)构建系统发育树,采用自举法(bootstrap)进行1000次重复检验(Tamura etal.,2013)。
具体是以代表菌株HHCJ-4的基因组DNA为模板,扩增ITS和EF-1α部分基因序列,获得大小约为550bp和300bp的片段。将PCR产物纯化后测序,并将序列提交到至GenBank(登录号分别为:MN647064和 MN650204);BLASTN同源性比对显示,所测序列与NCBI中已知的F.armeniacum菌株序列具有99.8%到100%的核苷酸相似性。如 F.armeniacum voucherRIFA 180(登录号为KF624790)和F.armeniacum MRC 2190(登录号为MH582288)等。
此外,HHCJ-4的ITS和EF-1α序列同样与Fusarium-ID数据库中F. armeniacum菌株具有98.9%到99.8%的核苷酸相似性(Geiser et al.,2004)。
利用HHCJ-4以及镰刀菌属不同种菌株的EF-1α序列进行多序列比对及系统发育树构建。进化分析结果显示,HHCJ-4与F.armeniacum菌株聚集于一个分支,如图7所示。因此,基于病原菌形态特征、EF-1α序列同源性比对和系统发育分析将该黄花菜叶斑病病原菌鉴定为F.armeniacum。
以上所述,仅为本发明的具体实施方式,但本发明的保护范围并不局限于此,任何熟悉本技术领域的技术人员在本发明揭露的技术范围内,可轻易想到变化或替换,都应涵盖在本发明的保护范围之内。因此,本发明的保护范围应所述以权利要求的保护范围为准。
序列表
<110> 湖南农业大学
<120> 一种叶斑病病原菌LAMP检测引物、试剂盒及LAMP快速检测方法
<141> 2022-03-18
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
accagtgcgg tggtatcg 18
<210> 2
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
caagagcgcc tgtcactc 18
<210> 3
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
ggatcgatgg gaagaagggc gcaagcgaac catcgagaag t 41
<210> 4
<211> 42
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
catacgacga ctcgacaagc gcccaccacc caaaaaaacg ac 42
Claims (10)
1.一种叶斑病病原菌的LAMP检测引物,其特征在于:
所述引物包括正向外引物F3、反向外引物B3、正向内引物FIP和反向内引物BIP,所述引物系列如下:
F3:5’-ACCAGTGCGGTGGTATCG-3’;
B3:5’-CAAGAGCGCCTGTCACTC-3’;
FIP:5’-GGATCGATGGGAAGAAGGGCGCAAGCGAACCATCGAGAAGT-3’;
BIP:5’-CATACGACGACTCGACAAGCGCCCACCACCCAAAAAAACGAC-3’。
2.一种叶斑病病原菌的LAMP检测试剂盒,其特征在于,包括权利要求1所述的引物。
3.根据权利要求2所述的叶斑病病原菌的LAMP检测试剂盒,其特征在于:试剂盒还包括dNTPs、Bst DNA聚合酶、10×isothermal amplificationbuffer和MgSO4。
4.一种包含权利要求1所述引物的叶斑病病原菌的LAMP检测反应体系,其特征在于:
反应体系包括:10uM的FIP和BIP引物各4μL,10uM F3和B3引物各0.5μL,10mM dNTPs2.5μL,8U/μL Bst DNA聚合酶1μL,50mM MgSO44μL,10×isothermal amplificationbuffer2.5μL,用灭菌超纯水补平至25μL。
5.一种基于权利要求4所述反应体系的叶斑病病原菌的LAMP检测方法,其特征在于,包括以下步骤:
S1:获取叶斑病病原菌的EF-1α基因保守区利用Primer Explore V5在线设计引物;
S2:制备25μL的权利要求4中的检测反应体系;
S3:将待测样品总DNA于S2所述的检测反应体系中进行LAMP扩增,反应结束后,取反应产物;
S4:将反应产物用琼脂糖凝胶电泳检测;或/和用SYBR GreenI染料观察LAMP反应液变化;
S5:LAMP反应产物在琼脂糖凝胶电泳出现连续呈弥散状条带为阳性,存在叶斑菌病原菌;和/或反应液呈现翠绿色为阳性,存在叶斑病病原菌,淡橙色为阴性,不存在叶斑病病原菌。
6.根据权利要求5所述的LAMP检测方法,其特征在于,所述反应体系中的反应条件为:温度为60℃、反应时间为60min。
7.根据权利要求5所述的LAMP检测方法,其特征在于,
所述S1中获取叶斑病病原菌的操作步骤如下:
步骤一:剪取罹病叶片病健交界部分的组织进行消毒,再用无菌水漂洗,用无菌滤纸吸干水分后将其放置于含有链霉素的PDA平板上,26℃黑暗培养;
步骤二:将长出的菌落转接至PDA培养基,通过单孢分离进行纯化后用PDA斜面保存于4℃备用,得到分离纯化的菌株。
8.根据权利要求7所述的LAMP检测方法,其特征在于,还包括对叶斑病病原菌的鉴定,包括以下步骤:
Sa:将分离纯化的菌株接种于PDA培养基中培养出菌丝,然后提取真菌DNA;
Sb:通过引物ITS4/ITS5和EF1-728F/EF1-986R扩增核糖体转录间区和翻译延长因子基因部分序列;
Sc:构建PCR反应体系,该PCR反应体系为2×Es Taq Master Mix25μL、总DNA模板2μL、10μM上下游引物各2μL、ddH2O 19μL,扩展反应取反应产物;
Sd:反应产物经琼脂糖凝胶电泳检测后,进行序列测定,获序列在NCBI数据库进行BLASTN比对分析,利用MEGA6软件中邻接法构建系统发育树。
9.根据权利要求8所述的LAMP检测方法,其特征在于,PCR反应条件94℃预变性5min;94℃变性30s,56℃退火30s,72℃延伸1min,共35个循环;最后72℃延伸10min,4℃保存。
10.根据权利要求2所述的试剂盒在植物叶斑菌病原菌鉴定中的应用,所述植物为黄花菜。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210272056.7A CN114480722A (zh) | 2022-03-18 | 2022-03-18 | 一种叶斑菌病原菌lamp检测引物、试剂盒及lamp快速检测方法 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210272056.7A CN114480722A (zh) | 2022-03-18 | 2022-03-18 | 一种叶斑菌病原菌lamp检测引物、试剂盒及lamp快速检测方法 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN114480722A true CN114480722A (zh) | 2022-05-13 |
Family
ID=81488301
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202210272056.7A Pending CN114480722A (zh) | 2022-03-18 | 2022-03-18 | 一种叶斑菌病原菌lamp检测引物、试剂盒及lamp快速检测方法 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN114480722A (zh) |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR20170046925A (ko) * | 2015-10-22 | 2017-05-04 | 대한민국(농촌진흥청장) | 후자리움 아르메니아쿰 검출용 프라이머쌍 및 이를 이용한 검출방법 |
CN108676910A (zh) * | 2018-07-09 | 2018-10-19 | 山西农业大学 | 一种层出镰刀菌的lamp检测引物及其应用 |
-
2022
- 2022-03-18 CN CN202210272056.7A patent/CN114480722A/zh active Pending
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR20170046925A (ko) * | 2015-10-22 | 2017-05-04 | 대한민국(농촌진흥청장) | 후자리움 아르메니아쿰 검출용 프라이머쌍 및 이를 이용한 검출방법 |
CN108676910A (zh) * | 2018-07-09 | 2018-10-19 | 山西农业大学 | 一种层出镰刀菌的lamp检测引物及其应用 |
Non-Patent Citations (5)
Title |
---|
L. W. BURGESS 等: "Characterization and Distribution of Fusarium Acuminatum Subsp. Armeniacum Subsp. Nov.", 《MYCOLOGIA》, vol. 85, no. 1, pages 119 - 124 * |
OKELLO, P.N. 等: "MH822071.1 Fusarium armeniacum isolate 14-OP-FUS-018 translation elongation factor (TEF1-alpha) gene, partial cds", 《GENBANK》, pages 1 - 2 * |
TOMOMI SANO 等: "First report of Fusarium armeniacum isolated from rice in Aichi Prefecture", 《JSM MYCOTOXINS》, vol. 72, no. 1, pages 1 - 3 * |
ZHONG, J. 等: "MN650204.1 Fusarium armeniacum isolate HHCJ-4 translation elongation factor 1- alpha (TEF1-alpha) gene, partial cds", 《GENBANK》, pages 1 - 2 * |
朱俊子 等: "黄花菜叶斑病菌鉴定、生物学特性分析、防治药剂筛选及LAMP检测体系的建立", 《国家自然科学基金大数据成果检索平台》, pages 1 * |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Strayer-Scherer et al. | Recombinase polymerase amplification assay for field detection of tomato bacterial spot pathogens | |
Engelbrecht et al. | Development of a nested quantitative real-time PCR for detecting Phytophthora cinnamomi in Persea americana rootstocks | |
Rayatpanah et al. | Pathogenic and genetic diversity among Iranian isolates of Macrophomina phaseolina | |
Basım et al. | Identification and characterization of Didymella bryoniae causing gummy stem blight disease of watermelon (Citrullus lanatus) in Turkey | |
CN110484478A (zh) | 一种枯草芽孢杆菌jz2-1-12及其应用 | |
Latifah et al. | Identification of Phytophthora spp. from perennial crops in Malaysia, its pathogenicity and cross-pathogenicity | |
CN112029665B (zh) | 橡胶树炭疽菌抗药性菌株HcgHNQZ1736及其在抗药性研究中的应用 | |
CN106635839B (zh) | 一种从土壤中分离长喙壳菌的方法 | |
CN110331223B (zh) | 一种用于鉴别不同茭白类型的分子标记、引物对、试剂盒及方法 | |
Ellis et al. | Association of putative fungal effectors in Fusarium oxysporum with wilt symptoms in soybean | |
CN114480722A (zh) | 一种叶斑菌病原菌lamp检测引物、试剂盒及lamp快速检测方法 | |
Kadir et al. | Morphological and molecular identification of Fusarium spp. and Colletotrichum spp. isolated from infected vanilla orchid | |
CN115960723A (zh) | 一种烟管菌及其应用 | |
Lima et al. | Mucor irregularis, a first record for South America | |
JP4261366B2 (ja) | 食物を毒する細菌を検出するためのプライマー及びその使用 | |
Yasouri et al. | The Effect of Environmental Stresses on Gene Expression in Pathogenic spp. through Real-Time PCR | |
Tadja | Morphological and genotypic characterization of fungi associated with the Ascochyta blight complex in western regions of Algeria | |
Iyanyi et al. | Biochemical and molecular characterization of bacteria associated with Cnidoscolus aconitifolius (Mill.) IM Johnston | |
CN108531646A (zh) | 生姜茎基腐病群结腐霉的lamp检测引物及检测方法 | |
Ghuffar et al. | Characterization of Pectobacterium Carotovoratum Subsp. Carotovorum causing Bacterial Soft Rot Disease of Turnip in Pakistan | |
Anitha et al. | Characterization of Ralstonia solanacearum (Smith) race 1, causing bacterial wilt of brinjal. | |
Ab Kadir et al. | Morphological and molecular identification of Fusarium spp. and Colletotrichum spp. isolated from infected vanilla orchid. | |
CN102559670B (zh) | 辅助鉴定尖镰孢菌菜豆专化型的引物对及其应用 | |
Nandan et al. | Molecular characterization and phylogenetic analysis of Rhizoctonia solani Kuhn. infecting tomato. | |
Abbas et al. | Detection of Pathogenic Fungi Causing Root and Crown Rot Diseases in Strawberry Fragaria ananassa Duch |
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 |