CN114959116B - Primer group, kit and method for detecting pepper light mottle virus and anthrax - Google Patents
Primer group, kit and method for detecting pepper light mottle virus and anthrax Download PDFInfo
- Publication number
- CN114959116B CN114959116B CN202210596142.3A CN202210596142A CN114959116B CN 114959116 B CN114959116 B CN 114959116B CN 202210596142 A CN202210596142 A CN 202210596142A CN 114959116 B CN114959116 B CN 114959116B
- Authority
- CN
- China
- Prior art keywords
- anthrax
- primer
- detection
- pepper
- raa
- 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
- 241000193738 Bacillus anthracis Species 0.000 title claims abstract description 156
- 235000002566 Capsicum Nutrition 0.000 title claims abstract description 51
- 241000700605 Viruses Species 0.000 title claims abstract description 43
- 239000006002 Pepper Substances 0.000 title claims abstract description 41
- 235000016761 Piper aduncum Nutrition 0.000 title claims abstract description 41
- 235000017804 Piper guineense Nutrition 0.000 title claims abstract description 41
- 235000008184 Piper nigrum Nutrition 0.000 title claims abstract description 41
- 238000000034 method Methods 0.000 title claims abstract description 32
- 244000203593 Piper nigrum Species 0.000 title abstract 2
- 238000001514 detection method Methods 0.000 claims abstract description 179
- 241000723824 Pepper mild mottle virus Species 0.000 claims abstract description 150
- 230000003321 amplification Effects 0.000 claims abstract description 137
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 137
- 239000000523 sample Substances 0.000 claims abstract description 99
- 241000196324 Embryophyta Species 0.000 claims abstract description 51
- 238000012360 testing method Methods 0.000 claims description 76
- 241000722363 Piper Species 0.000 claims description 46
- 239000006166 lysate Substances 0.000 claims description 45
- 239000012472 biological sample Substances 0.000 claims description 32
- 239000012634 fragment Substances 0.000 claims description 23
- 238000003908 quality control method Methods 0.000 claims description 17
- 238000004587 chromatography analysis Methods 0.000 claims description 14
- 201000010099 disease Diseases 0.000 claims description 13
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 13
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 10
- 238000012986 modification Methods 0.000 claims description 7
- 230000004048 modification Effects 0.000 claims description 7
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 claims description 5
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims description 4
- 238000005516 engineering process Methods 0.000 claims description 4
- 230000026731 phosphorylation Effects 0.000 claims description 4
- 238000006366 phosphorylation reaction Methods 0.000 claims description 4
- 238000005336 cracking Methods 0.000 claims description 3
- LTMHDMANZUZIPE-AMTYYWEZSA-N Digoxin Natural products O([C@H]1[C@H](C)O[C@H](O[C@@H]2C[C@@H]3[C@@](C)([C@@H]4[C@H]([C@]5(O)[C@](C)([C@H](O)C4)[C@H](C4=CC(=O)OC4)CC5)CC3)CC2)C[C@@H]1O)[C@H]1O[C@H](C)[C@@H](O[C@H]2O[C@@H](C)[C@H](O)[C@@H](O)C2)[C@@H](O)C1 LTMHDMANZUZIPE-AMTYYWEZSA-N 0.000 claims description 2
- 229960002685 biotin Drugs 0.000 claims description 2
- 235000020958 biotin Nutrition 0.000 claims description 2
- 239000011616 biotin Substances 0.000 claims description 2
- LTMHDMANZUZIPE-PUGKRICDSA-N digoxin Chemical compound C1[C@H](O)[C@H](O)[C@@H](C)O[C@H]1O[C@@H]1[C@@H](C)O[C@@H](O[C@@H]2[C@H](O[C@@H](O[C@@H]3C[C@@H]4[C@]([C@@H]5[C@H]([C@]6(CC[C@@H]([C@@]6(C)[C@H](O)C5)C=5COC(=O)C=5)O)CC4)(C)CC3)C[C@@H]2O)C)C[C@@H]1O LTMHDMANZUZIPE-PUGKRICDSA-N 0.000 claims description 2
- 229960005156 digoxin Drugs 0.000 claims description 2
- LTMHDMANZUZIPE-UHFFFAOYSA-N digoxine Natural products C1C(O)C(O)C(C)OC1OC1C(C)OC(OC2C(OC(OC3CC4C(C5C(C6(CCC(C6(C)C(O)C5)C=5COC(=O)C=5)O)CC4)(C)CC3)CC2O)C)CC1O LTMHDMANZUZIPE-UHFFFAOYSA-N 0.000 claims description 2
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 claims description 2
- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 claims description 2
- 238000003745 diagnosis Methods 0.000 claims 4
- 108020004707 nucleic acids Proteins 0.000 abstract description 31
- 102000039446 nucleic acids Human genes 0.000 abstract description 31
- 150000007523 nucleic acids Chemical class 0.000 abstract description 31
- 241000894006 Bacteria Species 0.000 abstract description 5
- 102000018120 Recombinases Human genes 0.000 abstract description 4
- 108010091086 Recombinases Proteins 0.000 abstract description 4
- 230000001404 mediated effect Effects 0.000 abstract description 3
- 239000000047 product Substances 0.000 description 64
- 238000006243 chemical reaction Methods 0.000 description 54
- 239000013612 plasmid Substances 0.000 description 53
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 34
- 230000035945 sensitivity Effects 0.000 description 31
- 239000013642 negative control Substances 0.000 description 28
- 239000000243 solution Substances 0.000 description 21
- 239000000203 mixture Substances 0.000 description 19
- 238000010790 dilution Methods 0.000 description 16
- 239000012895 dilution Substances 0.000 description 16
- 240000008574 Capsicum frutescens Species 0.000 description 14
- 239000006228 supernatant Substances 0.000 description 14
- 230000009977 dual effect Effects 0.000 description 12
- 108010033040 Histones Proteins 0.000 description 10
- 238000011529 RT qPCR Methods 0.000 description 10
- 239000001390 capsicum minimum Substances 0.000 description 10
- 235000013399 edible fruits Nutrition 0.000 description 10
- 238000002156 mixing Methods 0.000 description 10
- 241000222199 Colletotrichum Species 0.000 description 9
- 238000004458 analytical method Methods 0.000 description 9
- 239000003153 chemical reaction reagent Substances 0.000 description 9
- 238000001962 electrophoresis Methods 0.000 description 9
- 238000011156 evaluation Methods 0.000 description 9
- 239000003795 chemical substances by application Substances 0.000 description 8
- 238000000605 extraction Methods 0.000 description 8
- 208000015181 infectious disease Diseases 0.000 description 7
- 238000002372 labelling Methods 0.000 description 7
- 239000000463 material Substances 0.000 description 7
- 108090000623 proteins and genes Proteins 0.000 description 7
- 230000003247 decreasing effect Effects 0.000 description 6
- 244000052769 pathogen Species 0.000 description 6
- 238000003757 reverse transcription PCR Methods 0.000 description 6
- 101150083464 CP gene Proteins 0.000 description 5
- 238000012197 amplification kit Methods 0.000 description 5
- 239000000872 buffer Substances 0.000 description 5
- 238000010586 diagram Methods 0.000 description 5
- 238000007654 immersion Methods 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- 238000002360 preparation method Methods 0.000 description 5
- 241000894007 species Species 0.000 description 5
- 208000024891 symptom Diseases 0.000 description 5
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 4
- 241000222239 Colletotrichum truncatum Species 0.000 description 4
- 241000724252 Cucumber mosaic virus Species 0.000 description 4
- 241000723877 Tobacco mild green mosaic virus Species 0.000 description 4
- 239000000443 aerosol Substances 0.000 description 4
- 238000000246 agarose gel electrophoresis Methods 0.000 description 4
- 238000005119 centrifugation Methods 0.000 description 4
- 238000013461 design Methods 0.000 description 4
- 238000011161 development Methods 0.000 description 4
- 239000008176 lyophilized powder Substances 0.000 description 4
- 238000013207 serial dilution Methods 0.000 description 4
- 229910021642 ultra pure water Inorganic materials 0.000 description 4
- 239000012498 ultrapure water Substances 0.000 description 4
- 238000002965 ELISA Methods 0.000 description 3
- 241000755266 Kathetostoma giganteum Species 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 238000010170 biological method Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- 238000005457 optimization Methods 0.000 description 3
- 230000001717 pathogenic effect Effects 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 238000005096 rolling process Methods 0.000 description 3
- 238000005070 sampling Methods 0.000 description 3
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 235000000832 Ayote Nutrition 0.000 description 2
- 241000372028 Broad bean wilt virus Species 0.000 description 2
- 241001291881 Broad bean wilt virus 2 Species 0.000 description 2
- 101710132601 Capsid protein Proteins 0.000 description 2
- 241001005127 Chilli ringspot virus Species 0.000 description 2
- 101710094648 Coat protein Proteins 0.000 description 2
- 241001345409 Colletotrichum aenigma Species 0.000 description 2
- 241001639769 Colletotrichum fructicola Species 0.000 description 2
- 241001529387 Colletotrichum gloeosporioides Species 0.000 description 2
- 241001001916 Colletotrichum scovillei Species 0.000 description 2
- 241001639768 Colletotrichum siamense Species 0.000 description 2
- 240000004244 Cucurbita moschata Species 0.000 description 2
- 235000009854 Cucurbita moschata Nutrition 0.000 description 2
- 235000009804 Cucurbita pepo subsp pepo Nutrition 0.000 description 2
- 230000004544 DNA amplification Effects 0.000 description 2
- 102100021181 Golgi phosphoprotein 3 Human genes 0.000 description 2
- 102000006947 Histones Human genes 0.000 description 2
- 238000007397 LAMP assay Methods 0.000 description 2
- 101710125418 Major capsid protein Proteins 0.000 description 2
- 244000061176 Nicotiana tabacum Species 0.000 description 2
- 235000002637 Nicotiana tabacum Nutrition 0.000 description 2
- 101710141454 Nucleoprotein Proteins 0.000 description 2
- 241001429284 Pepper ringspot virus Species 0.000 description 2
- 241000068834 Pepper yellows virus Species 0.000 description 2
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 2
- 101710083689 Probable capsid protein Proteins 0.000 description 2
- 240000006677 Vicia faba Species 0.000 description 2
- 235000010749 Vicia faba Nutrition 0.000 description 2
- 235000002098 Vicia faba var. major Nutrition 0.000 description 2
- 241000961586 Virgaviridae Species 0.000 description 2
- 239000011543 agarose gel Substances 0.000 description 2
- 244000052616 bacterial pathogen Species 0.000 description 2
- 238000004166 bioassay Methods 0.000 description 2
- UDSAIICHUKSCKT-UHFFFAOYSA-N bromophenol blue Chemical compound C1=C(Br)C(O)=C(Br)C=C1C1(C=2C=C(Br)C(O)=C(Br)C=2)C2=CC=CC=C2S(=O)(=O)O1 UDSAIICHUKSCKT-UHFFFAOYSA-N 0.000 description 2
- 238000011109 contamination Methods 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 230000014509 gene expression Effects 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- 238000012125 lateral flow test Methods 0.000 description 2
- 239000012160 loading buffer Substances 0.000 description 2
- 238000011068 loading method Methods 0.000 description 2
- 230000002934 lysing effect Effects 0.000 description 2
- 230000036244 malformation Effects 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000011880 melting curve analysis Methods 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 230000017074 necrotic cell death Effects 0.000 description 2
- 235000015136 pumpkin Nutrition 0.000 description 2
- 239000013062 quality control Sample Substances 0.000 description 2
- 238000003762 quantitative reverse transcription PCR Methods 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000013518 transcription Methods 0.000 description 2
- 230000035897 transcription Effects 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- 210000003462 vein Anatomy 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 241000221198 Basidiomycota Species 0.000 description 1
- 235000009024 Ceanothus sanguineus Nutrition 0.000 description 1
- 229920002160 Celluloid Polymers 0.000 description 1
- 208000003322 Coinfection Diseases 0.000 description 1
- 241001123536 Colletotrichum acutatum Species 0.000 description 1
- 241000606071 Colletotrichum nigrum Species 0.000 description 1
- 241000514658 Colletotrichum spinosum Species 0.000 description 1
- 241000123288 Cryptoporus Species 0.000 description 1
- 241000219112 Cucumis Species 0.000 description 1
- 235000015510 Cucumis melo subsp melo Nutrition 0.000 description 1
- 240000008067 Cucumis sativus Species 0.000 description 1
- 235000010799 Cucumis sativus var sativus Nutrition 0.000 description 1
- 241000219104 Cucurbitaceae Species 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 241000220485 Fabaceae Species 0.000 description 1
- 241000710831 Flavivirus Species 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 240000003553 Leptospermum scoparium Species 0.000 description 1
- 235000015459 Lycium barbarum Nutrition 0.000 description 1
- 235000007688 Lycopersicon esculentum Nutrition 0.000 description 1
- 206010027146 Melanoderma Diseases 0.000 description 1
- 208000031888 Mycoses Diseases 0.000 description 1
- 108020005089 Plant RNA Proteins 0.000 description 1
- 238000002123 RNA extraction Methods 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- 241000208292 Solanaceae Species 0.000 description 1
- 240000003768 Solanum lycopersicum Species 0.000 description 1
- 241000723848 Tobamovirus Species 0.000 description 1
- 235000010726 Vigna sinensis Nutrition 0.000 description 1
- 244000042314 Vigna unguiculata Species 0.000 description 1
- FJJCIZWZNKZHII-UHFFFAOYSA-N [4,6-bis(cyanoamino)-1,3,5-triazin-2-yl]cyanamide Chemical compound N#CNC1=NC(NC#N)=NC(NC#N)=N1 FJJCIZWZNKZHII-UHFFFAOYSA-N 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 208000022338 anthrax infection Diseases 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 238000004891 communication Methods 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 238000013211 curve analysis Methods 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 241001493065 dsRNA viruses Species 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000005562 fading Methods 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 1
- 230000002538 fungal effect Effects 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000001788 irregular Effects 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 230000008635 plant growth Effects 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 238000012113 quantitative test Methods 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 238000005464 sample preparation method Methods 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000007790 scraping Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000012772 sequence design Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 230000000405 serological effect Effects 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 108700012359 toxins Proteins 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
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
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
-
- 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
- C12Q1/6804—Nucleic acid analysis using immunogens
-
- 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
-
- 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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/16—Primer sets for multiplex assays
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Analytical Chemistry (AREA)
- Immunology (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Genetics & Genomics (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Virology (AREA)
- Botany (AREA)
- Mycology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to the technical field of biological detection of plant viruses and bacteria, in particular to a primer group, a kit and a method for detecting pepper light mottle virus and anthrax based on a recombinase-mediated isothermal nucleic acid amplification (RT-RAA). The sequence of the forward primer PM-F2 for detecting PMMoV designed in the invention is shown as SEQ ID No.1, the sequence of the reverse primer PM-R2 is shown as SEQ ID No.2, the sequence of the probe primer PM-P3 is shown as SEQ ID No.3, the sequence of the forward primer CT-F1 for detecting anthrax bacteria is shown as SEQ ID No.4, the sequence of the reverse primer CT-R1 is shown as SEQ ID No.5, and the sequence of the probe primer CT-P1 is shown as SEQ ID No. 6.
Description
Technical Field
The invention relates to the technical field of biological detection of plant viruses and bacteria, in particular to a primer group, a kit and a method for detecting pepper light mottle virus and anthrax based on a recombinase-mediated isothermal nucleic acid amplification (RT-RAA).
Background
The pepper light mottle virus (Peppermild mottlevis, PMMoV) is a non-enveloped positive single stranded RNA virus of the genus Tobamovirus of the family Virgaviridae (Virgaviridae), which can cause the pepper fruit to fade, mottle and deformity, causing serious economic loss to pepper production worldwide. After being infected, the capsicum leaves show no obvious symptoms or fade mottle and flowers She Zhengzhuang appear; the plant growth is obviously slowed down, the earlier the disease is, the more the growth vigor is affected; the symptoms of the infected fruits are obvious, and the symptoms are shown as small fruits, fading and mottle on the surfaces of the fruits, concave-convex spots, and even malformation and necrosis. PMMoV was first reported in the united states in 1964, and its distribution range was expanding year by year as seed trade and germplasm resource communication became more frequent. PMMoV has great potential transmissibility, can be transmitted through juice friction and seed transmission, enters a human body along with the diseased chilli, and still has infection activity after being digested and discharged out of the human body. The positive detection rate of commercial pepper commodity seeds in Beijing area in China is as high as 61.11%. Besides capsicum, PMMoV can naturally infect tomato of Solanaceae, pumpkin, cucumber, pumpkin, melon, cowpea of Leguminosae and other crops, and has a detection rate of 83.33% in Jiangsu cucurbitaceae vegetable samples, and has high frequency and wide range of complex infection with other viruses and great harm. Currently, the detection methods for PMMoV mainly comprise biological methods, electron microscope technology, serological assays (enzyme-linked immunosorbent assay) and nucleic acid-based molecular biological assays (RT-PCR and real-time fluorescent RT-PCR), and the enzyme-linked immunosorbent assay (ELISA) and RT-PCR are most commonly used. In addition, methods for detecting PMMoV by reverse transcription in combination with Loop-mediated isothermal amplification (Loop-mediated isothermal amplification, LAMP) have been established.
The pepper anthracnose is a global fungal disease caused by different species of the genus Colletotrichum (Colletotrichum) of the subdivision Deuteromycetin. The anthrax is hidden in the seed or on the surface to overwinter with mycelium or conidium, and is spread to host plants by means of rainwater, insects or air flow, and invades through a wound of the host to cause primary infection, becomes a primary infection source for the next season disease, and then grows out conidium to cause secondary infection. The disease can be developed after bacteria invade for 3 to 5 days. The high temperature and the high humidity are favorable for disease occurrence, and can cause the large-area yield reduction of the capsicum. The pepper anthracnose is mainly harmful to leaves and fruits. The leaf infection mainly manifests symptoms and mostly occurs on mature leaves, so that water-immersed yellow brown circular spots are generated, and brown circular or irregular spots are formed on fruits. The anthracnose of the capsicum occurs worldwide, and the capsicum is often reduced by 30% -40% in areas such as China, india, indonesia and Korea, and the most serious year of the reduction of the capsicum is up to 80%. The reported pathogenic bacteria of pepper anthracnose are mainly colletotrichum gloeosporides, black anthracnose (Colletotrichum nigrum), black spot anthracnose (Colletotrichum capsici), acuminate anthracnose (Colletotrichum acutatum) and the like. As the detection method of anthrax, there are mainly biological methods and nucleic acid-based molecular biological assays (PCR, real-time fluorescent PCR and LAMP), and PCR and LAMP are most commonly used.
Recombinase-mediated isothermal nucleic acid amplification (Recombinase AidedAmplification) is a recently developed technique for amplifying DNA with high specificity at a constant low temperature (39 ℃) and at a detectable level within minutes. The RT-RAA/RAA products can be detected by agarose gel electrophoresis. Lateral flow test strips (Lateral flow strip, LFS), lateral immunochromatographic assays are useful for detecting a variety of biological substances including antibiotics, toxins, proteins, polypeptides, DNA, and the like, which utilize the ability to purify antibodies, to bind highly specifically and move cooperatively across a membrane, and compete with or are captured by detection segments on the membrane. The lateral flow test strip is one of the current common rapid detection methods, has the advantages of simple operation, small sample amount, rapid detection, low cost and the like, and is widely applied to a plurality of fields such as medicine, food, environment and the like.
Disclosure of Invention
Problems to be solved
At present, molecular biological methods for pepper light mottle virus (Peppermild mottlevis, PMMoV) and pepper anthracnose are either highly dependent on trained laboratory technicians, or require complex and expensive laboratory instruments or have complicated detection operation processes, and are difficult to separate from laboratory environments and apply under field conditions. In addition, the two pathogens can infect capsicum and various common pathogens of crops, but most of the existing detection methods take one of the pathogens as an identification target, so that the PMMoV and the anthrax are difficult to be identified rapidly and accurately in production practice, and a rapid, simple and accurate dual pathogen detection method is needed to be established. If the RT-RAA/RAA dual isothermal nucleic acid amplification system of PMMoV and anthrax can be established and combined with lateral chromatography test strip detection, the urgent requirements of farmers on virus type determination and targeted disease control can be met, and new technical support can be provided for national agricultural technical service departments, plant protection and plant quarantine departments.
Technical proposal
In order to solve the technical problems, the invention aims to provide a primer group, a kit and a method for detecting PMMoV and anthrax based on RT-RAA.
In one aspect, the invention provides an RT-RAA detection primer set for detecting pepper light mottle virus (PMMoV) and anthrax, wherein the primer set comprises a pepper light mottle virus detection primer, an anthrax detection primer and pepper
Light mottle virus detection probe PM-P3 and anthrax detection probe CT-P1; wherein,,
the primer for detecting the pepper mild mottle virus comprises a forward primer PM-CPF and a reverse primer PM-CPR,
the sequence of the forward primer PM-F2 is 5'-CTGTGTACTTCGGCGTTAGGCAATCAG-3' (SEQ ID NO. 1);
the sequence of the reverse primer PM-R2 is 5'-CATTCATGAGGTTACTTATACTGGCCC-3' (SEQ ID NO. 2);
the anthrax detection primer comprises a forward primer CT-F1 and a reverse primer CT-R1,
the forward primer CT-F1 has the sequence of 5'-GTGAGATCCGTCGCTACCAGAAG-3' (SEQ ID NO. 4);
the reverse primer CT-R1 has the sequence of 5'-GCATAGGTTGGTGTCCTCAAAGAG-3' (SEQ ID NO. 5).
Preferably, the primer group of the invention, the pepper light mottle virus detection probe PM-P3 has a sequence of 5'-GCACTTCTCGGAGCCTTTGATACTAGGHACAGGATAATAGAAGTT-3' (SEQ ID NO. 3); the anthrax detection probe CT-P1 has a sequence of 5'-CTTCAAGTCCGATCTCCGCTTCCAGTCHTCCGCCATCGGTGCCCTTC-3' (SEQ ID NO. 6); FITC is modified at the 5' end of the reverse primer PM-R2 of the PMMoV, biotin is modified at the 5' end of the detection probe PM-P3, phosphorylation modification is performed at the 3' end of the PM-P3, and H is tetrahydrofuran; digoxin is modified at the 5' end of the anthrax reverse primer CT-R1, rhodamine is modified at the 5' end of the probe primer CT-P1, phosphorylation modification is performed at the 3' end of the CT-P1, and H is tetrahydrofuran.
In one aspect, the invention provides a kit for detecting pepper mild mottle virus (PMMoV) and anthrax, comprising the primer set.
In one aspect of the invention, the RT-RAA detection primer set for detecting pepper light mottle virus (PMMoV) and anthrax provided by the invention can be used for preparing a kit for judging whether a sample to be detected contains PMMoV and/or anthrax.
In one aspect of the invention, the kits of the invention may further comprise other reagents, including RNA extraction reagents, DNA extraction reagents, sample lysates, buffers, PCR sample amplification reagents, kit instructions.
In one aspect of the invention, the RT-RAA detection primer set for detecting pepper light mottle virus (PMMoV) and anthrax provided by the invention can be used for judging whether a sample to be detected contains PMMoV and/or anthrax.
In one aspect of the invention, the kit provided by the invention can be used for detecting whether PMMoV and/or anthrax is contained in a biological sample to be detected.
In one aspect, the invention provides a method for detecting PMMoV and/or anthrax using a kit, comprising the steps of:
1) Extracting total RNA of a biological sample to be detected, or carrying out cracking treatment on a plant sample;
2) Using the total RNA or the plant lysate extracted in the step 1) as a template, and adopting a forward primer and a reverse primer in a primer group to carry out RT-RAA amplification to obtain an amplification product;
3) Detecting the amplified product, and judging whether PMMoV and/or anthrax is contained or not based on the detection result of the amplified product.
Preferably, in the invention, if the amplified product contains a 316bp DNA fragment, the biological sample to be detected contains PMMoV, and if the amplified product does not contain a 316bp DNA fragment, the biological sample to be detected does not contain PMMoV; if the amplified product contains a DNA fragment of 243bp, the biological sample to be detected contains anthrax, and if the amplified product does not contain a DNA fragment of 243bp, the biological sample to be detected does not contain anthrax.
In one aspect, the invention provides a method for detecting whether a biological sample to be detected contains PMMoV and/or anthrax, comprising:
detecting whether a lysate or a total RNA amplification product of the biological sample to be detected contains specific DNA fragments, if the lysate or the total RNA amplification product contains the specific DNA fragments, the biological sample to be detected contains PMMoV and/or anthrax, and if the lysate or the total RNA amplification product does not contain the specific DNA fragments, the biological sample to be detected does not contain PMMoV and/or anthrax; wherein the specific DNA fragment is the target sequence of the forward primer and the reverse primer.
In one aspect, the invention provides a method for detecting PMMoV and/or anthrax based on an RT-RAA amplification mode.
Preferably, the method comprises the steps of:
1) Lysing the plant sample according to the method provided by the nucleic acid releasing agent; or extracting total RNA according to TRIzol reagent instruction, and dissolving in TE solution or pure water;
2) Taking the total RNA or the plant lysate extracted in the step 1) as a template, adopting a forward primer and a reverse primer in a primer group to carry out RT-RAA amplification, if an amplification product contains a DNA fragment of 316bp, the biological sample to be detected contains PMMoV, and if the amplification product does not contain the DNA fragment of 316bp, the biological sample to be detected does not contain PMMoV; if the amplified product contains a DNA fragment of 243bp, the biological sample to be detected contains anthrax, and if the amplified product does not contain a DNA fragment of 243bp, the biological sample to be detected does not contain anthrax.
The forward primer PM-F2 has the sequence of 5'-CTGTGTACTTCGGCGTTAGGCAATCAG-3' (SEQ ID NO. 1),
the sequence of the reverse primer PM-R2 is 5'-CATTCATGAGGTTACTTATACTGGCCC-3' (SEQ ID NO. 2);
the forward primer CT-F1 has the sequence of 5'-GTGAGATCCGTCGCTACCAGAAG-3' (SEQ ID NO. 4),
The reverse primer CT-R1 has the sequence of 5'-GCATAGGTTGGTGTCCTCAAAGAG-3' (SEQ ID NO. 5).
Preferably, the reaction system for RT-RAA amplification is as follows:
| composition of the components | Volume of |
| Plant sample lysate or total RNA solution | 2μL |
| Base buffer | 29.4μL |
| PM-F2(5μM) | 0.29μL |
| PM-R2(5μM) | 0.4μL |
| CT-F1(5μM) | 0.9μL |
| CT-R1(5μM) | 1.26μL |
| ddH 2 O | To a total system of 47.5. Mu.L |
Preferably, the reaction conditions for the RAA amplification are set as follows: after completion of the preparation of the reaction solution, 2.5. Mu.L of 280mM MgAc was added to each of the 0.2mL eppendorf tubes 2 Fully and uniformly mixing; the eppendorf tube (0.2 mL) was placed on a PCR apparatus with a thermal cover function, and incubated at 39deg.C for 25min.
Preferably, the amplified product can be detected by agarose gel electrophoresis, and the RT-RAA amplified product has a minimum copy number of 10 as the initial amplified template for detecting the target sequence by agarose gel electrophoresis 1 And copies.
The detection process of the RT-RAA amplification product comprises the following steps: after the completion of the RT-RAA reaction, the reaction tube was taken out. Each reaction tube was charged with 100. Mu.L of phenol/chloroform (1:1), thoroughly homogenized by shaking, and centrifuged at 12000rpm for 10min (this step was vigorously mixed by shaking using a vortex shaker). Mixing 10 μL supernatant with 2 μL 6×loading Buffer, loading onto 1.5% agarose gel, and electrophoresis at 200V for 15min; ending electrophoresis when bromophenol blue moves to 2/3 of the position under gel; EB staining for 5min, observing under an ultraviolet lamp and photographing.
In one aspect, the invention provides a method for detecting whether a biological sample to be detected contains PMMoV or/and anthrax.
Preferably, the method comprises: detecting whether a lysate of a biological sample to be detected or an amplification product of a nucleic acid sample contains specific DNA fragments, if the lysate contains the specific DNA fragments, the biological sample to be detected contains PMMoV or/and anthrax, and if the lysate does not contain the specific DNA fragments, the biological sample to be detected does not contain PMMoV or/and anthrax; wherein the specific DNA fragment is the target sequence of the primer group in the total DNA of the biological sample to be detected.
In one aspect, the invention provides a method for detecting PMMoV or/and anthrax based on RT-RAA-lateral flow chromatography.
Preferably, the method comprises the steps of:
1) Lysing the plant sample according to the method provided by the nucleic acid releasing agent; or extracting total RNA according to TRIzol reagent instruction, and dissolving in TE solution or pure water;
2) Using the plant lysate obtained in the step 1) or the extracted total RNA or the template, and adopting a primer group to carry out RT-RAA amplification;
the forward primer PM-F2 has the sequence of 5'-CTGTGTACTTCGGCGTTAGGCAATCAG-3' (SEQ ID NO. 1),
The sequence of the reverse primer PM-R2 is 5'-CATTCATGAGGTTACTTATACTGGCCC-3' (SEQ ID NO. 2);
the pepper light mottle virus detection probe PM-P3 has a sequence of 5'-GCACTTCTCGGAGCCTTTGATACTAGGHACAGGATAATAGAAGTT-3' (SEQ ID NO. 3),
the forward primer CT-F1 has the sequence of 5'-GTGAGATCCGTCGCTACCAGAAG-3' (SEQ ID NO. 4),
the reverse primer CT-R1 has the sequence of 5'-GCATAGGTTGGTGTCCTCAAAGAG-3' (SEQ ID NO. 5).
The celluloid bacteria detection probe CT-P1,
the sequence is 5'-CTTCAAGTCCGATCTCCGCTTCCAGTCHTCCGCCATCGGTGCCCTTC-3' (SEQ ID NO. 6);
preferably, the reaction system for RT-RAA amplification is as follows:
| composition of the components | Volume of |
| Plant sample lysate or total RNA solution | 2μL |
| Base buffer | 29.4μL |
| PM-CPF(5μM) | 0.29μL |
| PM-CPR(5μM) | 0.4μL |
| PM-CPP(1μM) | 0.4μL |
| CT-HISF(5μM) | 0.9μL |
| CT-HISR(5μM) | 1.26μL |
| CT-HISP(1μM) | 1.26μL |
| ddH 2 O | To a total system of 47.5. Mu.L |
Preferably, the reaction conditions for the RAA amplification are set as follows: after completion of the preparation of the reaction solution, 2.5. Mu.L of 280mM MgAc was added to each of the 0.2mL eppendorf tubes 2 Fully and uniformly mixing; the eppendorf tube (0.2 mL) was placed on a PCR apparatus with a thermal cover function, and incubated at 39℃for 16min.
3) Detecting an amplification product by using a lateral flow chromatography test strip, and if the test strip only has one strip in a quality control area and the detection area has no strip, determining that the result is negative, and indicating that the sample does not contain PMMoV or anthrax; if two strips appear on the test strip, one strip is positioned in the quality control area and the other strip is positioned in the detection area T1, the result is positive, and the sample contains anthrax; if two strips appear on the test strip, one strip is positioned in the quality control area and the other strip is positioned in the detection area T2, the result is positive, and the test strip shows that the sample contains PMMoV; if three strips appear on the test strip, one strip is positioned in the quality control area, one strip is positioned in the detection area T1, and one strip is positioned in the detection area T2, the result is positive, and the sample contains anthrax and PMMoV; if the quality control line of the test strip does not develop color, the test is invalid, and the test strip needs to be replaced for re-detection.
The specific detection process can be as follows: the eppendorf tube lid containing 50. Mu.L of RT-RAA amplification product was opened and 2.5. Mu.L of amplification product was taken to a new 1.5mL eppendorf tube and diluted 20-fold. A new nucleic acid lateral chromatography test strip binding pad end is directly inserted into an eppendorf tube filled with diluted products, and the liquid level does not exceed the immersion upper limit mark of a sample pad. After the read zone is fully infiltrated, the test paper is laid flat for 1 minute, and the color development result is observed and recorded within 10 minutes. At least one quality control line, with or without detection lines, appears for each test sample. If the test strip only has one strip in the quality control area and the detection area has no strip, the result is negative, which indicates that the sample does not contain PMMoV or anthrax; if two strips appear on the test strip, one strip is positioned in the quality control area and the other strip is positioned in the detection area T1, the result is positive, and the sample contains anthrax; if two strips appear on the test strip, one strip is positioned in the quality control area and the other strip is positioned in the detection area T2, the result is positive, and the test strip shows that the sample contains PMMoV; if three strips appear on the test strip, one strip is positioned in the quality control area, one strip is positioned in the detection area T1, and one strip is positioned in the detection area T2, the result is positive, and the sample contains anthrax and PMMoV; if the quality control line of the test strip does not develop color, the test is invalid, and the test strip needs to be replaced for re-detection.
In one aspect, the invention provides a kit for detecting PMMoV or/and anthrax based on an RT-RAA-lateral flow chromatography technology, which comprises a primer group for detecting PMMoV and anthrax, and a lateral flow chromatography test strip, wherein the lateral flow chromatography test strip is provided with a detection line T1, a detection line T2 and a quality control area, and can detect amplification products of the primer group. Preferably, the sequences of the PMMoV and anthrax primer set are shown in SEQ ID NO. 1-6.
Advantageous effects
The invention provides a specific primer group for detecting PMMoV and anthrax, and establishes a dual rapid detection method for PMMoV and anthrax. The primer designed in the invention can effectively amplify target genes, has higher specificity and sensitivity, has no cross reaction with other pathogenic bacteria, and has 94 copies of the lowest copy number of the PMMoV initial amplification template detected by each reaction of the RT-RAA-lateral flow chromatography technology, and 41 copies of the lowest copy number of the anthrax target sequence initial amplification template. The invention can be used for on-site rapid detection of PMMoV and anthrax, and has important significance for effective control of diseases.
Drawings
FIG. 1A is an electrophoretogram of amplification using PMMoV amplification primer sets 1-4 RTRAA;
FIG. 1B is an electrophoretogram of 5-8 RTRAA amplification using anthrax amplification primer set;
FIG. 2A shows that 10-fold serial dilutions of PMMoV amplification primers PM-F1 and PM-R1 amplification product transformed plasmids were used as RTRAA templates, and that PM-F1 and PM-R1 amplification could be detected as low as 10 by analyzing the detection sensitivity of primer set 1PM-F1 and PM-R1RT-RAA 4 PMMoV CP gene plasmid of each copy. 50 mu L RT RAA amplified initial template copy number is from 10 6 Copy to 10 0 Copying and gradually decreasing; wherein M:labeling Plus DNA; "-": with ddH 2 O replaces the negative control of the same volume plasmid.
FIG. 2B shows that 10-fold serial dilutions of PMMoV amplification primers PM-F2 and PM-R2 amplification product transformed plasmids were used as RT RAA templates, and that PM-F2 and PM-R2RT-RAA amplification was detected as low as 10 by analysis of the detection sensitivity of primer sets 2PM-F2 and PM-R2 1 PMMoV CP gene plasmid of each copy. 50 mu L RT RAA amplified initial template copy number is from 10 6 Copy to 10 0 Copying and gradually decreasing; wherein M:labeling Plus DNA; "-": with ddH 2 O replaces the negative control of the same volume plasmid.
In FIG. 3A, 10-fold serial gradient dilutions of the transformation plasmid of the amplification products of anthrax bacteria amplification primers CT-F1 and CT-R1 are used as RT RAA templates, and the detection sensitivity detection results of the analysis primer set 5CT-F1 and CT-R1 are analyzed, and the detection of CT-F1 and CT-R1RT-RAA can be reduced to 10 1 Anthrax HIS gene plasmid in each copy. 50 mu L RT RAA amplified initial template copy number is from 10 6 Copy to 10 0 Copying and gradually decreasing; wherein M:labeling Plus DNA; "-": with ddH 2 O replaces the negative control of the same volume plasmid.
In FIG. 3, B is a 10-fold serial gradient dilution of plasmid transformed with anthrax amplification primers CT-F2 and CT-R2 as RT RAA template, and the detection sensitivity of the analysis primer set 6CT-F2 and CT-R2 is as low as 10 in CT-F2 and CT-R2RT-RAA amplification 2 Anthrax HIS gene plasmid in each copy. 50 mu L RT RAA amplified initial template copy number is from 10 6 Copy to 10 0 Copying and gradually decreasing; wherein M:labeling Plus DNA; "-": with ddH 2 O replaces the negative control of the same volume plasmid.
In FIG. 3, C is a 10-fold serial gradient dilution of plasmid transformed with anthrax amplification primers CT-F3 and CT-R3 as RT RAA template, and the detection sensitivity of the analysis primer set 7CT-F3 and CT-R3 was detected as low as 10 in CT-F3 and CT-R3RT-RAA amplification 1 Anthrax HIS gene plasmid in each copy. 50 mu L RT RAA amplified initial template copy number is from 10 6 Copy to 10 0 Copying and gradually decreasing; wherein M:labeling Plus DNA; "-": with ddH 2 O replaces the negative control of the same volume plasmid.
FIG. 3D shows a 10-fold serial dilutions of plasmid transformed with anthrax amplification primers CT-F4 and CT-R4 amplification products as RT RAA moldsThe plate analyzes the detection sensitivity detection results of the primer group 8CT-F4 and CT-R4, and the amplification of CT-F4 and CT-R4RT-RAA can detect as low as 10 3 Anthrax HIS gene plasmid in each copy. 50 mu L RT RAA amplified initial template copy number is from 10 6 Copy to 10 0 Copying and gradually decreasing; wherein M:labeling Plus DNA; "-": with ddH 2 O replaces the negative control of the same volume plasmid.
FIG. 4 is a schematic diagram of the structure and detection result of a single-target disposable nucleic acid detection test strip (JY 0201);
FIG. 5 shows the results of RT-RAA amplification and product test strip detection of positive plasmid and negative control using PMMoV primer set A, primer set B, primer set C, and primer set D;
FIG. 6 shows the results of RT-RAA amplification and product test strip detection of positive plasmid and negative control using anthrax primer set E, primer set F, primer set G, and primer set H;
FIG. 7 sensitivity detection of PMMoV primer set A RT RAA-LFS;
FIG. 8 sensitivity detection of PMMoV primer set B RT RAA-LFS;
FIG. 9 sensitivity detection of PMMoV primer set D RT RAA-LFS;
FIG. 10 sensitivity detection of anthrax primer set ERT RAA-LFS;
FIG. 11 sensitivity detection of anthrax primer set H RT RAA-LFS;
FIG. 12PMMoV primer set A RT RAA-LFS detection primer specificity evaluation;
FIG. 13PMMoV primer set D RT RAA-LFS detection primer specificity evaluation;
FIG. 14 anthrax primer set ERT RAA-LFS detection primer specificity evaluation;
FIG. 15 anthrax primer set H RT RAA-LFS detection primer specificity evaluation;
FIG. 16 is a diagram showing the structure and detection result of a dual-target disposable nucleic acid detection test strip (JY 0209);
FIG. 17 shows the results of double RTRAA-LFS detection of PMMoV primer set and anthrax primer set using plasmid as template;
FIG. 18 qPCR melting curve analysis of PMMoV primer set D (PM-F2 and PM-R2, panel A), anthrax primer set E2 (CT-F1 and CT-R1, panel B);
FIG. 19PMMoV primer set D (PM-F2 and PM-R2, panel A), anthrax primer set E2 (CT-F1 and CT-R1, panel B) qPCR correspond to absolute quantitative amplification curve analysis of gradient diluted plasmids;
FIG. 20 RTRAA-LFS detection results of plant leaf lysate of PMMoV primer set D and anthrax primer set E2, PMMoV primer set D and anthrax primer set H2;
FIG. 21 plant leaf lysate PMMoV, anthrax primer set D and primer set E2 double RT RAA-LFS reaction primer optimization detection result;
FIG. 22 plant leaf lysate PMMoV, anthrax primer set D and primer set H2 double RT RAA-LFS reaction primer optimization detection result;
FIG. 23 depicts the results of RTRAA-LFS rapid detection performed by random field sampling of pepper plants;
FIG. 24PMMoV infected pepper plant leaves and flat head anthrax mycelium RT RAA-LFS limit assay (1: sample with 100 ng/. Mu.L for total RNA after extraction, 2: sample with 10 ng/. Mu.L for total RNA after extraction, 3: sample with 1 ng/. Mu.L for total RNA after extraction, 4: sample with 100 pg/. Mu.L for total RNA after extraction, 5: sample with 10 pg/. Mu.L for total RNA after extraction, 6: sample with 1 pg/. Mu.L for total RNA after extraction, 7: sample with 100 fg/. Mu.L for total RNA after extraction, "-" ddH "was used 2 O instead of negative control of the same volume plasmid).
Detailed Description
The following describes the embodiments of the present invention in detail with reference to the drawings.
The experimental methods in the following examples are conventional, unless otherwise specified. The experimental materials described in the examples below, unless otherwise specified, were not purchased from conventional biochemical reagent stores. The quantitative tests in the following examples were all set up in triplicate and the results averaged.
The invention relates to a virus sample source:
The virus sample, the anthrax isolate and the corresponding total RNA or cDNA are all stored in a plant virology research institute of Ningbo university virus and host interaction research laboratory.
Wherein, the capsicum plant sample is collected in Ningbo city of Zhejiang province in 2020, the plant is delayed in development, the leaves are variegated and flowers She Zhengzhuang appear, the fruits are small, the fruit surfaces are variegated, concave-convex spots and inwards concave black brown disease spots appear, and even malformation and necrosis occur. Samples were snap frozen in liquid nitrogen after collection and stored at-70 ℃. Wherein, in the PMMoV RT-RAA primer specificity screening test, the total RNA of the plant infected by cucumber mosaic virus (cucumber mosaic virus, CMV), pepper ringspot virus (chilli ringspot virus, chiRSV), broad bean wilt virus2 (broadbean will virus, BBWV 2), pepper vein flaviviruses (pepper vein yellows virus, peVYV) and tobacco light green mosaic virus (tobacco mild green mosaic virus, TMGMV) which belongs to the same genus as the PMMoV is used as a template as a control template; pepper anthracnose is caused by a variety of pathogens, different anthrax species including colletotrichum gloeosporioides, colletotrichum glorioides, colletotrichum (Colletotrichum siamense), colletotrichum glomeratum (Colletotrichum fructicola), colletotrichum truncatum (Colletotrichum truncatum), colletotrichum spinosum (Colletotrichum scovillei), and colletotrichum cryptoporus (Colletotrichum aenigma).
The sample preparation method related by the invention comprises the following steps:
viral or fungal nucleic acids are extracted from PMMoV, anthrax infected pepper leaves or fruits using TRIzol reagent (Thermo Fisher Scientific). Total RNA from pepper leaves or seeds was extracted according to TRIzol reagent (Thermo Fisher Scientific) instructions.The nucleic acid lateral chromatography test strip is from Beijing Baozhen Yinghui biotechnology Co., ltd.
To assess whether the RT-RAA-LFS system used was useful for rapid detection of PMMoV, anthrax infection in plants, to collect fromThe capsicum plant samples infected with PMMoV and anthrax at the experimental base of the university of Ningbo plant virology research institute are used as test objects, and healthy capsicum plant leaves are used as negative control. Will beFresh pepper leaf sample is put into the container containing 100 mu L +.>The corresponding plant sample lysate was obtained in 1.5mL eppendorf tubes with a tip number of light weight (Tiosbio, beijing Baozhen Yinghui Biotechnology Co., ltd.). 1 mu L of plant sample lysate or dilution thereof is used as an amplification template of RT-RAA. The plant sample lysate can be tested immediately or stored for more than 3 months at-70 ℃.
Experimental example 1: primer design and screening for PMMoV and anthrax detection
The group of the invention uses PMMoV Coat Protein (CP) gene and anthrax histone H3 (HIS) gene as target genes through a large number of sequence analysis, and designs a species-specific amplification Primer by using Primer Premier 5.0 software after analyzing the characteristics of the target gene sequences. Primer design follows the following principle.
(1) The length of the amplified primer is about 20-30 bp, and the annealing temperatures of different primers are as consistent or similar as possible; (2) the length of the amplified product is about 200-500 bp, and the amplified product has species specificity.
The initial identification of primer amplification specificity was accomplished by alignment of primer amplification segment sequences using BLAST function of the National Center for Biotechnology Information (NCBI). The primers identified were synthesized by general biosystems (Anhui) Inc., purity grade HPLC. The specific sequences of the primers are shown in Table 1 below:
primers designed in Table 1 for PMMoV and anthrax detection
UsingRT RAA nucleic acid amplification kit (basic model JY 0203) takes total plant RNA or pepper plant leaf sample lysate infected with PMMoV and anthrax as a template to respectively carry out RT-RAA amplification of forward primers and reverse primers of groups 1-2 and 5-8, and an amplification system (single sample/reaction) is as follows:
| composition of the components | Volume of |
| Plant sample lysate or total RNA solution | 2μL |
| Base buffer | 29.4μL |
| Forward primer (5. Mu.M) | 2.1μL |
| Reverse primer (5. Mu.M) | 2.1μL |
| ddH 2 O | To a total system of 47.5. Mu.L |
After completion of the preparation of the reaction solution, 2.5. Mu.L of 280mM MgAc was added to each of the 0.2mL eppendorf tubes 2 Fully and uniformly mixing; will beThe eppendorf tube (0.2 mL) was placed on a PCR apparatus with a thermal cover function, and incubated at 39deg.C for 30min.
After the completion of the RT-RAA reaction, the reaction tube was taken out. Each reaction tube was charged with 100. Mu.L of phenol/chloroform (1:1), thoroughly homogenized by shaking, and centrifuged at 12000rpm for 10min (this step was vigorously mixed by shaking using a vortex shaker). Mixing 10 μL supernatant with 2 μL 6×loading Buffer, loading onto 1.5% agarose gel, and electrophoresis at 200V for 15min; ending electrophoresis when bromophenol blue moves to 2/3 of the position under gel; EB staining for 5min, observing under an ultraviolet lamp and photographing.
As shown in fig. 1, on the premise of adding 2 μl of plant total RNA solution or sample lysate, the RT-RAA amplification product electrophoresis bands of PMMoV amplification primer set 1 (PM-F1 and PM-R1) and PMMoV amplification primer set 2 (PM-F2 and PM-R2) are clear and bright, have no impurity band, and have amplification band sizes consistent with expectations; the PMMoV amplification primer group 3 (PM-F3 and PM-R3) and the PMMoV amplification primer group 4 (PM-F4 and PM-R4) have weak electrophoresis bands and poor amplification efficiency, and cannot be used as primers for detecting PMMoV; the primer group 5 to the primer group 8 for amplifying the anthrax can be used for detecting the anthrax, the electrophoresis band of the amplified product is clear and specific, no impurity band exists, and the size of the amplified band is consistent with the expected size. The amplified products of each primer pair are cloned to a T vector, and after sequencing, the amplified products are determined to be target sequences.
Experimental example 2: PMMoV and anthrax test sample comparison disc preparation and composition
PMMoV amplification primer set 1 (PM-F1 and PM-R1), PMMoV amplification primer set 2 (PM-F2 and PM-R2) CP gene amplification products and anthrax amplification primer set 5-primer set 8HIS gene amplification products clone and sequence correct plasmids, respectively transform and shake bacteria overnight, then carry out plasmid extraction, measure recombinant plasmid OD260, OD280 and OD260/OD280 values by an ultraviolet spectrophotometer, and repeat for 3 times to determine plasmid DNA concentration and purity.
The copy number of the plasmid was obtained according to the following formula:
copy number = plasmid concentration x 6.02 x 10 23 /(660X total length of plasmid)
Calculate copy number and dilute to 1×10 8 Copy/. Mu.L, store at-20deg.C for later use. Diluting the recombinant plasmid with a fixed value to 1X 10 6 Copy/. Mu.L, serial 10-fold dilution to obtain 1X 10 0 Copy/. Mu.L, 1X 10 1 Copy/. Mu.L, 1X 10 2 Copy/. Mu.L, 1X 10 3 Copy/. Mu.L, 1X 10 4 Copy/. Mu.L, 1X 10 5 Copy/. Mu.L and 1X 10 6 The copy/. Mu.L plasmid dilution was used as the template for subsequent amplifications.
Experimental example 3: detection of primer sensitivity for PMMoV and anthrax detection
To further examine the sensitivity of PMMoV amplification primer set 1 (PM-F1 and PM-R1), PMMoV amplification primer set 2 (PM-F2 and PM-R2), and B.anthracis amplification primer set 5 (CT-F1 and CT-R1), primer set 6 (CT-F2 and CT-R2), primer set 7 (CT-F3 and CT-R3), primer set 8 (CT-F4 and CT-R4), 10-fold serial dilutions (10 per microliter) of the respective amplification product plasmid DNA were performed 6 、10 5 、10 4 、10 3 、10 2 、10 1 、10 0 Copy) was used as template for RT-RAA using ddH 2 O was used as a negative control template. The same template was compared by 1.5% agarose gel electrophoresis to amplify the different primers RT-RAA, respectively.
As shown in FIG. 2, the amplification result of the RT-RAA of the PMMoV amplification primer set shows that the amplification of the RT-RAA of the primer set 1 (PM-F1 and PM-R1) can be detected as low as 10 4 A PMMoV CP gene plasmid of each copy; primer set 2 (PM-F2 and PM-R2) RT-RAA amplification was detectable as low as 10 1 A PMMoV CP gene plasmid with individual copies.
As shown in FIG. 3, the amplification results of the anthrax bacteria amplification primer set 5-8 RT-RAA show that the primer set 5 (CT-F1 and CT-R1) RT-RAA amplification can be detected as low as 10 1 The copy number of anthrax HIS gene plasmid and the primer group 6 (CT-F2 and CT-R2) RT-RAA amplification can detect as low as 10 2 Copy number of anthrax HIS gene plasmid; primer set 7 (CT-F3 and CT-R3) RT-RAA amplification was detectable as low as 10 1 Copy number of anthrax HIS gene plasmid; primer set 8 (CT-F4 and CT-R4) RT-RAA amplification was detectable as low as 10 3 And a copy of anthrax HIS gene plasmid.
Experimental example 4: PMMoV, anthrax RT-RAA-LFS detection
For the amplification segments of Primer sets 1 to 8 in example 1, a Primer Premier 5.0 software was used to design a species-specific probe sequence located in the middle segment of the amplification Primer, modified with tetrahydrofuran. Primer design follows the following principle.
(1) The probe length is about 30-45 bp; (2) the probe sequences are species specific.
The initial identification of primer amplification specificity was accomplished by alignment of primer amplification segment sequences using BLAST function of the National Center for Biotechnology Information (NCBI). The primers identified were synthesized by general biosystems (Anhui) Inc., purity grade HPLC. The specific primer sequences, probes, and amplified downstream primer labels are shown in Table 2 below:
/>
PMMoV, anthrax and Probe primers designed in Table 2
1X 10 amplification products corresponding to the upstream and downstream primers of each primer set 6 Copy/. Mu.L recombinant plasmid solution as template, usingThe RT RAA nucleic acid amplification kit (test strip method) (JY 0204) is used for preparing a RAA reaction system (single sample/reaction) of a primer group A to a primer group H, and the system is as follows:
the sample addition sequence was negative control samples (1X 10) 6 Copy/. Mu.L of recombinant plasmid solution was replaced with the same volume of ultrapure water), 1X 10 6 Copy/μl recombinant plasmid solution, and the tube cap is required to be closed immediately after each sample is added, avoiding aerosol contamination.And uniformly mixing the reaction systems, and adding the mixture into a basic reaction unit. The lyophilized powder was sufficiently dissolved, and note that this step was not possible to mix by vigorous shaking using a vortex shaker. The reaction unit was opened and 2.5. Mu.L of 280mM MgAc was added to each 0.2mL eppendorf tube 2 Thoroughly mixed and collected by centrifugation. Note that this step cannot be mixed by vigorous shaking with a vortex shaker.
The reaction tube was placed at 39℃for 16min. After the RAA reaction is finished, the eppendorf tube is opened, the amplified product is sucked into a new eppendorf tube, marked and diluted by 20-50 times.
A single-target disposable nucleic acid detection test strip (JY 0201) is shown in a structural schematic diagram in FIG. 4, the end of an immersion area (marked with a blue arrow upwards) of the test strip is inserted into an eppendorf tube, the liquid level cannot exceed the MAX indication line of the immersion area, the area to be interpreted is fully immersed (about 30-60 sec is needed), the test strip is flatly placed for 1min, and a red strip is waited to appear. And directly reading the detection result according to the color development condition of the test strip. The results were observed within 10min, and the interpretation was not valid after 10 min.
As shown in fig. 5, the detection result of the test strip after the PMMoV primer group C negative control RT RAA amplification product is diluted 20 times is positive, and the test strip can not be used for the sensitivity detection of the subsequent RT RAA nucleic acid amplification test strip; primer group A, primer group B and primer group D are negative in test strip detection result after the negative control RT RAA amplified product is diluted by 20 times, and the detection result is 1 multiplied by 10 6 The copy/mu L recombinant plasmid solution RTRAA detection result is positive, and can be used for the sensitivity detection of the subsequent RT RAA nucleic acid amplification test strip.
As shown in FIG. 6, the detection results of the test strips of the primer group F and the primer group G negative control RT RAA amplified products diluted by 20 times are positive, and the primer group G negative control RT RAA amplified products cannot be used for the sensitivity detection of the subsequent RT RAA nucleic acid amplification test strips; primer combination E and primer combination H are negative in test strip detection result after dilution of negative control RT RAA amplified product by 20 times, and 1 multiplied by 10 6 The copy/mu L recombinant plasmid solution RTRAA detection result is positive, and can be used for the sensitivity detection of the subsequent RT RAA nucleic acid amplification test strip.
Experimental example 5: comparison of detection sensitivity of different primer groups RT-RAA-LFS of PMMoV and anthrax
The different primer combinations 1×10 were completed by using the RT RAA amplification system in experimental example 4 with the plasmids corresponding to the corresponding amplified fragments of the different amplification primer sets of PMMoV primer set A, primer set D and anthrax primer set E and primer set H as templates 6 Copy/. Mu.L, 1X 10 5 Copy/. Mu.L, 1X 10 4 Copy/. Mu.L, 1X 10 3 Copy/. Mu.L, 1X 10 2 Copy/. Mu.L, 1X 10 1 Copy/. Mu.L and 1X 10 0 RT RAA amplification of copy/mu L plasmid diluent, and detection of the amplified products by a test strip after 20-time dilution, and determination of detection sensitivity of different primer combinations.
The sensitivity detection result of the PMMoV primer set ART RAA-LFS is shown in FIG. 7, and the minimum detection threshold of the PMMoV primer set A RT RAA-LFS is 10 1 And copies.
As shown in FIG. 8, the detection sensitivity of BRT RAA-LFS of PMMoV primer set is shown in the specification, and the lowest detection threshold of BRT RAA-LFS of PMMoV primer set is 10 4 And copies.
As shown in FIG. 9, the detection sensitivity of the DRT RAA-LFS of the PMMoV primer set shows that the minimum detection threshold of the D RT RAA-LFS of the PMMoV primer set is 10 1 And copies.
As shown in FIG. 10, the detection sensitivity of the ERT RAA-LFS of the anthrax primer set shows that the minimum detection threshold of the ERT RAA-LFS of the anthrax primer set is 10 1 And copies.
As shown in FIG. 11, the detection sensitivity of the primer set HRT RAA-LFS of anthrax bacteria shows that the minimum detection threshold of the primer set H RT RAA-LFS of anthrax bacteria is 10 1 And copies.
And finally determining the PMMoV primer group A, PMMoV primer group D, the anthrax primer group E and the anthrax primer group H to be used for primer specificity detection and subsequent dual RTRAA-LFS detection.
Example 6: specificity evaluation for PMMoV and anthrax detection primers
In this example, RT-PCR was used to verify that the total RNA of plants infected by cucumber mosaic virus (cucumber mosaic virus, CMV), pepper ring spot virus (chilli ringspot virus, chiRSV), broad bean wilt virus2 (broadbean will virus2, BBWV 2), pepper vein yellow virus (pepper vein yellows virus, peVYV), pepper light mottle virus (pepper mildmottle virus, PMMoV) and tobacco light green mosaic virus (tobacco mild green mosaic virus, TMGMV) of the same genus as PMMoV were used as templates, RT-RAA amplification was performed on the total RNA with the viruses by using the primer PMMoV primer set A, PMMoV primer set D screened in example 4, RT RAA amplification was completed for the total RNA of different virus plants, the amplified products were diluted 20 times and then subjected to test strip detection, and the detection sensitivity of different primer combinations, amplification system and test strip detection method were determined as described in example 4.
The pepper anthracnose is caused by various pathogens, different anthracnose including red anthracnose (colletotrichgloosporioides), siamesed basidiomycetes (Colletotrichum siamense), tea tree anthracnose (colletotrichum fructicola), flat-head anthracnose (Colletotrichum truncatum), colletotrichum glomerata (Colletotrichum scovillei) and colletotrichum clockii (Colletotrichum aenigma) standard substances are respectively used as amplification templates, the primer PMMoV primer set A, PMMoV primer set D, the anthracnose primer set E and the anthracnose primer set G screened in the embodiment 4 are used for respectively carrying out RT-RAA amplification on different total RNAs, RTRAA amplification on different total RNAs is completed, and test strip detection is carried out after 20 times dilution on the amplification products, so that the detection sensitivity of different primer combinations is determined, and the amplification system and the test strip detection mode are as described in the embodiment 4.
The specific evaluation results of the PMMoV primer set A RT RAA-LFS primer are shown in FIG. 12, and the specific evaluation results of the PMMoV primer set DRT RAA-LFS primer are shown in FIG. 13, so that the LFS detection results of the amplified products of the two primer sets only show positive in total RNA of PMMoV infected plants, and cross reaction with other viruses is not found, which indicates that the primer pair has good specificity and is suitable for RT RAA-LFS detection of PMMoV.
The specific evaluation result of the anthrax primer set E RT RAA-LFS primer is shown in figure 14, and the specific evaluation result of the anthrax primer set H RT RAA-LFS primer is shown in figure 15, so that the LFS detection results of the two primer amplification products of the anthrax show positive in the total RNA of infected plants in the detected pathogenic anthrax, and the primers are suitable for RTRAA-LFS detection of various pathogenic anthrax.
Experimental example 7: establishment of PMMoV and anthrax different primer sets dual RT-RAA-LFS detection system
The primers used in Experimental example 7 were synthesized by general biosystems (Anhui) Inc., and were of purity grade HPLC. The specific primer sequences, probes and amplified downstream primer labels are shown in Table 3 below:
TABLE 3 primer sequences in a double RT-RAA-LFS detection System
Respectively carrying out double RT RAA amplification of the primer group A and the primer group E2, the primer group A and the primer group H2, the primer group D and the primer group E2, and the primer group D and the primer group H2, wherein the amplification products are 1 multiplied by 10 corresponding to the upstream primer and the downstream primer of each primer group 6 Copy/. Mu.L plasmid dilution as template, useThe RT RAA nucleic acid amplification kit (test strip method) (JY 0204) is used for preparing a PMMoV and anthrax dual RT RAA reaction system (single sample/reaction) as follows: / >
The sample addition sequence was negative control samples (1X 10) 6 Copy/. Mu.L of recombinant plasmid solution was replaced with the same volume of ultrapure water), 1X 10 6 Copy/μl recombinant plasmid solution, and the tube cap is required to be closed immediately after each sample is added, avoiding aerosol contamination. And uniformly mixing the reaction systems, and adding the mixture into a basic reaction unit. The lyophilized powder was sufficiently dissolved, and note that this step was not possible to mix by vigorous shaking using a vortex shaker. Opening the reaction unit, in each0.2mL eppendorf tube was charged with 2.5. Mu.L of 280mM MgAc 2 Thoroughly mixed and collected by centrifugation. Note that this step cannot be mixed by vigorous shaking with a vortex shaker.
The reaction tube was placed at 39℃for 16min. After the RAA reaction is finished, the eppendorf tube is opened, the amplified product is sucked into a new eppendorf tube, marked and diluted by 20-50 times.
A structural schematic diagram of a double-target disposable nucleic acid detection test strip (JY 0209) is shown in FIG. 16, the end of an immersion area (marked with a blue arrow upwards) of the test strip is inserted into an eppendorf tube, the liquid level cannot exceed the MAX indication line of the immersion area, the area to be interpreted is fully immersed (about 30-60 sec is needed), the test strip is flatly placed for 1min, and a red strip is waited to appear. And directly reading the detection result according to the color development condition of the test strip. The results were observed within 10min, and the interpretation was not valid after 10 min.
The detection results of the double RT RAA-LFS of different primer plasmids and negative control are shown in FIG. 17, wherein the detection results of the test strips after the primer group A and the primer group E2, the primer group A and the primer group H2 negative control amplification products are diluted 20 times are positive, and the detection results cannot be used for detecting the leaf lysate of the subsequent double RT RAA-LFS; the detection results of the test strips after 20 times of dilution of the primer group D and the primer group E2 and the primer group D and the primer group H2 negative control amplification products are negative, and the detection results of the test strips after 20 times of dilution of the plasmid amplification products are positive, so that the method can be used for detecting the leaf lysate by the follow-up dual RTRAA-LFS.
Experimental example 8: drawing absolute quantitative standard curve of PMMoV and anthrax primer set D and primer set E2
Real-time qPCR was performed using the alignment plate in example 2, the sensitivity of the primers was verified using the PMMoV primer set D and the forward and reverse primers in the anthrax primer set E2, and absolute quantification was made of copy number.
As can be seen from FIG. 18, after qPCR, the melting curve analysis of PMMoV primer set D (PM-F2 and PM-R2, FIG. 18A) and anthrax primer set E2 (CT-F1 and CT-R1, FIG. 18B) were all sharp single peaks, indicating that the amplification specificity and the amplification efficiency of the PMMoV primer set D (PM-F2 and PM-R2) and the anthrax primer set E2 (CT-F1 and CT-R1) screened in this example were both high.
FIG. 19 shows that the above primer PMMoV primer set D (PM-F2 and PM-R2, FIG. 19A) and anthrax primer set E2 (CT-F1 and CT-R1, FIG. 19B) can successfully detect 10 1 ～10 6 The initial template of the copy, spanning a dynamic detection range of 6 log values, exhibited similar Cq differences between concentration gradients for each template, indicating a 10-fold difference in dilution gradient for each template.
PMMoV primer set D (PM-F2 and PM-R2) Cq values and log-transformed copy numbers (y= -4.349dx+37.412, R) 2 >0.998 Anthrax primer set E2 (CT-F1 and CT-R1) Cq values and log-transformed copy numbers (y= -4.5169x+39.047, R) 2 >0.973 The primers designed in the embodiment have good amplification specificity and sensitivity, and can be used for PMMoV and anthrax RT-PCR and RT-RAA analysis.
Experimental example 9: establishment of PMMoV and anthrax different primer sets plant material lysate dual RT RAA-LFS detection system
PMMoV positive (about 4.8X10) as identified by RT-qPCR 6 Individual virus copies/mg leaf), anthrax positive (about 4.8X10 6 Individual virus copies/mg leaf) plant leaf samples, 20mg plant leaf samples and 100. Mu.L were takenAnd (3) a cool flash nucleic acid releasing agent (BT 0068), rolling the material to be detected by a gun head for a plurality of times, centrifuging, and taking 2 mu L of blade lysate supernatant to be detected.
The primers of Experimental example 7 were used for double RT RAA amplification of primer set D and primer set E2, and primer set D and primer set H2, using plant leaf lysate supernatant as template, usingThe RT RAA nucleic acid amplification kit (test strip method) (JY 0204) is used for preparing a PMMoV and anthrax dual RT RAA reaction system (single sample/reaction) as follows:
the sample adding sequence is a negative quality control sample (the supernatant of the plant leaf lysate is replaced by ultrapure water with the same volume) and the supernatant of the plant leaf lysate, and a tube cover is required to be buckled immediately after each sample is added, so that aerosol pollution is avoided. And uniformly mixing the reaction systems, and adding the mixture into a basic reaction unit. The lyophilized powder was sufficiently dissolved, and note that this step was not possible to mix by vigorous shaking using a vortex shaker. The reaction unit was opened and 2.5. Mu.L of 280mM MgAc was added to each 0.2mL eppendorf tube 2 Thoroughly mixed and collected by centrifugation. Note that this step cannot be mixed by vigorous shaking with a vortex shaker.
The reaction tube was placed at 39℃for 16min. After the RAA reaction is finished, the eppendorf tube is opened, the amplified product is sucked into a new eppendorf tube, marked and diluted by 20-50 times.
The results of the detection conditions of the double RT RAA-LFS of different primer plasmids and negative control are shown in figure 20, wherein the detection results of PMMoV are positive after the primer group D and the primer group E2 negative control amplification products are diluted by 20 times; and the detection results of the test strip PMMoV and the anthrax bacteria after the primer group D and the primer group H2 negative control amplification products are diluted by 20 times are positive. Therefore, the primer group D and E2, the primer group D and the primer group H2 primer addition amount need to be optimized, and detection false positives caused by primer dimers in the negative control amplification result are reduced, so that the primer group D and the primer group H2 primer addition amount are used for RTRAA double amplification of plant leaf lysate.
Experimental example 10: optimization of PMMoV and anthrax plant material lysate dual RT-RAA-LFS detection system
The PMMoV primer set D, the anthrax primer set E2 and the anthrax primer set H2 in the example 7 are configured as mix, so as to be beneficial to optimizing the primer addition amount in the RTRAA double amplification system, and the primer mix comprises the following components:
plant leaf samples positive for PMMoV and anthrax identified by RT-PCR are taken to obtain 100 mu LAnd (3) a cool flash nucleic acid releasing agent (BT 0068), rolling the material to be detected for a plurality of times by using a gun head, centrifuging, and taking supernatant to be detected.
Double RT RAA amplification of primer set D and primer set E2, primer set D and primer set H2 was performed using the primers in Experimental example 7 to about 4.8X10 6 Plant leaf with individual virus copies/mg leaf was used as a test sample, 20mg plant leaf sample and 100. Mu.L were takenAnd (3) a cool flash nucleic acid releasing agent (BT 0068), rolling the material to be detected by a gun head for a plurality of times, centrifuging, and taking 2 mu L of blade lysate supernatant to be detected. Use->The RT RAA nucleic acid amplification kit (test strip method) (JY 0204) is used for preparing a PMMoV and anthrax dual RT RAA reaction system (single sample/reaction) as follows:
the sample adding sequence is a negative quality control sample (the supernatant of the plant leaf lysate is replaced by ultrapure water with the same volume) and the supernatant of the plant leaf lysate, and a tube cover is required to be buckled immediately after each sample is added, so that aerosol pollution is avoided. And uniformly mixing the reaction systems, and adding the mixture into a basic reaction unit. The lyophilized powder was sufficiently dissolved, and note that this step was not possible to mix by vigorous shaking using a vortex shaker. The reaction unit was opened and 2.5. Mu.L of 280mM MgAc was added to each 0.2mL eppendorf tube 2 Thoroughly mixed and collected by centrifugation. Note that this step cannot be mixed by vigorous shaking with a vortex shaker.
The reaction tube was placed at 39℃for 16min. After the RT RAA reaction is finished, the eppendorf tube is opened, the amplified product is sucked into a new eppendorf tube, marked and diluted by 20-50 times.
As shown in fig. 21, the detection result shows that when the primer set Mix in the 50 μl RT RAA amplification system is Mix 5 in combination with the RAA amplification primer of the primer set E2, the detection result of the test strip after 20-fold dilution of the amplification product of the negative control (the template is replaced by an equivalent volume of lysate) in the detection system is negative, and the detection result of the supernatant of the plant leaf lysate is positive for both PMMoV and anthrax.
As shown in fig. 22, when the primer set D is combined with the RAA amplification primer of the primer set H2 and the primer set Mix is added in the 50 μl RT RAA amplification system at Mix 4, the detection result of the test strip after the amplification product of the negative control (template is replaced by equivalent volume of lysate) in the detection system is diluted 20 times is negative, the detection result of the plant leaf lysate supernatant on PMMoV is positive, and the detection result of anthrax is negative; when the adding amount of the primer group Mix in the 50 mu L RT RAA amplification system is Mix 5, the detection result of a test strip after the amplification product of a negative control (the template is replaced by an equivalent-volume lysate) in the detection system is diluted by 20 times is negative, and the detection results of plant leaf lysate supernatant liquid on PMMoV and anthrax are both negative.
Therefore, a RT RAA double amplification primer combination of the primer group D and the primer group E2 is selected, the Mix addition amount of the primer group is set as Mix 5, and the subsequent sensitivity test of the supernatant of the plant lysate and the detection of a field sample are carried out.
Experimental example 10: dual RT RAA-LFS field rapid detection of pepper plant PMMoV and anthrax
RTRAA double amplification is carried out by using the primer group D, the primer group E2 sequence and the labeling mode determined in the experimental example 8, and the primer concentration is reduced so as to increase the primer adding volume and avoid influencing the detection effect due to high primer concentration and small adding volume. Randomly sampling in a field to perform field detection on pepper plants, taking 40mg of each sample, extracting total RNA from 20mg of the samples, performing RT qPCR analysis, measuring Ct value, and calculating the copy number of the samples; another 20mg sample was prepared as followsThe method provided by the cool flash nucleic acid releasing agent is used for completing the splitting of the leaf, fruit and seed samples of the capsicum, and carrying out RT-RAA amplification reaction, wherein the system is as follows:
| composition of the components | Volume of |
| Plant sample lysate or total RNA solution | 2μL |
| Base buffer | 29.4μL |
| PM-F2(5μM) | 0.29μL |
| PM-R2(5μM) | 0.4μL |
| PM-P3(1μM) | 0.4μL |
| CT-F1(5μM) | 0.9μL |
| CT-R1(5μM) | 1.26μL |
| CT-P1(1μM) | 1.26μL |
| ddH 2 O | To a total system of 47.5. Mu.L |
The reaction conditions for the RAA amplification were set as follows: after completion of the preparation of the reaction solution, 2.5. Mu.L of 280mM MgAc was added to each of the 0.2mL eppendorf tubes 2 Fully and uniformly mixing; the eppendorf tube (0.2 mL) was placed on a PCR apparatus with a thermal cover function, and incubated at 39℃for 16min. After the RTRAA reaction is completed, the eppendorf tube is opened, the amplified product is sucked into a new eppendorf tube, marked and diluted 20 times.
The results of RT RAA-LFS detection and RT-qPCR on different sampling positions of 4 samples obtained in the field are shown in the following Table 4:
the RTRAA-LFS detection result diagram of each sample is shown in figure 23, which shows that the system can realize the field rapid detection of PMMoV and anthrax by means of the rapid lysis of the samples.
Experimental example 11: copy number and detection limit analysis of PMMoV, anthrax primer set D and primer set E2 absolute quantitative actual measurement samples
Dividing 50mg of pepper plant leaves with PMMoV infection symptoms into two parts according to a plasmid absolute quantitative standard curve obtained by the comparison disc, extracting total RNA from one part of pepper plant leaves, carrying out gradient dilution after transcription, completing qPCR analysis, and calculating the copy number of a sample; another equivalent sample was prepared according toThe method provided by the cool flash nucleic acid releasing agent completes the splitting of the capsicum leaf and seed sample, and after the capsicum leaf and seed sample splitting solution is combined with the flat head anthrax sample splitting solution,the RT-RAA double amplification reaction was completed according to the system shown in Experimental example 10.
Scraping 20mg of the colletotrichum gloeosporioides mycelium sample on a separation inoculation tray, extracting 10mg of total RNA, carrying out gradient dilution after transcription, completing qPCR analysis, and calculating the copy number of the sample; another equivalent sample was prepared according toThe method provided by the cool flash nucleic acid releasing agent is used for completing the cleavage of the mycelium sample, combining the mycelium sample with the PMMoV-infected pepper sample lysate, and completing the RT RAA double amplification reaction according to the system shown in the experimental example 10. After the reaction was completed, the reaction solution was diluted 20 times, LFS detection was performed, the detection results are shown in fig. 24, and the RT qPCR detection results are shown in table 5 below, showing that 94 copies of PMMoV and 41 copies of anthrax bacteria were detected at the minimum for each 50 μl of the established RT-RAA reaction system. />
TABLE 5 detection limits for the detection of PMMoV and anthrax by RT RAA-LFS of the present application
In conclusion, the primer group capable of simultaneously detecting PMMoV and anthrax is screened through a series of verification, the primer group can effectively amplify target genes, has high specificity and sensitivity, has no cross reaction with other viruses, can be used for on-site rapid detection of plant PMMoV and anthrax, and has important significance for effectively preventing and treating diseases.
The application shown and described herein may be practiced without any of the elements, limitations specifically disclosed herein. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, and it is recognized that various modifications are possible within the scope of the application. It is therefore to be understood that while the present application has been specifically disclosed by various embodiments and optional features, modification and variation of the concepts herein described may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this application as defined by the appended claims.
The contents of the articles, patents, patent applications, and all other documents and electronically available information described or documented herein are incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. Applicants reserve the right to incorporate any and all materials and information from any such articles, patents, patent applications, or other documents.
Claims (9)
1. The RT-RAA detection primer group for detecting pepper mild mottle virus and anthrax is characterized by comprising pepper mild mottle virus detection primers, anthrax detection primers, pepper mild mottle virus detection probes PM-P3 and anthrax detection probes CT-P1; wherein,,
the primer for detecting the pepper light mottle virus comprises a forward primer PM-F2 and a reverse primer PM-R2,
the sequence of the forward primer PM-F2 is 5 '-CTGTGTACTTCGGCGTTAGGCAATCAG-3',
the sequence of the reverse primer PM-R2 is 5 '-CATTCATGAGGTTACTTATACTGGCCC-3';
the anthrax detection primer comprises a forward primer CT-F1 and a reverse primer CT-R1,
the forward primer CT-F1 has the sequence of 5 '-GTGAGATCCGTCGCTACCAGAAG-3',
the sequence of the reverse primer CT-R1 is 5 '-GCATAGGTTGGTGTCCTCAAAGAG-3';
The pepper light mottle virus detection probe PM-P3,
its sequence is 5' -GCACTTCTCGGAGCCTTTGATACTAGGHACAGGA
TAATAGAAGTT-3´；
The anthrax detection probe CT-P1 has a sequence of 5' -CTTCAAGTCCGA
TCTCCGCTTCCAGTCHTCCGCCATCGGTGCCCTTC-3´；
FITC is modified at the 5 ' -end of a reverse primer PM-R2 of the pepper light mottle virus, biotin is modified at the 5 ' -end of a detection probe PM-P3, phosphorylation modification is performed at the 3 ' -end of the PM-P3, and H is tetrahydrofuran; digoxin is modified at the 5 ' -end of the anthrax reverse primer CT-R1, rhodamine is modified at the 5 ' -end of the probe CT-P1, phosphorylation modification is performed at the 3 ' -end of the probe CT-P1, and H is tetrahydrofuran.
2. A kit for detecting pepper mild mottle virus and anthrax, comprising the primer set of claim 1.
3. The use of the primer set according to claim 1 for the non-disease diagnosis purpose in a kit for detecting pepper mild mottle virus and/or anthrax in a biological sample to be detected or for preparing a kit for detecting whether pepper mild mottle virus and/or anthrax is contained in a biological sample to be detected.
4. Use of a kit according to claim 2 for the purpose of non-disease diagnosis in the detection of the presence or absence of pepper mild mottle virus and/or anthrax in a biological sample to be tested.
5. A method for detecting the purpose of non-disease diagnosis of pepper mild mottle virus and/or anthrax using a kit, comprising the steps of:
1) Extracting total RNA of a biological sample to be detected, or carrying out cracking treatment on a plant sample;
2) Using the total RNA or plant lysate extracted in the step 1) as a template, and adopting the forward primer and the reverse primer in the primer group of claim 1 to carry out RT-RAA amplification to obtain an amplification product;
3) Detecting the amplified product, and judging whether the pepper light mottle virus and/or the anthrax are contained or not based on the detection result of the amplified product.
6. The method of claim 5, wherein if the amplified product contains a 316bp DNA fragment, the biological sample to be tested contains the pepper light mottle virus, and if the amplified product does not contain a 316bp DNA fragment, the biological sample to be tested does not contain the pepper light mottle virus; if the amplified product contains a DNA fragment of 243bp, the biological sample to be detected contains anthrax, and if the amplified product does not contain a DNA fragment of 243bp, the biological sample to be detected does not contain anthrax.
7. A method for detecting whether a biological sample to be tested contains pepper mild mottle virus and/or anthrax for the purpose of non-disease diagnosis, comprising:
Detecting whether a lysate or a total RNA amplification product of the biological sample to be detected contains specific DNA fragments, if so, the biological sample to be detected contains pepper light mottle virus and/or anthrax, and if not, the biological sample to be detected does not contain pepper light mottle virus and/or anthrax; wherein the specific DNA fragment is the target sequence of the forward primer and the reverse primer of claim 1.
8. A method for detecting the non-disease diagnostic purpose of pepper light mottle virus and/or anthrax based on RT-RAA-lateral flow chromatography technology, characterized by comprising the steps of:
1) Carrying out cracking treatment on the sample, or extracting total RNA of the biological sample to be detected;
2) Using the sample lysate or the extracted total RNA obtained in the step 1) as a template, and carrying out RT-RAA amplification by using the primer set of claim 1;
3) Detecting an amplification product by using a lateral flow chromatography test strip, and if the test strip only has one strip in a quality control area and the detection area has no strip, determining that the result is negative, and indicating that the sample does not contain pepper light mottle virus and anthrax; if two strips appear on the test strip, one strip is positioned in the quality control area, and the other strip is positioned in the detection line T1 or the detection line T2, the result is positive, and the sample contains anthrax bacteria or PMMoV; if three strips appear on the test strip, one strip is positioned in the quality control area, one strip is positioned in the detection area T1, and one strip is positioned in the detection area T2, the result is positive, and the sample contains pepper light mottle virus and anthrax; if the quality control line of the test strip does not develop color, the test is invalid, and the test strip needs to be replaced for re-detection.
9. The kit for detecting pepper light mottle virus and anthracnose is characterized by comprising the primer set as claimed in claim 1, and a lateral flow chromatography test strip, wherein the lateral flow chromatography test strip is provided with a detection line T1, a detection line T2 and a quality control area, and the lateral flow chromatography test strip can detect amplification products of the primer set.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210596142.3A CN114959116B (en) | 2022-05-30 | 2022-05-30 | Primer group, kit and method for detecting pepper light mottle virus and anthrax |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210596142.3A CN114959116B (en) | 2022-05-30 | 2022-05-30 | Primer group, kit and method for detecting pepper light mottle virus and anthrax |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114959116A CN114959116A (en) | 2022-08-30 |
| CN114959116B true CN114959116B (en) | 2023-09-01 |
Family
ID=82957065
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210596142.3A Active CN114959116B (en) | 2022-05-30 | 2022-05-30 | Primer group, kit and method for detecting pepper light mottle virus and anthrax |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114959116B (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104830984A (en) * | 2015-05-01 | 2015-08-12 | 新疆出入境检验检疫局检验检疫技术中心 | Fluorescence PCR detection method of Colletetrichum orbicalare, and primers and probes used for method |
| CN110283938A (en) * | 2019-06-20 | 2019-09-27 | 华南农业大学 | A kind of dual RT-RAA detection primer group, kit and the application of PEDV and PDCoV |
| CN111057796A (en) * | 2020-01-18 | 2020-04-24 | 广西壮族自治区农业科学院 | PCR primer group and kit for detecting pepper mild mottle virus and application thereof |
| CN113151601A (en) * | 2021-04-26 | 2021-07-23 | 山东农业大学 | Primer and probe combination for RAA-LFD detection of chicken Marek's virus wild strain and SC9-1 vaccine strain and application thereof |
| CN113736897A (en) * | 2021-09-18 | 2021-12-03 | 上海海洋大学 | Primer group, kit and method for detecting vibrio parahaemolyticus and vibrio cholerae based on dual RAA-LFD technology |
-
2022
- 2022-05-30 CN CN202210596142.3A patent/CN114959116B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104830984A (en) * | 2015-05-01 | 2015-08-12 | 新疆出入境检验检疫局检验检疫技术中心 | Fluorescence PCR detection method of Colletetrichum orbicalare, and primers and probes used for method |
| CN110283938A (en) * | 2019-06-20 | 2019-09-27 | 华南农业大学 | A kind of dual RT-RAA detection primer group, kit and the application of PEDV and PDCoV |
| CN111057796A (en) * | 2020-01-18 | 2020-04-24 | 广西壮族自治区农业科学院 | PCR primer group and kit for detecting pepper mild mottle virus and application thereof |
| CN113151601A (en) * | 2021-04-26 | 2021-07-23 | 山东农业大学 | Primer and probe combination for RAA-LFD detection of chicken Marek's virus wild strain and SC9-1 vaccine strain and application thereof |
| CN113736897A (en) * | 2021-09-18 | 2021-12-03 | 上海海洋大学 | Primer group, kit and method for detecting vibrio parahaemolyticus and vibrio cholerae based on dual RAA-LFD technology |
Non-Patent Citations (1)
| Title |
|---|
| 黑龙江省高粱炭疽病病原菌鉴定及品种抗性机制分析;杨霞;《中国优秀硕士学位论文全文数据库 农业科技辑》;摘要部分，第2.3.1.2部分及表1 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114959116A (en) | 2022-08-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Jarraud et al. | Identification of Legionella in clinical samples | |
| CN115852052A (en) | Real-time fluorescent quantitative PCR primer probe combination for detecting CymRSV and method thereof | |
| CN110628944A (en) | Duck type-3 adenovirus and duck novel reovirus differential diagnosis kit | |
| CN108624713B (en) | Method and kit for identifying and detecting porcine pseudorabies vaccine virus and wild virus | |
| WO2024078420A1 (en) | Isothermal nucleic acid amplification raa primer probe combination for detecting candida auris and use thereof | |
| CN114959116B (en) | Primer group, kit and method for detecting pepper light mottle virus and anthrax | |
| KR20190037027A (en) | Primer set for detection of SFTSV and SFTSV detection method using the same | |
| CN116814857A (en) | Cat parvovirus and kit thereof and fluorescent recombinase polymerase amplification method | |
| CN110964848A (en) | RAA amplification primer and probe for rapidly detecting carp herpesvirus II, detection kit and use method | |
| CN106521038B (en) | A kind of real-time fluorescence quantitative PCR detection methods of highly sensitive BHV 2 and kit | |
| JP5999817B2 (en) | Exogenous internal positive control | |
| CN116004909A (en) | Multiplex fluorescence PCR detection primer and probe for monkey pox virus, and detection method and application thereof | |
| CN112176080B (en) | Nested PCR primer group, kit and detection method for specifically detecting purple sisal leaf roll disease phytoplasma | |
| CN116287427A (en) | Primer and probe combination for detecting duck tembusu virus by RAA-LFD and application thereof | |
| CN110257560B (en) | Reagent for bluetongue virus type 8 detection, detection method and application | |
| KR101493910B1 (en) | Primer set for detecting strawberry latent ringspot virus and use thereof | |
| CN117165719A (en) | Primer group, kit and method for detecting tomato brown wrinkle fruit virus and tobacco mosaic virus | |
| CN107604101A (en) | One breeding pigeon New-type adenovirus real-time fluorescence quantitative PCR detection kit | |
| Youssef et al. | Detection and identification of Apple stem pitting virus and Apple stem grooving virus affecting apple and pear trees in Egypt | |
| CN111118213B (en) | Primer group, kit and method for detecting astragalus sinicus dwarf virus based on PCR and RPA | |
| CN112575122B (en) | Dual PCR primer set for rapidly detecting duck type 2 adenovirus and duck circovirus, and detection method and application thereof | |
| CN113322350B (en) | Kit for simultaneously detecting porcine circovirus type 2 and porcine circovirus type 3 viruses and application | |
| KR101434832B1 (en) | Primers of polymerase chain reactions for the detection of Phytophthora species broken out on kind of fruit tree or seedling, and detection kits and methods thereof | |
| CN114540525B (en) | Primer composition for detecting or assisting in detecting chlamydia psittaci and application thereof | |
| CN116411136B (en) | Primer probe combination and kit for simultaneously detecting bovine norovirus and bovine rotavirus and application of primer probe combination and kit |
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 |