CN114958835A - Combination product and kit for detecting bacterial rice blight bacteria - Google Patents
Combination product and kit for detecting bacterial rice blight bacteria Download PDFInfo
- Publication number
- CN114958835A CN114958835A CN202210578688.6A CN202210578688A CN114958835A CN 114958835 A CN114958835 A CN 114958835A CN 202210578688 A CN202210578688 A CN 202210578688A CN 114958835 A CN114958835 A CN 114958835A
- Authority
- CN
- China
- Prior art keywords
- kit
- combination product
- nucleic acid
- recombinase
- bacterial
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 239000013066 combination product Substances 0.000 title claims abstract description 17
- 229940127555 combination product Drugs 0.000 title claims abstract description 17
- 230000001580 bacterial effect Effects 0.000 title abstract description 57
- 235000007164 Oryza sativa Nutrition 0.000 title abstract description 31
- 235000009566 rice Nutrition 0.000 title abstract description 31
- 240000007594 Oryza sativa Species 0.000 title abstract 2
- 241000894006 Bacteria Species 0.000 title description 14
- 230000003321 amplification Effects 0.000 claims abstract description 29
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 29
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 23
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 22
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 22
- 239000000523 sample Substances 0.000 claims abstract description 22
- 238000001514 detection method Methods 0.000 claims abstract description 19
- 238000011144 upstream manufacturing Methods 0.000 claims abstract description 7
- 238000006243 chemical reaction Methods 0.000 claims description 20
- 239000003153 chemical reaction reagent Substances 0.000 claims description 17
- 102000018120 Recombinases Human genes 0.000 claims description 16
- 108010091086 Recombinases Proteins 0.000 claims description 16
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 16
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 claims description 14
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 claims description 14
- 239000007788 liquid Substances 0.000 claims description 13
- 125000003729 nucleotide group Chemical group 0.000 claims description 10
- DRBBFCLWYRJSJZ-UHFFFAOYSA-N N-phosphocreatine Chemical compound OC(=O)CN(C)C(=N)NP(O)(O)=O DRBBFCLWYRJSJZ-UHFFFAOYSA-N 0.000 claims description 8
- 239000002981 blocking agent Substances 0.000 claims description 7
- 238000005516 engineering process Methods 0.000 claims description 6
- 102000004169 proteins and genes Human genes 0.000 claims description 6
- 108090000623 proteins and genes Proteins 0.000 claims description 6
- 208000035657 Abasia Diseases 0.000 claims description 5
- 230000008929 regeneration Effects 0.000 claims description 5
- 238000011069 regeneration method Methods 0.000 claims description 5
- 102000052510 DNA-Binding Proteins Human genes 0.000 claims description 4
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 4
- 229950007002 phosphocreatine Drugs 0.000 claims description 4
- 239000000843 powder Substances 0.000 claims description 4
- 102000004420 Creatine Kinase Human genes 0.000 claims description 3
- 108010042126 Creatine kinase Proteins 0.000 claims description 3
- 101710116602 DNA-Binding protein G5P Proteins 0.000 claims description 3
- JLVVSXFLKOJNIY-UHFFFAOYSA-N Magnesium ion Chemical compound [Mg+2] JLVVSXFLKOJNIY-UHFFFAOYSA-N 0.000 claims description 3
- 101710162453 Replication factor A Proteins 0.000 claims description 3
- 101710176758 Replication protein A 70 kDa DNA-binding subunit Proteins 0.000 claims description 3
- 101710176276 SSB protein Proteins 0.000 claims description 3
- 101710126859 Single-stranded DNA-binding protein Proteins 0.000 claims description 3
- 229910001425 magnesium ion Inorganic materials 0.000 claims description 3
- 239000013642 negative control Substances 0.000 claims description 3
- 238000010791 quenching Methods 0.000 claims description 3
- 230000000171 quenching effect Effects 0.000 claims description 3
- 102000002281 Adenylate kinase Human genes 0.000 claims description 2
- 108020000543 Adenylate kinase Proteins 0.000 claims description 2
- 101100048967 Enterobacteria phage T4 uvsY gene Proteins 0.000 claims description 2
- 102100034343 Integrase Human genes 0.000 claims description 2
- 102000009609 Pyrophosphatases Human genes 0.000 claims description 2
- 108010009413 Pyrophosphatases Proteins 0.000 claims description 2
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 claims description 2
- 102000001218 Rec A Recombinases Human genes 0.000 claims description 2
- 108010055016 Rec A Recombinases Proteins 0.000 claims description 2
- 229930006000 Sucrose Natural products 0.000 claims description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 2
- 108020000005 Sucrose phosphorylase Proteins 0.000 claims description 2
- 230000027455 binding Effects 0.000 claims description 2
- 238000010494 dissociation reaction Methods 0.000 claims description 2
- 230000005593 dissociations Effects 0.000 claims description 2
- 238000009826 distribution Methods 0.000 claims description 2
- 108010052305 exodeoxyribonuclease III Proteins 0.000 claims description 2
- 238000000605 extraction Methods 0.000 claims description 2
- 230000002349 favourable effect Effects 0.000 claims description 2
- NZBSAAMEZYOGBA-UHFFFAOYSA-N luminogren Chemical compound C12=NC3=CC=CC=C3N2C(=O)C2=CC=CC3=CC=CC1=C23 NZBSAAMEZYOGBA-UHFFFAOYSA-N 0.000 claims description 2
- 239000003002 pH adjusting agent Substances 0.000 claims description 2
- 239000013641 positive control Substances 0.000 claims description 2
- 230000035484 reaction time Effects 0.000 claims description 2
- 238000005215 recombination Methods 0.000 claims description 2
- 230000006798 recombination Effects 0.000 claims description 2
- 230000002441 reversible effect Effects 0.000 claims description 2
- 239000005720 sucrose Substances 0.000 claims description 2
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 claims description 2
- 238000003556 assay Methods 0.000 claims 1
- 150000002500 ions Chemical class 0.000 claims 1
- 230000035945 sensitivity Effects 0.000 abstract description 7
- 239000000047 product Substances 0.000 abstract 1
- 241000209094 Oryza Species 0.000 description 30
- 238000000034 method Methods 0.000 description 20
- 108020004414 DNA Proteins 0.000 description 16
- 125000006850 spacer group Chemical group 0.000 description 13
- 238000012360 testing method Methods 0.000 description 12
- 239000008223 sterile water Substances 0.000 description 11
- 238000002474 experimental method Methods 0.000 description 9
- 238000012986 modification Methods 0.000 description 8
- 230000004048 modification Effects 0.000 description 8
- 239000000243 solution Substances 0.000 description 7
- 241001272684 Xanthomonas campestris pv. oryzae Species 0.000 description 6
- 239000002773 nucleotide Substances 0.000 description 5
- 238000003752 polymerase chain reaction Methods 0.000 description 5
- 238000000926 separation method Methods 0.000 description 5
- SUYVUBYJARFZHO-RRKCRQDMSA-N dATP Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-RRKCRQDMSA-N 0.000 description 4
- SUYVUBYJARFZHO-UHFFFAOYSA-N dATP Natural products C1=NC=2C(N)=NC=NC=2N1C1CC(O)C(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-UHFFFAOYSA-N 0.000 description 4
- 239000006916 nutrient agar Substances 0.000 description 4
- -1 Aqua Phluor593 Chemical compound 0.000 description 3
- 241001453380 Burkholderia Species 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 244000052616 bacterial pathogen Species 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 235000018102 proteins Nutrition 0.000 description 3
- SGTNSNPWRIOYBX-UHFFFAOYSA-N 2-(3,4-dimethoxyphenyl)-5-{[2-(3,4-dimethoxyphenyl)ethyl](methyl)amino}-2-(propan-2-yl)pentanenitrile Chemical compound C1=C(OC)C(OC)=CC=C1CCN(C)CCCC(C#N)(C(C)C)C1=CC=C(OC)C(OC)=C1 SGTNSNPWRIOYBX-UHFFFAOYSA-N 0.000 description 2
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 2
- 238000007400 DNA extraction Methods 0.000 description 2
- 238000007399 DNA isolation Methods 0.000 description 2
- 230000006820 DNA synthesis Effects 0.000 description 2
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 2
- 108091034117 Oligonucleotide Proteins 0.000 description 2
- 238000011529 RT qPCR Methods 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 239000007983 Tris buffer Substances 0.000 description 2
- 150000001412 amines Chemical class 0.000 description 2
- 239000011324 bead Substances 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 235000013339 cereals Nutrition 0.000 description 2
- 239000012295 chemical reaction liquid Substances 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- DAEAPNUQQAICNR-RRKCRQDMSA-K dADP(3-) Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@H]1C[C@H](O)[C@@H](COP([O-])(=O)OP([O-])([O-])=O)O1 DAEAPNUQQAICNR-RRKCRQDMSA-K 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000006073 displacement reaction Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 235000013922 glutamic acid Nutrition 0.000 description 2
- 239000004220 glutamic acid Substances 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 238000011901 isothermal amplification Methods 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 241000894007 species Species 0.000 description 2
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- KHQCCEUMCUSZKM-KVQBGUIXSA-N (2r,3s,5r)-5-(6,8-diaminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-ol Chemical compound NC1=NC2=C(N)N=CN=C2N1[C@H]1C[C@H](O)[C@@H](CO)O1 KHQCCEUMCUSZKM-KVQBGUIXSA-N 0.000 description 1
- CFBILACNYSPRPM-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]acetic acid Chemical group OCC(N)(CO)CO.OCC(CO)(CO)NCC(O)=O CFBILACNYSPRPM-UHFFFAOYSA-N 0.000 description 1
- MWBWWFOAEOYUST-UHFFFAOYSA-N 2-aminopurine Chemical compound NC1=NC=C2N=CNC2=N1 MWBWWFOAEOYUST-UHFFFAOYSA-N 0.000 description 1
- UDGUGZTYGWUUSG-UHFFFAOYSA-N 4-[4-[[2,5-dimethoxy-4-[(4-nitrophenyl)diazenyl]phenyl]diazenyl]-n-methylanilino]butanoic acid Chemical compound COC=1C=C(N=NC=2C=CC(=CC=2)N(C)CCCC(O)=O)C(OC)=CC=1N=NC1=CC=C([N+]([O-])=O)C=C1 UDGUGZTYGWUUSG-UHFFFAOYSA-N 0.000 description 1
- WCKQPPQRFNHPRJ-UHFFFAOYSA-N 4-[[4-(dimethylamino)phenyl]diazenyl]benzoic acid Chemical compound C1=CC(N(C)C)=CC=C1N=NC1=CC=C(C(O)=O)C=C1 WCKQPPQRFNHPRJ-UHFFFAOYSA-N 0.000 description 1
- XSGZNYKZSOJIAM-XUOJEKSQSA-N 4-amino-1-[(2r,4s,5r)-4-hydroxy-5-(hydroxymethyl)oxolan-2-yl]pyrimidin-2-one;2-(10h-phenoxazin-1-yl)ethanamine Chemical compound O=C1N=C(N)C=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1.O1C2=CC=CC=C2NC2=C1C=CC=C2CCN XSGZNYKZSOJIAM-XUOJEKSQSA-N 0.000 description 1
- UHPMCKVQTMMPCG-UHFFFAOYSA-N 5,8-dihydroxy-2-methoxy-6-methyl-7-(2-oxopropyl)naphthalene-1,4-dione Chemical compound CC1=C(CC(C)=O)C(O)=C2C(=O)C(OC)=CC(=O)C2=C1O UHPMCKVQTMMPCG-UHFFFAOYSA-N 0.000 description 1
- OZFPSOBLQZPIAV-UHFFFAOYSA-N 5-nitro-1h-indole Chemical compound [O-][N+](=O)C1=CC=C2NC=CC2=C1 OZFPSOBLQZPIAV-UHFFFAOYSA-N 0.000 description 1
- MSSXOMSJDRHRMC-UHFFFAOYSA-N 9H-purine-2,6-diamine Chemical compound NC1=NC(N)=C2NC=NC2=N1 MSSXOMSJDRHRMC-UHFFFAOYSA-N 0.000 description 1
- WNDDWSAHNYBXKY-UHFFFAOYSA-N ATTO 425-2 Chemical compound CC1CC(C)(C)N(CCCC(O)=O)C2=C1C=C1C=C(C(=O)OCC)C(=O)OC1=C2 WNDDWSAHNYBXKY-UHFFFAOYSA-N 0.000 description 1
- PWZJEXGKUHVUFP-UHFFFAOYSA-N ATTO 590 meta-isomer Chemical compound [O-]Cl(=O)(=O)=O.C1=2C=C3C(C)=CC(C)(C)N(CC)C3=CC=2OC2=CC3=[N+](CC)C(C)(C)C=C(C)C3=CC2=C1C1=CC=C(C(O)=O)C=C1C(O)=O PWZJEXGKUHVUFP-UHFFFAOYSA-N 0.000 description 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 239000012103 Alexa Fluor 488 Substances 0.000 description 1
- 108091093088 Amplicon Proteins 0.000 description 1
- 244000063299 Bacillus subtilis Species 0.000 description 1
- 235000014469 Bacillus subtilis Nutrition 0.000 description 1
- 108091026890 Coding region Proteins 0.000 description 1
- HMFHBZSHGGEWLO-SOOFDHNKSA-N D-ribofuranose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H]1O HMFHBZSHGGEWLO-SOOFDHNKSA-N 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 108010017826 DNA Polymerase I Proteins 0.000 description 1
- 102000004594 DNA Polymerase I Human genes 0.000 description 1
- 230000004543 DNA replication Effects 0.000 description 1
- 108700020911 DNA-Binding Proteins Proteins 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241000223218 Fusarium Species 0.000 description 1
- 108010014594 Heterogeneous Nuclear Ribonucleoprotein A1 Proteins 0.000 description 1
- 206010061217 Infestation Diseases 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 240000002582 Oryza sativa Indica Group Species 0.000 description 1
- 240000008467 Oryza sativa Japonica Group Species 0.000 description 1
- 244000194445 Oryzopsis hymenoides Species 0.000 description 1
- 235000000884 Oryzopsis hymenoides Nutrition 0.000 description 1
- PYMYPHUHKUWMLA-LMVFSUKVSA-N Ribose Natural products OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 description 1
- 241000191967 Staphylococcus aureus Species 0.000 description 1
- 108010006785 Taq Polymerase Proteins 0.000 description 1
- GYDJEQRTZSCIOI-UHFFFAOYSA-N Tranexamic acid Chemical compound NCC1CCC(C(O)=O)CC1 GYDJEQRTZSCIOI-UHFFFAOYSA-N 0.000 description 1
- 241000589634 Xanthomonas Species 0.000 description 1
- JFVJZFMWJVSZNC-KVQBGUIXSA-N [[(2r,3s,5r)-5-(2,6-diaminopurin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl] phosphono hydrogen phosphate Chemical compound C12=NC(N)=NC(N)=C2N=CN1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 JFVJZFMWJVSZNC-KVQBGUIXSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 125000003275 alpha amino acid group Chemical group 0.000 description 1
- HMFHBZSHGGEWLO-UHFFFAOYSA-N alpha-D-Furanose-Ribose Natural products OCC1OC(O)C(O)C1O HMFHBZSHGGEWLO-UHFFFAOYSA-N 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 238000012197 amplification kit Methods 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000000429 assembly Methods 0.000 description 1
- 230000000712 assembly Effects 0.000 description 1
- 238000004166 bioassay Methods 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000001917 fluorescence detection Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 125000004029 hydroxymethyl group Chemical group [H]OC([H])([H])* 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 239000008176 lyophilized powder Substances 0.000 description 1
- 210000001161 mammalian embryo Anatomy 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000003278 mimic effect Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- VYNDHICBIRRPFP-UHFFFAOYSA-N pacific blue Chemical compound FC1=C(O)C(F)=C2OC(=O)C(C(=O)O)=CC2=C1 VYNDHICBIRRPFP-UHFFFAOYSA-N 0.000 description 1
- 238000005191 phase separation Methods 0.000 description 1
- 244000000003 plant pathogen Species 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 210000001938 protoplast Anatomy 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 239000011535 reaction buffer Substances 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000010845 search algorithm Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 230000000405 serological effect Effects 0.000 description 1
- 238000004904 shortening Methods 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 108700014590 single-stranded DNA binding proteins Proteins 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 108010068698 spleen exonuclease Proteins 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000000153 supplemental effect Effects 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- ABZLKHKQJHEPAX-UHFFFAOYSA-N tetramethylrhodamine Chemical compound C=12C=CC(N(C)C)=CC2=[O+]C2=CC(N(C)C)=CC=C2C=1C1=CC=CC=C1C([O-])=O ABZLKHKQJHEPAX-UHFFFAOYSA-N 0.000 description 1
- MPLHNVLQVRSVEE-UHFFFAOYSA-N texas red Chemical compound [O-]S(=O)(=O)C1=CC(S(Cl)(=O)=O)=CC=C1C(C1=CC=2CCCN3CCCC(C=23)=C1O1)=C2C1=C(CCC1)C3=[N+]1CCCC3=C2 MPLHNVLQVRSVEE-UHFFFAOYSA-N 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000009966 trimming Methods 0.000 description 1
- 238000011179 visual inspection Methods 0.000 description 1
- 238000010792 warming Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to the technical field of molecular biology, in particular to a combination product and a kit for detecting bacterial rice blight germ. The combination product comprises an upstream primer, a downstream primer and a probe, and can be matched with an isothermal nucleic acid amplification system for use. The combined product and the kit provided by the invention have the advantages of high detection sensitivity and specificity, convenience in detection and short time consumption.
Description
Technical Field
The invention relates to the technical field of molecular biology, in particular to a combination product and a kit for detecting bacterial rice blight germ.
Background
Bacterial blight of rice, a gram-negative bacterium of the genus xanthomonas, is a pathogenic bacterium causing bacterial blight of rice (BPBR), which was first found in japan and currently spread to parts of south east asia, america, africa, (CABI). Bacterial rice blight bacteria are firstly detected in asymptomatic grains in China in 2007 (J.Luo et.al.,2007), and are firstly detected in symptomatic diseased leaves in southern paddy fields in 2020 (Y.Hou et.al.,2020), and the diseases are sporadically distributed in China at present. Bacterial blight can result in up to 75% yield loss in field inoculation trials; one U.S. study demonstrated that as global warming progresses, the area of bacterial blight infestation increases, resulting in increased yield loss, and that at 3 ℃ elevated temperatures, bacterial blight of rice is expected to cause an additional $ 2.04 million per year loss (Shew AM et al, 2019). The rice bacterial blight seriously threatens the grain safety, so the rice bacterial blight is listed as an imported plant quarantine harmful organism in China, and the rapid and accurate quarantine detection technology can effectively prevent the propagation of the rice bacterial blight and ensure the safe production of the rice.
At present, the detection of the bacterial rice blight germ is mainly a traditional bioassay, a Polymerase Chain Reaction (PCR) method real-time fluorescence PCR (qRT-PCR) method and a serological method of direct separation and molecular detection, but the PCR and ELISA methods have limited sensitivity and long time consumption, and qRT-PCR depends on expensive detection instruments and is sensitive to inhibition factors and pollution in the environment and easy to have false positive or false negative.
The Recombinase Polymerase Amplification (RPA) technique is a novel multi-enzyme isothermal nucleic acid amplification technique proposed in 2006, and can be used for rapid and convenient nucleic acid detection and analysis (pineburg et al, 2006). Under the constant temperature condition of 25-43 ℃, the RPA technology can realize the amplification of specific nucleic acid sequences within 5-20min and observe the result. The improved RPA technique, called the multi-enzyme isothermal nucleic acid rapid amplification technique (MIRA), used in this experiment has better stability and higher fluorescence at the same time (Hui Chenet al, 2021).
Disclosure of Invention
A first object of the present invention is to provide a combination product comprising:
an upstream primer: TCGCTCTCCCGAGGGAGATGACAGCCGCTACA, respectively;
a downstream primer: ACACGGAACACCTGGGTAGTCTCTGTAGGGAAG, respectively; and
and (3) probe: CCATCTCAAATAAGCGCTTCCGCTATCCACTXTTACTACTTCCAGAT;
wherein the probe is based on recombinase polymerase amplification technology, a 31 st base T marks a luminescent group, a 32 nd base X is an abasic nucleotide analogue, a 33 rd base T marks a quenching group, and a 3' end is modified by a blocking agent; the blocking agent is used to block polymerase extension of the probe.
It is a second object of the invention to provide a kit comprising a combination product as described above.
The third purpose of the invention is to provide the combination product as described above or the application of the kit as described above in detecting rice bacterial blight germ.
The combination product and the kit provided by the invention are used for detecting the bacterial rice blight germ, and have the advantages of higher detection sensitivity and specificity, convenience in detection and shorter time consumption.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments or the prior art descriptions will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIGS. 1 to 6 show the results of the detection of different plant pathogens of the genus Burkholderia according to an embodiment of the present invention;
FIG. 1: a is strain 2; b, strain 5; c, strain 8; d, strain 7; e, strain 6; f, strain 1; g, strain 3; h, strain 4;
FIG. 2: a is strain 9; b, strain 10; c, sterile water control;
FIG. 3: a is strain 2; B-G is strain 11-16; h, sterile water control;
FIG. 4: a is strain 2; B-G is strain 17-22; h, sterile water control;
FIG. 5: a is strain 2; b, strain 23; C-G is 24-28 of strain; h, sterile water control;
FIG. 6: a is strain 2; B-G is strain 29-343; h, sterile water control;
FIG. 7 shows the detection results of bacterial liquid of bacterial blight of rice at different concentrations according to one embodiment of the present invention;
a is 2.28X 10 8 CFU/ml bacterial blight strain 2; b with a concentration of 2.28X 10 7 CFU/ml bacterial blight strain 2; c with a concentration of 2.28X 10 6 CFU/ml bacterial blight strain 2; d, the concentration is 2.28 multiplied by 10 5 CFU/ml bacterial blight strain 2; e, concentration of 2.28X 10 4 CFU/ml bacterial blight strain 2; f with a concentration of 2.28X 10 3 CFU/ml bacterial blight strain 2; g is 2.28X 10 in concentration 2 CFU/ml bacterial blight strain 2; h, sterile water control;
FIG. 8 shows the result of detecting DNA of bacterial blight of rice in various concentrations according to an embodiment of the present invention;
a, bacterial blight strain 2 with the concentration of 9.66 ng/. mu.L; b with a concentration of 9.66X 10 -1 ng/mu L bacterial blight strain 2; c with a concentration of 2.28X 10 -2 ng/muL bacterial blight strain 2; d, the concentration is 2.28 multiplied by 10 -3 ng/muL bacterial blight strain 2; e, concentration of 2.28X 10 -4 ng/mu L bacterial blight strain 2; f with a concentration of 2.28X 10 -5 ng/mu L bacterial blight strain 2; g, the concentration is 2.28×10 -6 ng/mu L bacterial blight strain 2; h, sterile water control.
Detailed Description
Reference will now be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. For instance, features illustrated or described as part of one embodiment, can be used on another embodiment to yield a still further embodiment.
Unless otherwise defined, all terms (including technical and scientific terms) used to disclose the invention are to be interpreted as commonly understood by one of ordinary skill in the art to which this invention belongs. The following definitions serve to better understand the teachings of the present invention by way of further guidance. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
The term "and/or", "and/or" as used herein is intended to be inclusive of any one of the two or more items listed in association, and also to include any and all combinations of the items listed in association, including any two or more of the items listed in association, any more of the items listed in association, or all combinations of the items listed in association. It should be noted that when at least three items are connected by at least two conjunctive combinations selected from "and/or", "or" and/or ", it should be understood that in this application, the technical solutions unquestionably include the technical solutions all connected by" logical and ", and also unquestionably include the technical solutions all connected by" logical or ". For example, "A and/or B" includes A, B and A + B. For example, the embodiments of "a, and/or, B, and/or, C, and/or, D" include any of A, B, C, D (i.e., all embodiments using "logical or" connection "), any and all combinations of A, B, C, D (i.e., any two or any three of A, B, C, D), and four combinations of A, B, C, D (i.e., all embodiments using" logical and "connection).
As used herein, the terms "comprising," "including," and "comprising" are synonymous, inclusive or open-ended, and do not exclude additional, unrecited members, elements, or method steps.
The recitation of numerical ranges by endpoints herein includes all numbers and fractions subsumed within that range, as well as the recited endpoints.
The present invention relates to concentration values, which include fluctuations within a certain range. For example, it may fluctuate within a corresponding accuracy range. For example, 2%, may be allowed to fluctuate within 0.1%. For values that are larger or do not require more fine control, the meaning is also allowed to include greater fluctuations. For example, 100mM, may allow fluctuations within the range of. + -. 1%, + -2%, + -5%, etc.
In the present invention, the terms "plurality", and the like mean, unless otherwise specified, 2 or more in number.
In the present invention, the technical features described in the open type include a closed technical solution composed of the listed features, and also include an open technical solution including the listed features.
In the present invention, "preferably", "better" and "preferable" are only embodiments or examples with better description, and it should be understood that the scope of the present invention is not limited by them. In the present invention, "optionally", "optional" and "optional" refer to the presence or absence, i.e., to any one of two juxtapositions selected from "present" and "absent". If multiple optional parts appear in one technical scheme, if no special description exists, and no contradiction or mutual constraint relation exists, each optional part is independent.
The present invention relates to a combination product comprising:
an upstream primer: TCGCTCTCCCGAGGGAGATGACAGCCGCTACA, respectively;
a downstream primer: ACACGGAACACCTGGGTAGTCTCTGTAGGGAAG, respectively; and
and (3) probe: CCATCTCAAATAAGCGCTTCCGCTATCCACTXTTACTACTTCCAGAT;
wherein the probe is based on recombinase polymerase amplification technology, a 31 st base T marks a luminescent group, a 32 nd base X is an abasic nucleotide analogue, a 33 rd base T marks a quenching group, and a 3' end is modified by a blocking agent; the blocking agent is used to block polymerase extension of the probe.
In some embodiments, the abasic nucleotide analog is tetrahydrofuran.
In some embodiments, the blocking moiety is selected from the group consisting of a spacer, a phosphate group, biotin-TEG, or an amine (e.g., C6 amine).
In some embodiments, the Spacer is selected from any one of ethylene glycol, a C9 Spacer (Spacer 9), a C18 Spacer (Spacer 18), a dideoxyspacer [1 ', 2' -dideoxyspace (dspacer) ], a C3Spacer (C3 Spacer).
In some embodiments, the spacer modification is selected from the group consisting of C3 spacer.
The Spacer (Spacer) can provide the necessary spacing for oligonucleotide labeling to reduce the interaction between the labeling group and the oligonucleotide, and is mainly applied to the research of DNA hairpin structure and double-stranded structure. C3 spacers are used primarily to mimic the three-carbon spacing between the 3 'and 5' hydroxyl groups of ribose, or to "substitute" for unknown bases in a sequence. 3'-Spacer C3 was used to introduce a 3' Spacer to prevent the 3 'exonuclease and 3' polymerase from acting.
In some embodiments, the luminophore is selected from any one of AMCA, Pacific Blue, Atto 425, BODIPY FL, FAM, Alexa Fluor 488, TET, JOE, Yakima Yellow, VIC, HEX, Quasar 570, Cy3, NED, TAMRA, ROX, Aqua Phluor593, Texas Red, Atto 590, Cy5, Quasar 670, Cy5.5, and Cy5.5.
In some embodiments, the quencher group is selected from any one of BHQ1, BHQ2, BHQ3, Dabcyl, Eclipse and MGB.
In some specific embodiments, the luminogen is FAM and the quencher is BHQ.
In one aspect, useful primers and probes include nucleotide sequences having greater than 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the upstream primer, downstream primer, or probe of the particular sequences provided above. Such primer and probe modifications are also contemplated and may be made according to standard techniques.
The term "% identity" in the context of two or more nucleotide or amino acid sequences refers to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection. For example,% identity is relative to the entire length of the coding region of the sequences to be compared.
For sequence comparison, typically one sequence is used as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, the test sequence and the reference sequence are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence relative to the reference sequence based on the specified program parameters. Percent identity can be determined using search algorithms such as BLAST and PSI-BLAST (Altschul et al, 1990, J Mol Biol 215:3, 403-.
The primer and probe modification can be carried out by a known method. Modified versions of these primer and/or probe sequences may include, by way of non-limiting example, adding one or more nucleotides to the 5 'end, adding one or more nucleotides to the 3' end, adding one or more nucleotides to the 5 'and 3' ends, adding a tail, shortening the sequence, lengthening the sequence, moving the sequence several bases upstream and downstream, or any combination thereof. In addition to the modifications already mentioned above, other modifications which can be applied without significantly negatively affecting the primer/probe function, such as 3' P, 5-nitroindole, 2-aminopurine, 8-amino-2 ' -deoxyadenosine, C-5-propynyl-deoxycytidine, C-5-propynyl-deoxyuridine, 2-amino-2 ' -deoxyadenosine-5 ' -triphosphate, 2, 6-diaminopurine (2-amino-dA), inverted dT, inverted dideoxy-T, hydroxymethyl dC, iso-dC, 5-methyl dC, aminoethyl-phenoxazine-deoxycytidine, and locked nucleic acids (LNA's), and including at least one mismatched base at one of the bases, or replacing at least one of the bases with an RNA base, to achieve, for example, an increase in nucleic acid interactions at the 3' end of the mutant-specific primer to increase Tm. The addition of a double-stranded stable base modification has a positive effect on PCR, enabling it to be performed at higher temperatures, in the range where Taq polymerase is known to show the greatest activity. The modified probe should retain the ability to distinguish between the mutated site to be detected and the wild-type site.
The invention also relates to a kit comprising a combination product as described above.
The term "kit" refers to any article of manufacture (e.g., a package or container) comprising at least one device, the kit may further comprise instructions for use, supplemental reagents, and/or components or assemblies for use in the methods described herein or steps thereof.
In some embodiments, the kit further comprises one or more of a nucleic acid extraction reagent, a reagent for isothermal nucleic acid amplification, a positive control, and a negative control.
In some embodiments, the reagents used for isothermal nucleic acid amplification include one or more of a recombinase capable of binding single-stranded nucleic acids, single-stranded DNA binding proteins, strand displacement DNA polymerases, helper proteins, exonuclease III, reverse transcriptase, ATP, reagents for an ATP regeneration system, pH regulators, dntps, BSA and/or PEG of various molecular weight distributions, DTT, and water;
wherein the helper protein is used for changing the reversible reaction process of the dissociation and recombination of the recombinase-primer complex, so that the reaction is more favorable for isothermal nucleic acid amplification.
The pH adjusting agent may contain acids and bases that do not significantly affect the progress of the reaction, as well as buffer components (e.g., Tris and acetate, etc.). Further, the Tris buffer is Tris-tricine, and the working concentration thereof may be about 80mM to 120 mM.
In some embodiments, the recombinase is selected from uvsX and/or RecA;
in some embodiments, the single-stranded DNA binding protein is gp 32;
in some embodiments, the strand displacing DNA polymerase is selected from BSu DNA polymerase and/or Sau DNA polymerase. The DNA polymerases used in the recombinase-mediated isothermal amplification of nucleic acids are Bacillus subtilis Pol I (Bsu) or Staphylococcus aureus Pol I (Sau), both of which belong to the family of DNA polymerases I. The DNA polymerase I family is the polymerases responsible for damage repair during DNA replication, and most of the DNA polymerases in the family have low processivity, i.e., the polymerases in the family bind to the template and catalyze a small number of polymerization reactions at one time.
In some embodiments, the accessory protein is selected from uvsY;
in the case where a recombinase is used for the strand insertion step, the system may require an energy source. Most of these enzymes utilize ATP as an energy source, but because of the magnesium ions necessary for ATP trimming (collate) enzyme activity, it is advantageous to provide an additional ATP regeneration system rather than to increase the concentration of ATP. In some embodiments, the reagent used in the ATP regeneration system is selected from one or more of magnesium ions, phosphocreatine and its counterions, creatine kinase, myokinase, pyrophosphatase, sucrose, and sucrose phosphorylase.
From the above components, the kit of the present invention can adopt and preferably adopt a Recombinase Polymerase Amplification (RPA) method, but can also adopt a method improved on the technology, for example, a Recombinase-dependent Amplification (RDA) method.
In some embodiments, the reagents used for isothermal nucleic acid amplification are lyophilized powder reagents or mixed liquid reagents.
The components are preferably realized in lyophilized form, for example in the form of one or more so-called lyophilized beads. Lyophilized beads are generally understood to mean lyophilizates which are compressed into spherical form after production (after which the substance is generally present as a powder). Thus, the components required for the amplification reaction, in particular the various enzymes, the nucleic acid components and the reaction buffer components, may be provided in lyophilized form. In this way, the amplification process can be started directly in a very user-friendly manner by adding the sample to be quantified and optionally other desired components. In particular, the provision of a lyophilized form is highly advantageous for automated applications.
According to a further aspect of the invention, the invention also relates to the use of a combination product as described above, or a kit as described above, for detecting bacterial rice blight.
The reaction principle of the recombinase polymerase amplification reaction system provided by the invention is as follows: (1) in the reaction system, a recombinase-primer complex formed by combining a recombinase with a primer searches a target site in a double-stranded DNA template; (2) after the recombinase-primer complex recognizes a template specific sequence, positioning occurs and strand exchange is initiated, and the single-strand binding protein is combined with a D-Loop structure formed by the displaced DNA strands; (3) the dATP conformation in the recombinase-primer complex hydrolysis system is changed, the 3 'end of the primer is exposed and recognized by DNA polymerase after the recombinase is dissociated, and the DNA polymerase starts DNA synthesis at the 3' end of the primer according to a template sequence; (4) the DNA polymerase has a strand displacement function, continues to unwind the double-helix DNA structure of the template while the primer is extended, and the DNA synthesis process continues; (5) completing the amplification of the two primers to form a complete amplicon; (6) in the reaction system, dATP is hydrolyzed to supply energy to recombinase and then becomes dADP, phosphocreatine can transfer the phosphate group of the phosphocreatine into a dADP molecule under the catalysis of creatine kinase to form dATP, and therefore the level of dATP in the reaction system is restored. The above process is repeated continuously, and finally the high-efficiency amplification of nucleic acid is realized.
In some embodiments, the detection is performed under isothermal amplification conditions, the temperature is 37-42 ℃, and the reaction time is more than or equal to 20 min.
The sample to be tested which is suitable for use in the present invention may be any of the various components suspected of containing bacterial rice blight bacteria, for example: the water source, soil, etc. near the rice planting environment, of course, also includes rice plants and their corresponding tissues. The rice variety is not limited, and may be japonica rice, java rice, indica rice, glutinous rice, mountain rice, palea rice, or various introgression lines. The rice tissue may be a shoot, root, stem, cell, protoplast, leaf, pollen, embryo, cotyledon, hypocotyl, anther, flower and seed of rice.
In some embodiments, the use further comprises the step of isolating DNA (particularly genomic DNA) from a component suspected of containing bacterial blight of rice. Isolating the DNA fragments from the treated material may include using a separation solvent, such as methanol, ethanol, water, acetone, or a combination thereof. In some embodiments, kits of DNA isolation kits may be used, including, for example, DNA isolation protocols using the Dneasy Mericon food kit (Qiagen, Germantown, MD, USA) or cetyltrimethylammonium bromide (CTAB). Other separation techniques include lysis, heating, alcohol precipitation, salt precipitation, organic separation, solid phase separation, raw silica membrane separation, CSCL gradient purification, or any combination thereof.
Embodiments of the present invention will be described in detail with reference to examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures for the following examples, in which specific conditions are not specified, can be performed according to the instructions given in the present invention, according to the experimental manual or the conventional conditions in the art, according to other experimental procedures known in the art, or according to the conditions suggested by the manufacturer.
In the following specific examples, the measurement parameters relating to the components of the raw materials, if not specified otherwise, may be subject to slight deviations within the accuracy of the weighing. Temperature and time parameters are involved to allow for acceptable deviation due to instrument test accuracy or operational accuracy.
Example 1
Materials and instruments
Preparing a nutrient agar culture medium: weighing 23g of Nutrient Agar and 14g of Agar powder into a clean wide-mouth bottle, adding 1L of deionized water, mixing uniformly, sterilizing for 20min at 121 ℃ in an autoclave, pouring a flat plate in a sterile operating platform, cooling and solidifying into an NA solid culture medium, and inverting for later use. The TIANGEN bacterium genome DNA extraction kit (centrifugal column type), the Weifang Anpu future constant temperature fluorescence detector (WL-16-II) and the Weifang Anpu future constant temperature DNA rapid amplification kit (fluorescent type).
Second, Experimental methods
1. Primer specificity experiments: the required strains are shown in table 1, 1 to 10 are different rice bacterial glutamic acid bacterial strains, 11 to 23 are other bacteria in the same genus, namely Burkholderia, and 24 to 34 are other bacteria capable of infecting rice, 2 mu L of bacterial liquid is taken as a template, and fluorescent RPA detection is carried out by using a primer probe shown in table 2 according to the Anpu future kit.
TABLE 1 primer specificity test strains
TABLE 2 primer and Probe sequences for fluorescent RPA detection
2. Primer sensitivity experiment, diluting DNA extracted from bacterial rice blight ATCC33617 and its kit with 10 times of sterile water, and performing fluorescence RPA test with 2 μ L of each concentration.
3. The detection method adopted by the embodiment comprises the following steps:
(1) fluorescent RPA amplification: according to the method of the Anpu future kit,
add 29.4. mu. L A buffer to each dry powder reaction tube.
② 2 mul of upstream primer and 2 mul of downstream primer and 0.6 mul of probe are added into each reaction tube.
③ adding 11.5 mu L of ddH into the reaction tube in turn 2 O and 2. mu.L of nucleic acid template.
Finally, 2.5. mu. L B buffer solution was added to the reaction tube and the mixture was inverted 10 times and mixed well.
Fifthly, after mixing uniformly, throwing the reaction liquid to the bottom of the reaction tube (or centrifuging the reaction liquid to the bottom of the reaction tube), immediately putting the reaction tube into a fluorescence detection device, setting the fluorescence device to be constant at 39 ℃, collecting the fluorescence value of the FAM channel every 30 seconds, and reacting for 20 min.
(2) Primer specificity test experiment: adopting different bacterial strains of rice bacterial blight; other pathogenic bacteria of the same genus, i.e. Burkholderia; and other pathogenic bacteria of different species on rice 10, 19, 10 species are shown in Table 1, and 2. mu.L of each of them is taken as a template and added into the reaction system for fluorescent RPA amplification and signal collection.
(3) Sensitivity test experiment of bacterial blight bacterium liquid: the method comprises the steps of streaking and purifying rice bacterial blight ATCC33617 on a nutrient agar plate, culturing for 48 hours, washing the bacterial colony with sterile water, collecting bacterial liquid, vibrating the bacterial liquid uniformly, diluting the collected bacterial liquid with ten times of gradient of the sterile water to obtain an initial bacterial load of 0.1-10 -8 And (4) coating 40 mu L of the bacterial suspension on a nutrient agar plate, culturing for 48 hours, calculating the number of colonies, and converting to the number of colonies CFU. And (3) taking 7 groups of bacteria liquid with proper gradients to perform fluorescence RPA amplification and signal acquisition.
(4) Extracting bacterial blight bacterium DNA: extracting DNA of the rice bacterial glutamic acid bacterial strain ATCC33617 by using a bacterial genome DNA extraction kit, and performing tertiary concentration determination by using a Nano Drop2000 ultramicro ultraviolet spectrophotometer after sequencing identification. The results of 3 times were averaged to obtain the final concentration.
(5) Bacterial blight bacterium DNA sensitivity test experiment: extracting the above extractive solution with ddH 2 Dilution with a gradient of 10 times O to give an initial concentration of 10 -1 ～10 -6 And (5) carrying out fluorescence RPA amplification and signal acquisition by using the dilution liquid.
(6) And (4) judging a result: the fluorescence signal threshold was set at 100, the determination value was 2, the CT value was 35 or less, and the peak shape was a part of the sigmoid curve, and the result was determined to be positive.
Third, experimental results
Real-time fluorescent RPA detection of bacterial cerealsThe specificity test CT values of the fusarium wilt are shown in table 3, and corresponding fluorescence value signal graphs are shown in figures 1 to 6; the sensitivity test of the primers adopts bacterial liquid and DNA of the rice bacterial blight bacterium ATCC33617, the concentration of the corresponding bacterial liquid is obtained by conversion through a plate coating counting method, the negative control is sterile water, the CT value result is shown in table 4, and the corresponding fluorescence value signal diagram is shown in fig. 7 and 8. As shown in the table, the limit of detection of the real-time fluorescent RPA for detecting the bacterial bodies of the rice bacterial blight germ is 2.28 multiplied by 10 3 CFU/ml, DNA detection limit of 9.66X 10 -6 ng/μL。
TABLE 3 real-time fluorescent RPA specificity test results
TABLE 4 real-time fluorescent RPA amplification results of bacterial liquid and DNA of rice bacterial blight bacteria with different concentrations
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the patent of the present invention should be subject to the appended claims, and the description and the drawings can be used for explaining the contents of the claims.
Claims (11)
1. A combination product comprising:
an upstream primer: TCGCTCTCCCGAGGGAGATGACAGCCGCTACA, respectively;
a downstream primer: ACACGGAACACCTGGGTAGTCTCTGTAGGGAAG, respectively; and
and (3) probe: CCATCTCAAATAAGCGCTTCCGCTATCCACTXTTACTACTTCCAGAT;
wherein the probe is based on recombinase polymerase amplification technology, a 31 st base T marks a luminescent group, a 32 nd base X is an abasic nucleotide analogue, a 33 rd base T marks a quenching group, and a 3' end is modified by a blocking agent; the blocking agent is used to block polymerase extension of the probe.
2. The combination product of claim 1, wherein the abasic nucleotide analog is tetrahydrofuran.
3. Combination according to claim 1, the blocking agent being an inter arm, preferably a C3 inter arm.
4. The combination product of claim 1, the luminogen being a FAM and the quencher being BHQ.
5. A kit comprising the combination product of any one of claims 1 to 4.
6. The kit of claim 5, further comprising one or more of a nucleic acid extraction reagent, a reagent for isothermal nucleic acid amplification, a positive control, and a negative control.
7. The kit of claim 6, wherein the reagents for isothermal nucleic acid amplification comprise one or more of a recombinase capable of binding single-stranded nucleic acids, a single-stranded DNA binding protein, a strand-displacing DNA polymerase, a helper protein, exonuclease III, a reverse transcriptase, ATP, reagents for an ATP regeneration system, a pH adjuster, dNTPs, BSA and/or PEG of various molecular weight distributions, DTT, and water;
wherein the helper protein is used for changing the reversible reaction process of the dissociation and recombination of the recombinase-primer complex, so that the reaction is more favorable for isothermal nucleic acid amplification.
8. The kit of claim 7, wherein the recombinase is selected from uvsX and/or RecA;
optionally, the single-stranded DNA binding protein is gp 32;
optionally, the strand displacing DNA polymerase is selected from BSu DNA polymerase and/or Sau DNA polymerase;
optionally, the accessory protein is selected from uvsY;
optionally, the reagent used by the ATP regeneration system is selected from one or more of magnesium ion, phosphocreatine and its counter ion, creatine kinase, myokinase, pyrophosphatase, sucrose, and sucrose phosphorylase.
9. The kit according to any one of claims 6 to 8, wherein the reagent for isothermal nucleic acid amplification is a freeze-dried powder reagent or a mixed liquid reagent.
10. Use of a combination product according to any one of claims 1 to 4, or a kit according to any one of claims 5 to 9, in an assay.
11. The use of claim 1, wherein the detection is carried out under the condition of constant temperature amplification, the temperature is 37-42 ℃, and the reaction time is not less than 20 min.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210578688.6A CN114958835A (en) | 2022-05-25 | 2022-05-25 | Combination product and kit for detecting bacterial rice blight bacteria |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210578688.6A CN114958835A (en) | 2022-05-25 | 2022-05-25 | Combination product and kit for detecting bacterial rice blight bacteria |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN114958835A true CN114958835A (en) | 2022-08-30 |
Family
ID=82955560
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210578688.6A Pending CN114958835A (en) | 2022-05-25 | 2022-05-25 | Combination product and kit for detecting bacterial rice blight bacteria |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114958835A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116287354A (en) * | 2023-05-11 | 2023-06-23 | 三亚中国检科院生物安全中心 | Method and kit for detecting bacterial fusarium wilt of corn |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103498000A (en) * | 2013-10-11 | 2014-01-08 | 中国计量学院 | Primer group, kit and method for detecting rice quarantine pathogenic bacteria by multiplex PCR (polymerase chain reaction) method |
-
2022
- 2022-05-25 CN CN202210578688.6A patent/CN114958835A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103498000A (en) * | 2013-10-11 | 2014-01-08 | 中国计量学院 | Primer group, kit and method for detecting rice quarantine pathogenic bacteria by multiplex PCR (polymerase chain reaction) method |
Non-Patent Citations (3)
| Title |
|---|
| CHEN HUI等: "Rapid Detection of SARS-CoV-2 Using Duplex Reverse Transcription-Multienzyme Isothermal Rapid Amplification in a Point-of-Care Testing", FRONTIERS IN CELLULAR AND INFECTION MICROBIOLOGY, vol. 11, pages 1 - 11 * |
| 中国器材网: "多酶恒温核酸快速扩增技术MIRPA的引物与探针设计指导", pages 1 - 3, Retrieved from the Internet <URL:中国器材网> * |
| 刘国宪等: "MIRA技术在动物疫病防控领域应用研究", 科技成果, 31 December 2021 (2021-12-31), pages 1 - 6 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116287354A (en) * | 2023-05-11 | 2023-06-23 | 三亚中国检科院生物安全中心 | Method and kit for detecting bacterial fusarium wilt of corn |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111549182B (en) | Probe and kit for detecting SARS-CoV-2 | |
| CN106399490B (en) | LAMP primer group for detecting phytoplasma and kit and application thereof | |
| JP2010532665A (en) | Nucleic acid sequences and their combinations for sensitive amplification and detection of bacterial and fungal sepsis pathogens | |
| CN109072283B (en) | Compositions and methods for detecting mycoplasma genitalium | |
| KR101644773B1 (en) | Genetic Markers for Discrimination and Detection of Causative Bacteria of Edwardsiellosis and Streptococcosis, and Method of Discrimination and Detection of Causative Bacteria Using the Same | |
| CN116287354A (en) | Method and kit for detecting bacterial fusarium wilt of corn | |
| CN102643910B (en) | Application of asymmetric multicolor fluorescence hairpin probe chain reaction in pathogenic bacterium detection | |
| US8377656B2 (en) | Compositions and methods for detecting Cryptococcus neoformans | |
| CN111154739B (en) | Novel recombinase-dependent amplification method and kit | |
| CN111118219B (en) | RDA method and kit for rapidly detecting influenza A virus | |
| CN114958835A (en) | Combination product and kit for detecting bacterial rice blight bacteria | |
| CN115029470A (en) | Combination product and kit for detecting rice bacterial blight | |
| Galetto et al. | Real-time PCR diagnosis and quantification of phytoplasmas | |
| JP5210634B2 (en) | Detection, identification and differentiation of Serratia species using spacer regions | |
| CN104641000A (en) | Compositions and methods for detection of clostridium difficile | |
| WO2022194756A2 (en) | Compositions and methods for detecting severe acute respiratory syndrome coronavirus 2 (sars-cov-2) variants having spike protein mutations | |
| US20150140548A1 (en) | Extraction control for rna | |
| CN110804674B (en) | Primer probe composition and kit for detecting soybean root rot based on recombinase polymerase amplification method and application of primer probe composition and kit | |
| CN112301153A (en) | RDA method and kit for rapidly detecting Canine Coronavirus (CCV) | |
| CN113897456B (en) | Real-time fluorescence PCR detection primer probe combination and detection kit for four fir anthracnose pathogens and application of kit | |
| CN114350828B (en) | Specific primer for amplifying Pantoea ananatis and application thereof | |
| CN112301137B (en) | RDA method and kit for rapidly detecting ureaplasma urealyticum | |
| CN112301151B (en) | RDA method and kit for rapidly detecting Canine Distemper Virus (CDV) | |
| CN106755581A (en) | The method that one-step method real-time quantitative fluorescence PCR detects carnation mottle virus | |
| JP2006508670A (en) | Detection, identification and discrimination of eubacteria using hybridization assays |
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 |