WO2024058300A1 - Vhsv detection composition using gene scissors and loop-mediated isothermal amplification, kit, and vhsv detection method using same - Google Patents
Vhsv detection composition using gene scissors and loop-mediated isothermal amplification, kit, and vhsv detection method using same Download PDFInfo
- Publication number
- WO2024058300A1 WO2024058300A1 PCT/KR2022/014820 KR2022014820W WO2024058300A1 WO 2024058300 A1 WO2024058300 A1 WO 2024058300A1 KR 2022014820 W KR2022014820 W KR 2022014820W WO 2024058300 A1 WO2024058300 A1 WO 2024058300A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- vhsv
- gene
- composition
- isothermal amplification
- nucleic acid
- Prior art date
Links
- 239000000203 mixture Substances 0.000 title claims abstract description 38
- 238000001514 detection method Methods 0.000 title description 33
- 238000007397 LAMP assay Methods 0.000 title description 25
- 238000010457 gene scissor Methods 0.000 title description 10
- 241000711825 Viral hemorrhagic septicemia virus Species 0.000 claims abstract description 66
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 35
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 31
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 31
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 31
- 108010061100 Nucleoproteins Proteins 0.000 claims abstract description 12
- 102000011931 Nucleoproteins Human genes 0.000 claims abstract description 10
- 238000011901 isothermal amplification Methods 0.000 claims description 30
- 108020005004 Guide RNA Proteins 0.000 claims description 20
- 239000000523 sample Substances 0.000 claims description 20
- 101710163270 Nuclease Proteins 0.000 claims description 9
- 102000004169 proteins and genes Human genes 0.000 claims description 7
- 230000000295 complement effect Effects 0.000 claims description 6
- 108700004991 Cas12a Proteins 0.000 claims description 5
- 238000000034 method Methods 0.000 abstract description 20
- 208000015181 infectious disease Diseases 0.000 abstract description 11
- 239000013615 primer Substances 0.000 description 65
- 238000006243 chemical reaction Methods 0.000 description 36
- 239000000047 product Substances 0.000 description 23
- 108020004414 DNA Proteins 0.000 description 19
- 230000003321 amplification Effects 0.000 description 11
- 238000003199 nucleic acid amplification method Methods 0.000 description 11
- 102000004190 Enzymes Human genes 0.000 description 10
- 108090000790 Enzymes Proteins 0.000 description 10
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 8
- 241000251468 Actinopterygii Species 0.000 description 8
- 206010040047 Sepsis Diseases 0.000 description 8
- 238000003745 diagnosis Methods 0.000 description 8
- 230000002008 hemorrhagic effect Effects 0.000 description 8
- 230000003612 virological effect Effects 0.000 description 8
- 102000053602 DNA Human genes 0.000 description 7
- 108091034117 Oligonucleotide Proteins 0.000 description 7
- 241000700605 Viruses Species 0.000 description 7
- 239000003153 chemical reaction reagent Substances 0.000 description 7
- 238000003776 cleavage reaction Methods 0.000 description 7
- 239000002773 nucleotide Substances 0.000 description 7
- 125000003729 nucleotide group Chemical group 0.000 description 7
- 238000002474 experimental method Methods 0.000 description 6
- 238000000605 extraction Methods 0.000 description 6
- 230000035484 reaction time Effects 0.000 description 6
- 230000007017 scission Effects 0.000 description 6
- 229960005486 vaccine Drugs 0.000 description 6
- 108091079001 CRISPR RNA Proteins 0.000 description 5
- 108020004682 Single-Stranded DNA Proteins 0.000 description 5
- 108091028113 Trans-activating crRNA Proteins 0.000 description 5
- AUIINJJXRXMPGT-UHFFFAOYSA-K trisodium 3-hydroxy-4-[(2-hydroxy-4-sulfonatonaphthalen-1-yl)diazenyl]naphthalene-2,7-disulfonate Chemical compound [Na+].[Na+].[Na+].Oc1cc(c2ccccc2c1N=Nc1c(O)c(cc2cc(ccc12)S([O-])(=O)=O)S([O-])(=O)=O)S([O-])(=O)=O AUIINJJXRXMPGT-UHFFFAOYSA-K 0.000 description 5
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 4
- KWIUHFFTVRNATP-UHFFFAOYSA-N Betaine Natural products C[N+](C)(C)CC([O-])=O KWIUHFFTVRNATP-UHFFFAOYSA-N 0.000 description 4
- 108091033409 CRISPR Proteins 0.000 description 4
- 102100034343 Integrase Human genes 0.000 description 4
- JLVVSXFLKOJNIY-UHFFFAOYSA-N Magnesium ion Chemical compound [Mg+2] JLVVSXFLKOJNIY-UHFFFAOYSA-N 0.000 description 4
- KWIUHFFTVRNATP-UHFFFAOYSA-O N,N,N-trimethylglycinium Chemical compound C[N+](C)(C)CC(O)=O KWIUHFFTVRNATP-UHFFFAOYSA-O 0.000 description 4
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 4
- 229960003237 betaine Drugs 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 238000004737 colorimetric analysis Methods 0.000 description 4
- 238000002405 diagnostic procedure Methods 0.000 description 4
- 229910001425 magnesium ion Inorganic materials 0.000 description 4
- 244000052769 pathogen Species 0.000 description 4
- 238000003752 polymerase chain reaction Methods 0.000 description 4
- 229920000136 polysorbate Polymers 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- 239000011535 reaction buffer Substances 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 150000001875 compounds Chemical class 0.000 description 3
- 238000007796 conventional method Methods 0.000 description 3
- 238000005520 cutting process Methods 0.000 description 3
- 238000010438 heat treatment Methods 0.000 description 3
- 239000003550 marker Substances 0.000 description 3
- 239000011259 mixed solution Substances 0.000 description 3
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 3
- 239000002987 primer (paints) Substances 0.000 description 3
- 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 3
- COCMHKNAGZHBDZ-UHFFFAOYSA-N 4-carboxy-3-[3-(dimethylamino)-6-dimethylazaniumylidenexanthen-9-yl]benzoate Chemical compound C=12C=CC(=[N+](C)C)C=C2OC2=CC(N(C)C)=CC=C2C=1C1=CC(C([O-])=O)=CC=C1C(O)=O COCMHKNAGZHBDZ-UHFFFAOYSA-N 0.000 description 2
- 239000012099 Alexa Fluor family Substances 0.000 description 2
- 230000004544 DNA amplification Effects 0.000 description 2
- 241000191967 Staphylococcus aureus Species 0.000 description 2
- 241000193996 Streptococcus pyogenes Species 0.000 description 2
- 208000001449 Viral Hemorrhagic Septicemia Diseases 0.000 description 2
- 108020000999 Viral RNA Proteins 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 238000012790 confirmation Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 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 description 2
- 238000009396 hybridization Methods 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 229940126578 oral vaccine Drugs 0.000 description 2
- 238000006116 polymerization reaction Methods 0.000 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 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 238000013207 serial dilution Methods 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 230000008685 targeting Effects 0.000 description 2
- 238000002255 vaccination Methods 0.000 description 2
- 108700026220 vif Genes Proteins 0.000 description 2
- VGIRNWJSIRVFRT-UHFFFAOYSA-N 2',7'-difluorofluorescein Chemical compound OC(=O)C1=CC=CC=C1C1=C2C=C(F)C(=O)C=C2OC2=CC(O)=C(F)C=C21 VGIRNWJSIRVFRT-UHFFFAOYSA-N 0.000 description 1
- QCPFFGGFHNZBEP-UHFFFAOYSA-N 4,5,6,7-tetrachloro-3',6'-dihydroxyspiro[2-benzofuran-3,9'-xanthene]-1-one Chemical compound O1C(=O)C(C(=C(Cl)C(Cl)=C2Cl)Cl)=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 QCPFFGGFHNZBEP-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
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
- IDLISIVVYLGCKO-UHFFFAOYSA-N 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein Chemical compound O1C(=O)C2=CC=C(C(O)=O)C=C2C21C1=CC(OC)=C(O)C(Cl)=C1OC1=C2C=C(OC)C(O)=C1Cl IDLISIVVYLGCKO-UHFFFAOYSA-N 0.000 description 1
- BZTDTCNHAFUJOG-UHFFFAOYSA-N 6-carboxyfluorescein Chemical compound C12=CC=C(O)C=C2OC2=CC(O)=CC=C2C11OC(=O)C2=CC=C(C(=O)O)C=C21 BZTDTCNHAFUJOG-UHFFFAOYSA-N 0.000 description 1
- 206010002091 Anaesthesia Diseases 0.000 description 1
- 241000589941 Azospirillum Species 0.000 description 1
- 241000606125 Bacteroides Species 0.000 description 1
- 238000010354 CRISPR gene editing Methods 0.000 description 1
- 238000010453 CRISPR/Cas method Methods 0.000 description 1
- 241000589876 Campylobacter Species 0.000 description 1
- 241000589875 Campylobacter jejuni Species 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 241000186216 Corynebacterium Species 0.000 description 1
- 239000003155 DNA primer Substances 0.000 description 1
- 230000006820 DNA synthesis Effects 0.000 description 1
- 241000186394 Eubacterium Species 0.000 description 1
- 241000178967 Filifactor Species 0.000 description 1
- 241000589565 Flavobacterium Species 0.000 description 1
- 241000032681 Gluconacetobacter Species 0.000 description 1
- 241000186660 Lactobacillus Species 0.000 description 1
- 241000589248 Legionella Species 0.000 description 1
- 208000007764 Legionnaires' Disease Diseases 0.000 description 1
- 241000204031 Mycoplasma Species 0.000 description 1
- NULAJYZBOLVQPQ-UHFFFAOYSA-N N-(1-naphthyl)ethylenediamine Chemical compound C1=CC=C2C(NCCN)=CC=CC2=C1 NULAJYZBOLVQPQ-UHFFFAOYSA-N 0.000 description 1
- 241000588653 Neisseria Species 0.000 description 1
- 241000135938 Nitratifractor Species 0.000 description 1
- 206010029719 Nonspecific reaction Diseases 0.000 description 1
- 102000007999 Nuclear Proteins Human genes 0.000 description 1
- 108010089610 Nuclear Proteins Proteins 0.000 description 1
- 241001386753 Parvibaculum Species 0.000 description 1
- 241000269908 Platichthys flesus Species 0.000 description 1
- 241000605947 Roseburia Species 0.000 description 1
- CGNLCCVKSWNSDG-UHFFFAOYSA-N SYBR Green I Chemical compound CN(C)CCCN(CCC)C1=CC(C=C2N(C3=CC=CC=C3S2)C)=C2C=CC=CC2=[N+]1C1=CC=CC=C1 CGNLCCVKSWNSDG-UHFFFAOYSA-N 0.000 description 1
- 241000191940 Staphylococcus Species 0.000 description 1
- 241000194017 Streptococcus Species 0.000 description 1
- 241000194020 Streptococcus thermophilus Species 0.000 description 1
- 241000123710 Sutterella Species 0.000 description 1
- 241000589886 Treponema Species 0.000 description 1
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 1
- 230000037005 anaesthesia Effects 0.000 description 1
- 239000013060 biological fluid Substances 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 239000012502 diagnostic product Substances 0.000 description 1
- 238000006073 displacement reaction Methods 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 1
- 238000010362 genome editing Methods 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 229940031551 inactivated vaccine Drugs 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 239000000138 intercalating agent Substances 0.000 description 1
- 238000009830 intercalation Methods 0.000 description 1
- 150000002540 isothiocyanates Chemical class 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 229940039696 lactobacillus Drugs 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 239000002777 nucleoside Substances 0.000 description 1
- -1 nucleoside triphosphates Chemical class 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 230000008816 organ damage Effects 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 230000000379 polymerizing effect Effects 0.000 description 1
- 108091033319 polynucleotide Proteins 0.000 description 1
- 102000040430 polynucleotide Human genes 0.000 description 1
- 239000002157 polynucleotide Substances 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
- 230000000171 quenching effect Effects 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 230000036632 reaction speed Effects 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 241001507086 salmonid fish Species 0.000 description 1
- 230000005477 standard model Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- JGVWCANSWKRBCS-UHFFFAOYSA-N tetramethylrhodamine thiocyanate Chemical compound [Cl-].C=12C=CC(N(C)C)=CC2=[O+]C2=CC(N(C)C)=CC=C2C=1C1=CC=C(SC#N)C=C1C(O)=O JGVWCANSWKRBCS-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
- ANRHNWWPFJCPAZ-UHFFFAOYSA-M thionine Chemical compound [Cl-].C1=CC(N)=CC2=[S+]C3=CC(N)=CC=C3N=C21 ANRHNWWPFJCPAZ-UHFFFAOYSA-M 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- 239000001226 triphosphate Substances 0.000 description 1
- 235000011178 triphosphate Nutrition 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/22—Ribonucleases RNAses, DNAses
-
- 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
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]
-
- 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
- C12Q2527/00—Reactions demanding special reaction conditions
- C12Q2527/101—Temperature
Definitions
- the present invention relates to a composition for detecting Viral Hemorrhagic Septicemia Virus (VHSV) infection, a kit containing the composition, and a method for diagnosing VHSV infection using the same.
- VHSV Viral Hemorrhagic Septicemia Virus
- the present invention provides a composition for diagnosing VHSV infection containing a nucleic acid oligomer capable of specifically amplifying the N gene, a VHSV nucleoprotein coding gene, a kit containing the composition, and the same. This relates to a method of diagnosing VHSV infection using this method.
- Viral hemorrhagic septicemia is a disease that occurs in salmonid fish farmed in Europe and flounder farmed in East Asia. It is an aquatic organism infectious disease designated by the 'Aquatic Products Disease Control Act', and affects farmed fish species and wild fish. It can also spread to marine fish.
- VHSV vaccines developed to date are vaccines that use injections, and processes such as temporary anesthesia required for the vaccine treatment process can induce stress in fish.
- vaccination is expensive as it requires the work of skilled experts, and it is not technically easy for young fish that require vaccination as organ damage may occur during the injection process. Therefore, it is necessary to develop a low-cost, high-efficiency vaccine that does not cause stress to fish during the vaccine treatment process and can be applied to young fish.
- the most suitable vaccine for this is an oral vaccine that can be administered by mixing with feed. An oral vaccine that can induce immunity has not been developed.
- VHSV diagnosis is considered important.
- several diagnostic methods including cell culture, immune antibody detection, and molecular biological diagnostic methods.
- the inventors of the present application developed a new detection method targeting the nucleoprotein coding gene (N gene) of VHSV.
- the purpose of the present invention is to recognize the problems of the prior art mentioned above and to provide a composition, a kit, and a detection method using the same that can quickly provide VHSV detection results.
- the present invention is a VHSV comprising a nucleic acid oligomer capable of specifically isothermal amplification of the nucleoprotein coding gene (N gene) of Viral Hemorrhagic Septicemia Virus (VHSV). It relates to a composition for detection.
- the nucleic acid oligomer may be characterized by further comprising a probe capable of hybridizing complementary to the N gene product of VHSV specifically isothermally amplified by the nucleic acid oligomer.
- gRNA guide RNA
- the present invention also relates to a kit for detecting VHSV comprising the composition.
- the present invention furthermore includes the step of treating the sample with a nucleic acid oligomer capable of specifically isothermally amplifying the nucleoprotein coding gene (N gene) of Viral Hemorrhagic Septicemia Virus (VHSV).
- N gene nucleoprotein coding gene of Viral Hemorrhagic Septicemia Virus
- results can be confirmed quickly through an isothermal amplification-based detection method, making it possible to effectively detect VHSV. It is expected that a VHSV diagnostic system can be built more simply without complex heat treatment equipment.
- Figure 1 shows a portion of the VHSV nucleoprotein gene designated as the main target sequence in the present invention and the binding site of the primer set for loop-mediated isothermal amplification.
- the recognition site of the guide RNA (gRNA) which is the most important in the diagnosis of gene scissors performed on the isothermal amplification product, is also indicated.
- gRNA guide RNA
- Figure 2 shows the results of an experiment to confirm the specific reactivity of the new primer developed through the present invention. This is the result of an experiment to find the time conditions under which primers do not produce non-specific products by varying the time from 25 to 45 minutes.
- Figure 3 shows the results of an experiment to find the optimal temperature conditions when loop-mediated isothermal amplification of target viral RNA using a new primer. The reaction was confirmed by mixing the amplification product with the fluorescent marker SYBR Green I and using electrophoresis.
- Figure 4 shows the results of an experiment to confirm the detection limit when loop-mediated isothermal amplification using a new primer was performed. The experimental results were confirmed through electrophoresis and colorimetric analysis using hydroxy naphthol blue.
- Figure 5 shows the results of an experiment applying gene scissors diagnosis to the loop-mediated isothermal amplification product, and the detection limit was confirmed by measuring the fluorescence signal.
- the present invention utilizes Reverse Transcriptional Loop-mediated isothermal amplification (RT-LAMP) and gene scissors diagnostic technology (CRISPR/Cas), which can diagnose viral hemorrhagic sepsis virus (VHSV).
- R-LAMP Reverse Transcriptional Loop-mediated isothermal amplification
- CRISPR/Cas gene scissors diagnostic technology
- VHSV viral hemorrhagic sepsis virus
- the VHSV diagnosis method applying loop-mediated isothermal amplification and gene scissor technology can confirm molecular diagnostic test results within 40 minutes with only a simple heat treatment device, so the current real-time PCR test method requires only 2 amplification time. Although it takes about a while, it can provide test results quickly, so it can be useful in diagnosing VHSV infection.
- the present invention is a VHSV detection comprising a nucleic acid oligomer capable of specifically isothermal amplification of the nucleoprotein coding gene (N gene) of VHSV and a gRNA capable of specifically identifying the isothermal amplification product. It relates to a composition for use.
- nucleic acid oligomer may refer to a material containing two or more nucleotides produced by polymerizing nucleic acids as monomers.
- the nucleic acid oligomer may function as a primer or probe.
- 'primer' refers to a single-stranded oligonucleotide that can act as an initiation point for template-directed DNA synthesis under suitable conditions (four different nucleoside triphosphates and polymerization enzymes) at a suitable temperature and buffer. do.
- the primers may be designed to have “general” complementarity with each strand of the gene locus to be amplified. This means that the primer has sufficient complementarity to hybridize with the corresponding nucleic acid strand under the conditions for performing the polymerization reaction.
- the isothermal amplification refers to 'Loop-mediated isothermal amplification (LAMP)', and unlike the existing PCR (polymerase chain reaction) method, it is a method that can perform an amplification reaction under isothermal conditions. Basically, 4 types of primers (F3, B3, FIP, BIP) are required for the LAMP reaction, and to improve the reaction speed, 2 types of primers (LF, LB) are added to produce 6 different base sequences. Oligonucleotide primers may be required for the reaction.
- LAMP 'Loop-mediated isothermal amplification
- the four types of basic primers consist of two types of outer primers and two types of inner primers, and the “inner primer” is a single primer that can bind to template DNA and act as a starting point for new DNA chain synthesis. refers to a stranded oligonucleotide, and “outer primer” refers to a single-stranded oligonucleotide that binds to the template DNA from a position further outside than the position where the internal primer binds to the template DNA. After the DNA chain is extended, strand displacement occurs due to the combination of the external primer and the template DNA, causing the previously formed chain to separate.
- Loop primer is an initial stem loop structure chain formed by binding the inner primer and outer primer to the template DNA, which increases the number of loop structure creations and can ultimately accelerate the overall reaction. It refers to a single-stranded oligonucleotide that can act as a starting point for nucleotide synthesis.
- the outer primer consists of two types of primers, the forward outer (F3) primer and the reverse outer (B3) primer, and plays a role in unwinding the DNA double strand during the non-cyclic step of the reaction.
- the internal primer consists of two types, the forward inner primer (FIP) and the reverse inner primer (BIP), and is attached to the forward and reverse base sequences to create a loop essential for the loop-mediated isothermal amplification reaction. It is composed of the corresponding nucleotides.
- the additional two types of primers are composed of a forward loop (LF) primer and a backward loop (LB) primer, and attach to a base sequence that the inner primer does not bind to, thereby carrying out a ring-mediated isothermal amplification reaction. accelerate.
- the nucleic acid oligomer capable of specifically isothermal amplification of the N gene of VHSV may be a primer containing a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 6.
- the primers can be used simultaneously, for example, by pairing forward and reverse primers for amplification.
- the sequence of SEQ ID NO: 1 is F3, a forward external primer
- the sequence of SEQ ID NO: 2 is B3, a reverse external primer
- the sequence of SEQ ID NO: 3 is FIP, a forward internal primer
- the sequence of SEQ ID NO: 4 is BIP, a reverse internal primer.
- the sequence of SEQ ID NO: 5 may be LF, a forward loop primer
- SEQ ID NO: 6 may be LB, a reverse loop primer.
- the present invention may further include a probe capable of hybridizing complementary to the N gene amplification product of VHSV specifically isothermally amplified by the nucleic acid oligomer.
- the probe means an oligonucleotide capable of hybridizing with an amplification product under specific conditions.
- the conditions used to achieve a particular level of stringency vary depending on the nature of the nucleic acid being hybridized. For example, the length of the nucleic acid region to be hybridized, the degree of homology, the nucleotide sequence composition (e.g., GC/AT composition ratio), and the nucleic acid type (e.g., RNA, DNA) are considered in selecting hybridization conditions.
- the probe may include, for example, a portion of the sequence of SEQ ID NO:7.
- a fluorophore and/or quencher may bind to the primer or probe.
- fluorescent substances can be identified and reaction results can be monitored in real time.
- they may be labeled with radioisotopes, fluorescent compounds, bioluminescent compounds, chemiluminescent compounds, metal chelates, or enzymes. Labeling is a technique widely known in the field, and can be performed through conventional methods.
- the amount of the amplification product can be detected by a fluorescence signal.
- the intercalating method uses an intercalator that binds to the double-stranded DNA of the amplification product to which the probe is bound and displays fluorescence.
- the 5' end is labeled with a fluorophore and the 3' end is labeled with a quencher.
- the fluorophores include FAM (6-carboxyfluorescein), Texas Red, fluorescein, HEX (2',4',5',7'-tetrachloro-6-carboxy-4,7-dichlorofluorescein) , fluorescein chlorotriazinyl, rhodamine green, rhodamine red, tetramethylrhodamine, FITC (fluorescein isothiocyanate), Oregon green, Alexa Fluor (alexa fluor), JOE (6-Carboxy-4',5'-Dichloro-2',7'-Dimethoxyfluorescein), ROX (6-Carboxyl-X-Rhodamine), TET (Tetrachloro-Fluorescein), TRITC ( It may be any one selected from the group consisting of tertramethylrodamine isothiocyanate), TAMRA (6-carboxytetramethyl-rhodamine), NED (N-
- the quenching agent includes TAMRA (6-carboxytetramethyl-rhodamine), BHQ1 (black hole quencher 1), BHQ2 (black hole quencher 2), BHQ3 (black hole quencher 3), NFQ (nonfluorescent quencher), dabcyl, Eclipse, It may be any one selected from the group consisting of DDQ (Deep Dark Quencher), Blackberry Quencher, and Iowa black, but is not limited thereto.
- TAMRA 6-carboxytetramethyl-rhodamine
- BHQ1 black hole quencher 1
- BHQ2 black hole quencher 2
- BHQ3 black hole quencher 3
- NFQ nonfluorescent quencher
- dabcyl Eclipse
- It may be any one selected from the group consisting of DDQ (Deep Dark Quencher), Blackberry Quencher, and Iowa black, but is not limited thereto.
- it may further include gRNA and nuclease that specifically bind to the N gene of the VHSV or nucleic acid encoding the same, and reporter single-stranded DNA labeled with a fluorescence and a quencher.
- the gRNA may comprise a portion of the sequence UAAUUUCUACUAAGUGUAGAUUCCACCGAGUACUUGGUCAA of SEQ ID NO:7.
- the gRNA can recognize and specifically bind to the N gene part of VHSV or its complementary sequence, and when recognized through nuclease, it collaterally cleaves (collateral cleavage or trans-cleavage) all surrounding single-stranded DNA. It refers to the base sequence that allows The gRNA has sequence complementarity of at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% with the nucleotide sequence of the complementary strand of the DNA strand where the PAM sequence is located. It refers to a nucleotide sequence and can be combined with the nucleotide sequence of the complementary strand.
- a nuclease system that causes incidental cleavage such as Cas12, Cas13, or Cas14
- the surrounding single-stranded DNA is incidentally cut, so a single reporter labeled with fluorescence and a quencher is used.
- the strand of DNA is cut, and as a result, a quenched fluorescence signal is generated, which can be used as an indicator to recognize the isothermal amplification product.
- the gRNA may be one or more selected from the group consisting of CRISPR RNA (crRNA), trans-activating crRNA (tracrRNA), and single guide RNA (sgRNA).
- crRNA and tracrRNA are linked to each other. It may be a bound double-stranded crRNA:tracrRNA complex, or a single-stranded guide RNA (sgRNA) in which the crRNA or a portion thereof and the tracrRNA or a portion thereof are linked by an oligonucleotide linker.
- the nuclease may be a nucleic acid cutting enzyme capable of cutting DNA double helix.
- the nuclease may be a Cas protein that causes collateral cleavage, but is not limited thereto.
- the Cas protein may be a nuclease having a secondary cleavage function, such as Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas12g, Cas12h, Cas12i, Cas13a, Cas13b, Cas13c, Cas13d, Cas14, Csx17, Csx14, Csx10, Csx16, etc. It is not limited.
- the Cas protein is Corynebacter, Sutterella, Legionella, Treponema, Filifactor, Eubacterium, and Streptococcus (Streptococcus pyogenes) , Lactobacillus, Mycoplasma, Bacteroides, Flaviivola, Flavobacterium, Azospirillum, Gluconacetobacter , Neisseria, Roseburia, Parvibaculum, Staphylococcus (Staphylococcus aureus), Nitratifractor, Corynebacterium and Campylobacter. ), and may be simply isolated or recombinant from a microbial genus containing an ortholog of a Cas protein selected from the group consisting of.
- a PAM (protospacer associated motif) sequence is required to help the nucleic acid cutting enzyme recognize the target DNA.
- the PAM sequence is TTTV
- the Cas9 protein which does not cause collateral cleavage but is widely utilized in CRISPR gene editing technology, is from Streptococcus pyogenes
- the PAM sequence is 5'-NGG-3' (N is A, T, G, or C)
- the Cas9 protein is from Streptococcus thermophilus
- the PAM sequence is 5'-NNAGAAW- 3' (N is A, T, G, or C)
- the Cas9 protein is from Staphylococcus aureus
- the PAM sequence is 5'-NNGRRT-3' (N is A, T, G, or C and R is A or G)
- the Cas9 protein is from Campylobacter jejuni
- the PAM sequence is 5'-NNNNRYAC-3' ( N)
- the present invention relates to a kit containing the composition.
- the kit may include a compartmentalized carrier means for holding a sample, a container containing a reagent, and a container containing a nucleic acid oligomer.
- a container containing a probe for detecting each gene amplification product may be additionally included.
- the carrier means is suitable to contain one or more containers, such as bottles or tubes, each container containing independent components used in the method of the invention.
- containers such as bottles or tubes
- a person skilled in the art can easily dispense the required agent in the container.
- the kit according to the present invention may optionally include reagents necessary for carrying out an isothermal nucleic acid amplification reaction, such as enzymes and buffers.
- the kit according to the present invention may also further include various polynucleotide molecules, various buffers and reagents.
- the optimal amount of reagents, buffers or reactants used for a particular reaction in the kit can be determined by a person skilled in the art, and can be manufactured in separate packaging or compartments containing each of the aforementioned primers or probes. .
- the present invention also includes the step of treating the sample with a nucleic acid oligomer capable of specifically isothermally amplifying the nucleoprotein coding gene (N gene) of Viral Hemorrhagic Septicemia Virus (VHSV).
- N gene nucleoprotein coding gene of Viral Hemorrhagic Septicemia Virus
- the sample may include a wide range of biological fluids obtained from body fluids, cell lines, tissue cultures, etc. derived from an individual to be diagnosed, and is not limited as long as it is a sample containing the VHSV genome.
- a step of extracting nucleic acids from the sample may be further included, and extraction of nucleic acids may be performed using, for example, various commercially available kits or extraction reagents.
- Example 1 Isothermal amplification primer design for VHSV detection
- VHSV viral hemorrhagic sepsis virus
- LAMP loop-mediated isothermal amplification reaction
- VHSV viral hemorrhagic sepsis virus
- the reaction time at which the primer set targeting the base sequence region of the prepared nucleoprotein gene did not appear to be a non-specific reaction was confirmed. Since this example is to check for non-specific reactions between primer sets, only the primer set and isothermal reaction reagents without target RNA were added and the generation of amplification products over time was checked at 65 degrees, the general loop-mediated isothermal amplification reaction temperature.
- Viral RNA prepared through extraction and purification from the specimen was used as a template.
- RNA template prepared by extraction and purification from the specimen using a conventional method
- 2.5 ⁇ l of 10-fold concentration isothermal amplification reaction buffer (20mM Tris-HCl, 10mM ( NH4 ) 2SO4 , 50mM KCl, 2 mM MgSO 4 , 0,1 % Tween® 20, pH 8.8@25 °C), 100 mM MgSO 4 1.5 ⁇ l, 10 mM dNTP mix 3.5 ⁇ l, primer mix 5 ⁇ l (actual reaction concentration F3/B3 primer 1.6 M, FIP/ Mix BIP 0.2 ⁇ M, LF/LB 0.4 ⁇ M), isothermal amplification enzyme 1 ⁇ l (8 units), reverse transcriptase 0.5 ⁇ l (7.5 units), 8 M betaine 2.5 ⁇ l, and RNAse free water 7.5 ⁇ l for isothermal amplification in a final volume of 25 ⁇ l.
- a mixed solution for reaction was prepared.
- the mixture was reacted for 30 minutes at various temperatures of 56°C, 59°C, 62°C, 65°C, 68°C, 71°C, and 74°C, and then the final LAMP product was obtained.
- VHSV viral hemorrhagic sepsis virus
- RNA isolated from a positive or negative viral hemorrhagic sepsis sample was prepared by serial dilution from 50 pM to 50 aM, loop-mediated isothermal amplification reaction temperature and reaction time were 68°C and 30 minutes, and colorimetric analysis was performed. For this, hydroxy naphthol blue was added.
- Hydroxynaphthol blue is a marker that binds to magnesium ions used in loop-mediated isothermal amplification reactions. When bound to magnesium ions, it appears purple, and as magnesium ions are used in the loop-mediated isothermal amplification reaction, it binds to magnesium ions. When this decreases, the color changes to blue. Hydroxy naphthol blue is a marker that can easily check the pattern of loop-mediated isothermal amplification because it does not affect the loop-mediated isothermal amplification process even if it is added together with other buffer solutions or enzymes when adding the first reagent.
- RNA template prepared by extraction and purification, 2.5 ⁇ l of 10-fold concentration isothermal amplification reaction buffer (20mM Tris-HCl, 10mM ( NH4 ) 2SO4 , 50mM KCl , 2mMMgSO4 , 0,1 % Tween® 20, pH 8.8@25 °C), 100 mM MgSO 4 1.5 ⁇ l, 10 mM dNTP mix 3.5 ⁇ l, primer mix 5 ⁇ l (actual reaction concentration F3/B3 primer 1.6 ⁇ M, FIP/BIP 0.2 ⁇ M, LF/LB 0.4 ⁇ M), 1 ⁇ l (8 units) of isothermal amplification enzyme, 0.5 ⁇ l (7.5 units) of reverse transcriptase, 2.5 ⁇ l of 8 M betaine, 2.5 ⁇ l of 1.5 mM hydroxynaphthol blue, and 5 ⁇ l of RNAse free water, and mix isothermal in a final volume of 25 ⁇ l.
- a mixed solution for amplification reaction was
- a detection limit of 500 fM can be confirmed through loop-mediated isothermal amplification.
- LAMP loop-mediated isothermal amplification reaction
- Viral hemorrhagic sepsis virus RNA isolated by a conventional method was prepared by serial dilution from 5 fM to 50 aM, the loop-mediated isothermal amplification reaction temperature and reaction time were 68°C and 30 minutes, and the gene scissors reaction was performed at 37°C for 10 minutes. Minutes progressed.
- RNA template prepared by extraction and purification for loop-mediated isothermal amplification, 2.5 ⁇ l of 10-fold concentration isothermal amplification reaction buffer (20 mM Tris-HCl, 10 mM (NH 4 ) 2 SO 4 , 50 mM KCl, 2 mM MgSO 4 , 0,1 % Tween® 20, pH 8.8@25 °C), 1.5 ⁇ l of 100 mM MgSO 4 , 3.5 ⁇ l of 10 mM dNTP mix, 5 ⁇ l of primer mix (actual reaction concentration F3/B3 primer 1.6 M, FIP/ Mix BIP 0.2 ⁇ M, LF/LB 0.4 ⁇ M), isothermal amplification enzyme 1 ⁇ l (8 units), reverse transcriptase 0.5 ⁇ l (7.5 units), 8 M betaine 2.5 ⁇ l, and RNAse free water 7.5 ⁇ l for isothermal amplification in a final volume of 25 ⁇ l. A mixed solution for reaction was prepared.
- the detection limit could be lowered to 50 aM by adding the gene scissors reaction, which is 10,000 times lower than the detection limit of 500 fM obtained only with the existing loop-mediated isothermal amplification.
- VHSV diagnosis system can be constructed more simply without complex heat treatment equipment. do.
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Molecular Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biomedical Technology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Immunology (AREA)
- Analytical Chemistry (AREA)
- Virology (AREA)
- Plant Pathology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention relates to a composition for detecting the infection of viral hemorrhagic septicemia virus (VHSV), a kit comprising the composition, and a method for diagnosing the infection of VHSV using same. Specifically, the present invention relates to a composition for diagnosing the infection of VHSV, a kit comprising the composition, and a method for diagnosing the infection of VHSV using same, wherein the composition includes a nucleic acid oligomer that can target and specifically amplify the N gene, which is a coding gene for VHSV nucleoprotein.
Description
본 발명은 바이러스성출혈성패혈증바이러스(VHSV, Viral Hemorrhagic Septicemia Virus) 감염 여부를 검출하기 위한 조성물, 상기 조성물을 포함하는 키트 및 이를 이용하여 VHSV의 감염 여부를 진단하는 방법에 관한 것이다. 구체적으로, 본 발명은 VHSV 핵단백질 (Nucleoprotein) 코딩 유전자인 N 유전자를 타겟으로 하여 특이적으로 증폭할 수 있는 핵산 올리고머를 포함하는 VHSV 감염 여부를 진단하기 위한 조성물, 상기 조성물을 포함하는 키트 및 이를 이용하여 VHSV 감염 여부를 진단하는 방법에 관한 것이다.The present invention relates to a composition for detecting Viral Hemorrhagic Septicemia Virus (VHSV) infection, a kit containing the composition, and a method for diagnosing VHSV infection using the same. Specifically, the present invention provides a composition for diagnosing VHSV infection containing a nucleic acid oligomer capable of specifically amplifying the N gene, a VHSV nucleoprotein coding gene, a kit containing the composition, and the same. This relates to a method of diagnosing VHSV infection using this method.
바이러스성출혈성패혈증(VHS: Viral hemorrhagic septicemia)은 유럽에서 양식되고 있는 연어과 어류와 동아시아에서 양식되고 있는 넙치에서 발생하는 질병으로 '수산생물질병관리법'에서 지정하고 있는 수산생물전염병으로, 양식 어종 및 자연산 해수 어류에도 전파될 수 있다. Viral hemorrhagic septicemia (VHS: Viral hemorrhagic septicemia) is a disease that occurs in salmonid fish farmed in Europe and flounder farmed in East Asia. It is an aquatic organism infectious disease designated by the 'Aquatic Products Disease Control Act', and affects farmed fish species and wild fish. It can also spread to marine fish.
어류의 VHSV 감염 예방을 위해 불활성화 백신을 개발하여 대응하고자 하나, 현재까지 개발된 VHSV 백신은 주사를 이용하는 백신으로, 백신 처리과정에 필요한 일시적인 마취 등의 과정은 어류에 스트레스를 유도할 수 있다. 또한 백신 처리는 숙련된 전문가의 작업이 필요하여 비용이 많이 소요되며, 접종이 필요한 어린 물고기는 주사 과정에서 장기 손상 등이 발생할 수 있어 기술적으로 용이하지 않다. 따라서 백신 처리 과정에서 어류에 스트레스를 주지 않으며, 어린 물고기에도 적용이 가능한 저비용 고효율의 백신 개발이 필요하며, 이에 가장 적합한 백신은 사료와 혼합하여 투여할 수 있는 경구용 백신이나, 현재까지 VHSV에 대한 면역을 유도할 수 있는 경구용 백신은 개발되어 있지 않다.We are trying to respond by developing an inactivated vaccine to prevent VHSV infection in fish, but the VHSV vaccines developed to date are vaccines that use injections, and processes such as temporary anesthesia required for the vaccine treatment process can induce stress in fish. In addition, vaccination is expensive as it requires the work of skilled experts, and it is not technically easy for young fish that require vaccination as organ damage may occur during the injection process. Therefore, it is necessary to develop a low-cost, high-efficiency vaccine that does not cause stress to fish during the vaccine treatment process and can be applied to young fish. The most suitable vaccine for this is an oral vaccine that can be administered by mixing with feed. An oral vaccine that can induce immunity has not been developed.
이러한 상황에서 VHSV 진단이 중요하게 고려되고 있다. VHSV 검출을 위해, 세포배양법, 면역항체검출법, 분자생물학적 진단법 등 여러 가지 진단법이 제시되어 있다.In these situations, VHSV diagnosis is considered important. To detect VHSV, several diagnostic methods have been proposed, including cell culture, immune antibody detection, and molecular biological diagnostic methods.
진단에 사용할 병원체를 확보하기 어려운 상황이거나 병원체가 고위험으로 분류되어 해당 실험실에서 보유 또는 다루기 어려운 상황이라면 진단법 개발에 많은 제한이 따를 수밖에 없고, 각 검체마다 병원체의 양적 차이가 있으므로 검출 민감도에 대한 유효성 평가가 제대로 수행되기에는 한계가 있었다. 실제 병원체를 검출하는데 사용될 핵산 농도 부족 및 핵산추출물 내 PCR 저해제의 유입으로 이어져 제대로 된 진단이 어려울 수 있다. 더욱이, 같은 목적의 진단 제품을 생산한다고 하더라도 유전자 검출 부위가 각각 다르므로 그 검출 감도 또한 달라진다. 이와 같은 한계로 인하여, PCR의 검출한계에 대해 정확한 표준 모델이 없는 실정이다.In situations where it is difficult to secure pathogens to be used for diagnosis, or when pathogens are classified as high risk and difficult to possess or handle in the relevant laboratory, there are bound to be many limitations in the development of diagnostic methods, and since there are quantitative differences in the pathogens for each sample, the effectiveness of detection sensitivity is evaluated. There were limits to its ability to be carried out properly. Proper diagnosis may be difficult due to insufficient nucleic acid concentration used to detect actual pathogens and the introduction of PCR inhibitors into nucleic acid extracts. Moreover, even if diagnostic products for the same purpose are produced, the gene detection sites are different, so the detection sensitivity also varies. Due to these limitations, there is no accurate standard model for the detection limit of PCR.
이러한 기술적 배경하에서, 본 출원의 발명자들은 VHSV의 핵단백질 (Nucleoprotein) 코딩 유전자 (N 유전자)를 타겟으로 하는 새로운 검출방법을 개발하였다.Under this technical background, the inventors of the present application developed a new detection method targeting the nucleoprotein coding gene (N gene) of VHSV.
본 배경기술 부분에 기재된 상기 정보는 오직 본 발명의 배경에 대한 이해를 향상시키기 위한 것이며, 이에 본 발명이 속하는 기술분야에서 통상의 지식을 가지는 자에게 있어 이미 알려진 선행기술에 대한 정보는 포함하지 않을 수 있다.The above information described in this background section is only for improving the understanding of the background of the present invention, and therefore does not include information about prior art already known to those skilled in the art to which the present invention pertains. You can.
본 발명의 목적은 앞서 언급한 종래기술의 문제점을 인식하고, 신속하게 VHSV 검출 결과를 제공할 수 있는 조성물, 키트 및 이를 이용한 검출방법을 제공하는 것이다.The purpose of the present invention is to recognize the problems of the prior art mentioned above and to provide a composition, a kit, and a detection method using the same that can quickly provide VHSV detection results.
상기 목적을 달성하기 위하여 본 발명은 바이러스성출혈성패혈증바이러스(VHSV, Viral Hemorrhagic Septicemia Virus)의 핵단백질 (Nucleoprotein) 코딩 유전자 (N 유전자)를 특이적으로 등온증폭할 수 있는 핵산 올리고머를 포함하는, VHSV 검출용 조성물에 관한 것이다. In order to achieve the above object, the present invention is a VHSV comprising a nucleic acid oligomer capable of specifically isothermal amplification of the nucleoprotein coding gene (N gene) of Viral Hemorrhagic Septicemia Virus (VHSV). It relates to a composition for detection.
또한, 상기 핵산 올리고머에 의해 특이적으로 등온증폭된 VHSV의 N 유전자 산물에 상보적으로 혼성화할 수 있는 프로브를 추가로 포함하는 것을 특징으로 할 수 있다. In addition, it may be characterized by further comprising a probe capable of hybridizing complementary to the N gene product of VHSV specifically isothermally amplified by the nucleic acid oligomer.
상기 VHSV의 N 유전자에 특이적으로 결합하는 가이드 RNA (gRNA) 및 뉴클레아제 또는 이를 코딩하는 핵산을 추가로 포함할 수 있다. It may further include a guide RNA (gRNA) that specifically binds to the N gene of the VHSV and a nuclease or a nucleic acid encoding the same.
본 발명은 또한, 상기 조성물을 포함하는 VHSV 검출용 키트에 관한 것이다. The present invention also relates to a kit for detecting VHSV comprising the composition.
본 발명은 더욱이, 바이러스성출혈성패혈증바이러스(VHSV, Viral Hemorrhagic Septicemia Virus)의 핵단백질 (Nucleoprotein) 코딩 유전자 (N 유전자)를 특이적으로 등온증폭할 수 있는 핵산 올리고머를 샘플에 처리하는 단계를 포함하는 VHSV 검출을 위한 정보 제공 방법에 관한 것이다. The present invention furthermore includes the step of treating the sample with a nucleic acid oligomer capable of specifically isothermally amplifying the nucleoprotein coding gene (N gene) of Viral Hemorrhagic Septicemia Virus (VHSV). This relates to a method of providing information for VHSV detection.
본 발명을 통해, 등온증폭 기반의 검출방법을 통해 빠른 시간 내에 결과 확인이 가능하여 효과적으로 VHSV를 검출할 수 있다. 복잡한 열처리 장비 없이 보다 간단하게 VHSV 진단 시스템을 구축할 수 있을 것으로 기대된다. Through the present invention, results can be confirmed quickly through an isothermal amplification-based detection method, making it possible to effectively detect VHSV. It is expected that a VHSV diagnostic system can be built more simply without complex heat treatment equipment.
도 1은 본 발명에서 주요 타겟서열로 지정한 VHSV의 nucleoprotein gene의 일부 및 루프매개 등온증폭을 위한 프라이머 세트의 결합부위를 표현하고 있다. 아울러, 등온증폭 산물에 대해 진행하게 되는 유전자 가위 진단에 가장 핵심적인 가이드 RNA (gRNA)의 인식 부위 또한 표시하고 있어, 도 1을 통하여 표적 유전자와 이의 검출을 위한 프라이머, gRNA의 결합부위를 확인할 수 있다. Figure 1 shows a portion of the VHSV nucleoprotein gene designated as the main target sequence in the present invention and the binding site of the primer set for loop-mediated isothermal amplification. In addition, the recognition site of the guide RNA (gRNA), which is the most important in the diagnosis of gene scissors performed on the isothermal amplification product, is also indicated. Through Figure 1, the target gene, the primer for its detection, and the binding site of the gRNA can be identified. there is.
도 2는 본 발명을 통해 개발한 신규 프라이머의 특이적 반응성 여부를 확인하기 위한 실험 결과이다. 25분부터 45분까지 시간을 달리하여 프라이머가 비특이적 산물을 만들지 않는 시간 조건을 찾기 위한 실험 결과이다. Figure 2 shows the results of an experiment to confirm the specific reactivity of the new primer developed through the present invention. This is the result of an experiment to find the time conditions under which primers do not produce non-specific products by varying the time from 25 to 45 minutes.
도 3은 신규 프라이머를 이용하여 표적 바이러스 RNA를 루프매개 등온증폭 할 때의 최적의 온도 조건을 찾기 위한 실험 결과이다. 증폭 산물에 형광 표지자 SYBR Green I를 섞어서 반응여부를 확인하는 방법과 전기영동을 통하여 확인하였다. Figure 3 shows the results of an experiment to find the optimal temperature conditions when loop-mediated isothermal amplification of target viral RNA using a new primer. The reaction was confirmed by mixing the amplification product with the fluorescent marker SYBR Green I and using electrophoresis.
도 4는 신규 프라이머를 이용한 루프매개 등온증폭을 실시했을 때의 검출한계를 확인하기 위한 실험 결과이다. 실험결과는 전기영동과 하이드록시 나프톨 블루를 이용한 색도 분석법을 통하여 확인하였다. Figure 4 shows the results of an experiment to confirm the detection limit when loop-mediated isothermal amplification using a new primer was performed. The experimental results were confirmed through electrophoresis and colorimetric analysis using hydroxy naphthol blue.
도 5는 루프매개 등온증폭 산물에 대한 유전자 가위 진단을 적용한 실험 결과로 형광신호를 측정하여 검출한계를 확인하였다. Figure 5 shows the results of an experiment applying gene scissors diagnosis to the loop-mediated isothermal amplification product, and the detection limit was confirmed by measuring the fluorescence signal.
다른 식으로 정의되지 않는 한, 본 명세서에서 사용된 모든 기술적 및 과학적 용어들은 본 발명이 속하는 기술분야에서 숙련된 전문가에 의해서 통상적으로 이해되는 것과 동일한 의미를 갖는다. 일반적으로, 본 명세서에서 사용된 명명법은 본 기술분야에서 잘 알려져 있고 통상적으로 사용되는 것이다.Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by a person skilled in the art to which the present invention pertains. In general, the nomenclature used herein is well known and commonly used in the art.
본 발명은 바이러스성출혈성패혈증바이러스(VHSV)를 진단할 수 있는 역전사 고리매개 등온증폭반응(Reverse Transcriptional Loop-mediated isothermal amplification, RT-LAMP)과 유전자 가위 진단 기술 (CRISPR/Cas)을 활용한 것으로, VHSV의 핵단백질을 코딩하는 유전자 영역에 특이적인 등온증폭용 프라이머 세트와 등온증폭 산물을 특이적으로 식별할 수 있는 gRNA를 설계한 후 VHSV 의 시료를 대상으로 역전사 등온증폭반응 및 유전자 가위 진단 기술을 적용한 결과, 50 aM 수준의 VHSV의 RNA 핵산을 검출할 수 있었다.The present invention utilizes Reverse Transcriptional Loop-mediated isothermal amplification (RT-LAMP) and gene scissors diagnostic technology (CRISPR/Cas), which can diagnose viral hemorrhagic sepsis virus (VHSV). After designing a primer set for isothermal amplification specific to the gene region encoding the nuclear protein of VHSV and a gRNA that can specifically identify the isothermal amplification product, reverse transcription isothermal amplification reaction and gene scissors diagnostic technology were performed on VHSV samples. As a result of application, it was possible to detect RNA nucleic acid of VHSV at a level of 50 aM.
본 발명에 따르면, 루프 매개 등온증폭과 유전자 가위 기술을 적용한 VHSV 진단 방법은 간단한 열처리 기기 만으로 분자진단 검사 결과를 40분 이내에 확인할 수 있으므로, 현행 리얼타임 PCR (Real time PCR) 검사법이 증폭시간만 2시간 정도 소요되는데 비해 신속하게 검사 결과를 제공할 수 있어, VHSV 감염증의 진단에 유용하게 활용될 수 있다.According to the present invention, the VHSV diagnosis method applying loop-mediated isothermal amplification and gene scissor technology can confirm molecular diagnostic test results within 40 minutes with only a simple heat treatment device, so the current real-time PCR test method requires only 2 amplification time. Although it takes about a while, it can provide test results quickly, so it can be useful in diagnosing VHSV infection.
이러한 관점에서, 본 발명은 VHSV의 핵단백질 (Nucleoprotein) 코딩 유전자 (N 유전자)를 특이적으로 등온증폭할 수 있는 핵산 올리고머와 등온 증폭 산물을 특이적으로 식별할 수 있는 gRNA를 포함하는, VHSV 검출용 조성물에 관한 것이다. From this point of view, the present invention is a VHSV detection comprising a nucleic acid oligomer capable of specifically isothermal amplification of the nucleoprotein coding gene (N gene) of VHSV and a gRNA capable of specifically identifying the isothermal amplification product. It relates to a composition for use.
본 명세서에서 핵산 올리고머는 핵산을 단량체로 하여 중합하여 생성되는 2개 이상의 뉴클레오타이드를 포함하는 물질을 의미할 수 있다. 상기 핵산 올리고머는 프라이머 또는 프로브로 기능할 수 있다. As used herein, nucleic acid oligomer may refer to a material containing two or more nucleotides produced by polymerizing nucleic acids as monomers. The nucleic acid oligomer may function as a primer or probe.
본 명세서에서 '프라이머'는 적합한 온도 및 완충액 내에서 적합한 조건(4종의 다른 뉴클레오사이드 트리포스페이트 및 중합반응 효소) 하에서 주형-지시 DNA 합성의 개시점으로 작용할 수 있는 단일-가닥 올리고뉴클레오타이드를 의미한다. 상기 프라이머는 증폭될 유전자 로커스의 각 가닥과 "대체적으로" 상보성을 가지도록 제작될 수 있다. 이는 중합반응을 수행하는 조건에서 프라이머가 대응하는 핵산 가닥과 혼성화 되기에 충분한 상보성을 가지는 것을 의미한다.As used herein, 'primer' refers to a single-stranded oligonucleotide that can act as an initiation point for template-directed DNA synthesis under suitable conditions (four different nucleoside triphosphates and polymerization enzymes) at a suitable temperature and buffer. do. The primers may be designed to have “general” complementarity with each strand of the gene locus to be amplified. This means that the primer has sufficient complementarity to hybridize with the corresponding nucleic acid strand under the conditions for performing the polymerization reaction.
상기 등온증폭은 '고리매개등온증폭(Loop-mediated isothermal amplification, LAMP)'을 의미하고, 기존 PCR(polymerase chain reaction) 법과 달리 등온의 조건에서 증폭 반응을 수행할 수 있는 방법이다. LAMP 반응을 위해서는 기본적으로 4종의 프라이머(F3, B3, FIP, BIP)가 필요하고, 반응 속도를 향상시키기 위해 2종의 프라이머(LF, LB)를 추가하여 최종 6종의 각기 다른 염기서열로 이루어진 올리고뉴클레오타이드 프라이머가 반응에 필요할 수 있다. The isothermal amplification refers to 'Loop-mediated isothermal amplification (LAMP)', and unlike the existing PCR (polymerase chain reaction) method, it is a method that can perform an amplification reaction under isothermal conditions. Basically, 4 types of primers (F3, B3, FIP, BIP) are required for the LAMP reaction, and to improve the reaction speed, 2 types of primers (LF, LB) are added to produce 6 different base sequences. Oligonucleotide primers may be required for the reaction.
4종의 기본 프라이머는 외부(outer) 프라이머 2종과 내부(inner) 프라이머 2종으로 구성되며, "내부 프라이머(inner primer)"는 주형 DNA에 결합하여 새로운 DNA 사슬 합성의 시작점으로 작용할 수 있는 단일가닥 올리고뉴클레오타이드를 의미하고, "외부 프라이머(outer primer)"는 내부 프라이머가 주형 DNA에 결합한 위치보다 더 바깥쪽에서 주형 DNA에 결합하는 단일가닥 올리고뉴클레오타이드를 의미하는 것으로, 내부 프라이머가 주형 DNA에 결합하여 DNA 사슬이 신장된 이후, 외부 프라이머와 주형 DNA의 결합으로 사슬 변위(strand displacement)가 발생하여 먼저 형성된 사슬이 떨어져 나오게 된다. "고리 프라이머(loop primer)"는 상기 내부 프라이머 및 외부 프라이머가 주형 DNA에 결합하여 형성된 초기 줄기 고리(stem loop) 구조 사슬이 고리(loop) 구조 생성 횟수를 증가시켜 결과적으로 전체 반응을 가속화시킬 수 있도록 하는, 뉴클레오타이드 합성의 시작점으로서 작용할 수 있는 단일가닥 올리고뉴클레오타이드를 의미한다.The four types of basic primers consist of two types of outer primers and two types of inner primers, and the “inner primer” is a single primer that can bind to template DNA and act as a starting point for new DNA chain synthesis. refers to a stranded oligonucleotide, and "outer primer" refers to a single-stranded oligonucleotide that binds to the template DNA from a position further outside than the position where the internal primer binds to the template DNA. After the DNA chain is extended, strand displacement occurs due to the combination of the external primer and the template DNA, causing the previously formed chain to separate. “Loop primer” is an initial stem loop structure chain formed by binding the inner primer and outer primer to the template DNA, which increases the number of loop structure creations and can ultimately accelerate the overall reaction. It refers to a single-stranded oligonucleotide that can act as a starting point for nucleotide synthesis.
외부 프라이머는 정방향 외부(forward outer, F3) 프라이머와 역방향 외부(backward outer, B3) 프라이머 2종으로 구성되고 반응의 비순환기(non-cyclic step) 동안 DNA 이중 가닥을 풀어주는 역할을 한다. 내부 프라이머는 정방향 내부 프라이머(forward inner primer, FIP)와 역방향 내부 프라이머(backward inner primer, BIP) 2종으로 구성되고 고리 매개 등온증폭 반응에 필수적인 고리(loop)를 만들 수 있도록 정방향 및 역방향 염기서열에 해당하는 뉴클레오타이드로 구성된다. 추가 2종의 프라이머는 정방향 고리(forward loop, LF) 프라이머와 역방향 고리 (backward loop, LB) 프라이머 2종으로 구성되며 내부(inner) 프라이머가 결합하지 않는 염기서열에 부착하여 고리매개등온증폭 반응을 가속화시킨다.The outer primer consists of two types of primers, the forward outer (F3) primer and the reverse outer (B3) primer, and plays a role in unwinding the DNA double strand during the non-cyclic step of the reaction. The internal primer consists of two types, the forward inner primer (FIP) and the reverse inner primer (BIP), and is attached to the forward and reverse base sequences to create a loop essential for the loop-mediated isothermal amplification reaction. It is composed of the corresponding nucleotides. The additional two types of primers are composed of a forward loop (LF) primer and a backward loop (LB) primer, and attach to a base sequence that the inner primer does not bind to, thereby carrying out a ring-mediated isothermal amplification reaction. accelerate.
하나의 실시예에서, 상기 VHSV의 N 유전자를 특이적으로 등온증폭할 수 있는 핵산 올리고머는 서열번호 1 내지 서열번호 6으로 구성된 군에서 선택되는 서열을 포함하는 프라이머일 수 있다. In one embodiment, the nucleic acid oligomer capable of specifically isothermal amplification of the N gene of VHSV may be a primer containing a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 6.
상기 프라이머는 예를 들어, 증폭을 위해 정방향 및 역방향 프라이머를 쌍으로 만들어서 동시에 사용할 수 있다. 서열번호 1의 서열은 정방향 외부 프라이머인 F3이고, 서열번호 2의 서열은 역방향 외부 프라이머인 B3이고, 서열번호 3의 서열은 정방향 내부 프라이머인 FIP이며, 서열번호 4의 서열은 역방향 내부 프라이머인 BIP이며, 서열번호 5의 서열은 정방향 고리 프라이머인 LF이고, 서열번호 6은 역방향 고리 프라이머인 LB일 수 있다. The primers can be used simultaneously, for example, by pairing forward and reverse primers for amplification. The sequence of SEQ ID NO: 1 is F3, a forward external primer, the sequence of SEQ ID NO: 2 is B3, a reverse external primer, the sequence of SEQ ID NO: 3 is FIP, a forward internal primer, and the sequence of SEQ ID NO: 4 is BIP, a reverse internal primer. The sequence of SEQ ID NO: 5 may be LF, a forward loop primer, and SEQ ID NO: 6 may be LB, a reverse loop primer.
본 발명은 상기 핵산 올리고머에 의해 특이적으로 등온증폭된 VHSV의 N 유전자 증폭산물에 상보적으로 혼성화할 수 있는 프로브를 추가로 포함할 수 있다. The present invention may further include a probe capable of hybridizing complementary to the N gene amplification product of VHSV specifically isothermally amplified by the nucleic acid oligomer.
상기 프로브와 관련하여, 특정 조건에서 증폭 산물과 혼성화될 수 있는 올리고뉴클레오타이드를 의미한다.In relation to the probe, it means an oligonucleotide capable of hybridizing with an amplification product under specific conditions.
혼성화 반응에서, 엄격한 특정 수준을 달성하기 위하여 사용되는 조건은 혼성화 되는 핵산의 성질에 따라 다양하다. 예를 들면, 혼성화 되는 핵산 부위의 길이, 상동성 정도, 뉴클레오타이드 서열 조성(예를 들면, GC/AT 조성비) 및 핵산 타입(예를 들면, RNA, DNA)등이 혼성화 조건을 선택하는데 고려된다. In hybridization reactions, the conditions used to achieve a particular level of stringency vary depending on the nature of the nucleic acid being hybridized. For example, the length of the nucleic acid region to be hybridized, the degree of homology, the nucleotide sequence composition (e.g., GC/AT composition ratio), and the nucleic acid type (e.g., RNA, DNA) are considered in selecting hybridization conditions.
하나의 실시예에서, 상기 프로브는 예를 들어, 서열번호 7의 서열의 일부를 포함할 수 있다. In one embodiment, the probe may include, for example, a portion of the sequence of SEQ ID NO:7.
경우에 따라서, 상기 프라이머 또는 프로브에 형광자 및/또는 소광자가 결합할 수 있다. 이를 통해 형광 물질을 확인하여 반응 결과를 실시간으로 모니터링할 수 있다. 예를 들면 방사선 동위원소, 형광 화합물, 바이오 발광 화합물, 화학 발광 화합물, 금속 킬레이트 또는 효소로 표지될 수 있다. 표지하는 것은 당해 분야에서 널리 알려진 기술이며, 통상적인 방법을 통하여 수행할 수 있다.In some cases, a fluorophore and/or quencher may bind to the primer or probe. Through this, fluorescent substances can be identified and reaction results can be monitored in real time. For example, they may be labeled with radioisotopes, fluorescent compounds, bioluminescent compounds, chemiluminescent compounds, metal chelates, or enzymes. Labeling is a technique widely known in the field, and can be performed through conventional methods.
상기 증폭 산물의 양은 형광신호에 의해 검출할 수 있다. 프로브가 결합된 증폭산물의 이중 나선 DNA에 결합하여 형광을 나타내는 시약(intercalator)을 사용하는 인터컬레이팅(Intercalating)법, 5' 말단은 형광자, 3' 말단은 소광자(quencher)로 표지된 올리고뉴클레오타이드를 사용하는 방법 등이 있다. The amount of the amplification product can be detected by a fluorescence signal. The intercalating method uses an intercalator that binds to the double-stranded DNA of the amplification product to which the probe is bound and displays fluorescence. The 5' end is labeled with a fluorophore and the 3' end is labeled with a quencher. There is a method using oligonucleotides.
상기 형광자는 FAM(6-carboxyfluorescein), 텍사스 레드(texas red), 플루오레신(fluorescein), HEX(2',4',5',7'-tetrachloro-6-carboxy-4,7-dichlorofluorescein), 플루오레신 클로로트리아지닐(fluorescein chlorotriazinyl), 로다민 그린(rhodamine green), 로다민 레드(rhodamine red), 테트라메틸로다민(tetramethylrhodamine), FITC(fluorescein isothiocyanate), 오레곤 그린(oregon green), 알렉사 플루오로(alexa fluor), JOE(6-Carboxy-4',5'-Dichloro-2',7'-Dimethoxyfluorescein), ROX(6-Carboxyl-X-Rhodamine), TET(Tetrachloro-Fluorescein), TRITC(tertramethylrodamine isothiocyanate), TAMRA(6-carboxytetramethyl-rhodamine), NED(N-(1-Naphthyl) ethylenediamine), 시아닌(Cyanine) 계열 염료 및 씨아디카르보시아닌(thiadicarbocyanine)으로 구성된 군으로부터 선택되는 어느 하나일 수 있으나, 이에 제한되는 것은 아니다.The fluorophores include FAM (6-carboxyfluorescein), Texas Red, fluorescein, HEX (2',4',5',7'-tetrachloro-6-carboxy-4,7-dichlorofluorescein) , fluorescein chlorotriazinyl, rhodamine green, rhodamine red, tetramethylrhodamine, FITC (fluorescein isothiocyanate), Oregon green, Alexa Fluor (alexa fluor), JOE (6-Carboxy-4',5'-Dichloro-2',7'-Dimethoxyfluorescein), ROX (6-Carboxyl-X-Rhodamine), TET (Tetrachloro-Fluorescein), TRITC ( It may be any one selected from the group consisting of tertramethylrodamine isothiocyanate), TAMRA (6-carboxytetramethyl-rhodamine), NED (N-(1-Naphthyl) ethylenediamine), cyanine dyes, and thiadicarbocyanine. However, it is not limited to this.
상기 소광제는 TAMRA(6-carboxytetramethyl-rhodamine), BHQ1(black hole quencher 1), BHQ2(black hole quencher 2), BHQ3(black hole quencher 3), NFQ(nonfluorescent quencher), 답실(dabcyl), Eclipse, DDQ(Deep Dark Quencher), 블랙베리 퀸처(Blackberry Quencher), 아이오와 블랙(Iowa black)으로 구성된 군으로부터 선택되는 어느 하나일 수 있으나, 이에 제한되는 것은 아니다.The quenching agent includes TAMRA (6-carboxytetramethyl-rhodamine), BHQ1 (black hole quencher 1), BHQ2 (black hole quencher 2), BHQ3 (black hole quencher 3), NFQ (nonfluorescent quencher), dabcyl, Eclipse, It may be any one selected from the group consisting of DDQ (Deep Dark Quencher), Blackberry Quencher, and Iowa black, but is not limited thereto.
경우에 따라서, 상기 VHSV의 N 유전자에 특이적으로 결합하는 gRNA 및 뉴클레아제 또는 이를 코딩하는 핵산, 형광과 소광자가 표지된 리포터 단일 가닥 DNA 를 추가로 포함할 수 있다. gRNA는 서열번호 7의 서열 UAAUUUCUACUAAGUGUAGAUUCCACCGAGUACUUGGUCAA의 일부를 포함할 수 있다In some cases, it may further include gRNA and nuclease that specifically bind to the N gene of the VHSV or nucleic acid encoding the same, and reporter single-stranded DNA labeled with a fluorescence and a quencher. The gRNA may comprise a portion of the sequence UAAUUUCUACUAAGUGUAGAUUCCACCGAGUACUUGGUCAA of SEQ ID NO:7.
상기 gRNA는 VHSV의 N 유전자 부분 또는 이의 상보서열을 인식하여 특이적으로 결합할 수 있으며, 뉴클레아제를 통해 인식이 되면 주변의 모든 단일 가닥 DNA를 부수적으로 절단 (collateral cleavage 또는 trans-cleavage)할 수 있도록 하는 염기서열을 의미한다. 상기 gRNA는 PAM 서열이 위치하는 DNA 가닥의 상보적인 가닥의 뉴클레오타이드 서열과 90% 이상, 95% 이상, 96% 이상, 97% 이상, 98% 이상, 99% 이상, 또는 100%의 서열 상보성을 갖는 뉴클레오타이드 서열을 의미하는 것으로, 상기 상보적 가닥의 뉴클레오타이드 서열과 결합 가능하다. Cas12, Cas13, Cas14 등 부수적 절단을 유발하는 뉴클레아제 계통을 사용하는 경우 gRNA에 의해서 등온증폭 산물이 인식이 되면 주변에 있는 단일 가닥 DNA를 부수적으로 절단하게 되므로, 형광과 소광자가 표지된 리포터 단일가닥 DNA가 절단되고, 이에 따라 소광되었던 형광신호가 발생하게 되어 등온증폭산물을 인식할 수 있는 지표로 사용할 수 있게 된다. The gRNA can recognize and specifically bind to the N gene part of VHSV or its complementary sequence, and when recognized through nuclease, it collaterally cleaves (collateral cleavage or trans-cleavage) all surrounding single-stranded DNA. It refers to the base sequence that allows The gRNA has sequence complementarity of at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% with the nucleotide sequence of the complementary strand of the DNA strand where the PAM sequence is located. It refers to a nucleotide sequence and can be combined with the nucleotide sequence of the complementary strand. When using a nuclease system that causes incidental cleavage, such as Cas12, Cas13, or Cas14, when the isothermal amplification product is recognized by gRNA, the surrounding single-stranded DNA is incidentally cut, so a single reporter labeled with fluorescence and a quencher is used. The strand of DNA is cut, and as a result, a quenched fluorescence signal is generated, which can be used as an indicator to recognize the isothermal amplification product.
상기 gRNA는 예를 들어, CRISPR RNA (crRNA), trans-activating crRNA (tracrRNA), 및 단일 가이드 RNA (single guide RNA; sgRNA)로 이루어진 군에서 선택된 1종 이상일 수 있으며, 구체적으로 crRNA와 tracrRNA가 서로 결합된 이중 가닥 crRNA:tracrRNA 복합체, 또는 crRNA 또는 그 일부와 tracrRNA 또는 그 일부가 올리고뉴클레오타이드 링커로 연결된 단일 가닥 가이드 RNA (sgRNA)일 수 있다.For example, the gRNA may be one or more selected from the group consisting of CRISPR RNA (crRNA), trans-activating crRNA (tracrRNA), and single guide RNA (sgRNA). Specifically, crRNA and tracrRNA are linked to each other. It may be a bound double-stranded crRNA:tracrRNA complex, or a single-stranded guide RNA (sgRNA) in which the crRNA or a portion thereof and the tracrRNA or a portion thereof are linked by an oligonucleotide linker.
상기 뉴클레아제는 DNA 이중나선 절단 가능한 핵산절단효소일 수 있다. 상기 뉴클레아제는 부수적 절단을 유발하는 Cas 단백질일 수 있으나, 이에 제한되는 것은 아니다. 상기 Cas 단백질은 Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas12g, Cas12h, Cas12i, Cas13a, Cas13b, Cas13c, Cas13d, Cas14, Csx17, Csx14, Csx10, Csx16 등 부수적 절단 기능을 가지는 뉴클레아제일 수 있으나, 이에 제한되는 것은 아니다.The nuclease may be a nucleic acid cutting enzyme capable of cutting DNA double helix. The nuclease may be a Cas protein that causes collateral cleavage, but is not limited thereto. The Cas protein may be a nuclease having a secondary cleavage function, such as Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas12g, Cas12h, Cas12i, Cas13a, Cas13b, Cas13c, Cas13d, Cas14, Csx17, Csx14, Csx10, Csx16, etc. It is not limited.
상기 Cas 단백질은 코리네박터(Corynebacter), 수테렐라(Sutterella), 레지오넬라(Legionella), 트레포네마(Treponema), 피리팩터(Filifactor), 유박테리움(Eubacterium), 스트렙토코커스(Streptococcus: Streptococcus pyogenes), 락토바실러스(Lactobacillus), 미코플라즈마(Mycoplasma), 박터로이드(Bacteroides), 플라비플라비이볼라(Flaviivola), 플라보박테리움(Flavobacterium), 아조스피릴룸(Azospirillum), 글루코나세토박터(Gluconacetobacter), 나이세리아(Neisseria), 로세부리아(Roseburia), 파비바큐럼(Parvibaculum), 스타필로코커스(Staphylococcus: Staphylococcus aureus), 니트라티프랙터(Nitratifractor), 코리네박터리움(Corynebacterium) 및 캠필로박터(Campylobacter)로 이루어지는 군으로부터 선택되는 Cas 단백질의 오소로그(ortholog)를 포함하는 미생물 속으로부터 유래하고, 이들로부터 단순 분리된 것 또는 재조합된 것일 수 있다.The Cas protein is Corynebacter, Sutterella, Legionella, Treponema, Filifactor, Eubacterium, and Streptococcus (Streptococcus pyogenes) , Lactobacillus, Mycoplasma, Bacteroides, Flaviivola, Flavobacterium, Azospirillum, Gluconacetobacter , Neisseria, Roseburia, Parvibaculum, Staphylococcus (Staphylococcus aureus), Nitratifractor, Corynebacterium and Campylobacter. ), and may be simply isolated or recombinant from a microbial genus containing an ortholog of a Cas protein selected from the group consisting of.
상기 핵산절단효소가 타겟 DNA를 인지할 수 있도록 도와주는 PAM (protospacer associated motif) 서열이 필요하다. 부수적 절단을 유발하는 Cas12a의 경우, PAM 서열은 TTTV 이고, 부수적 절단을 유발하지는 않지만 CRISPR 유전자 가위 기술에서 많이 활용되는 Cas9 단백질이 스트렙토코커스 피요게네스 (Streptococcus pyogenes) 유래의 것인 경우, PAM 서열은 5'-NGG-3' (N은 A, T, G, 또는 C임)이고, 상기 Cas9 단백질이 스트렙토코커스 써모필러스 (Streptococcus thermophilus) 유래의 것인 경우, 상기 PAM 서열은 5'-NNAGAAW-3' (N은 A, T, G, 또는 C임)이고, 상기 Cas9 단백질이 스타필로코커스 아우레우스 (Staphylococcus aureus) 유래의 것인 경우, 상기 PAM 서열은 5'-NNGRRT-3' (N은 A, T, G, 또는 C이고, R은 A 또는 G)이고, 상기 Cas9 단백질이 캄필로박터 제주니 (Campylobacter jejuni) 유래의 것인 경우, 상기 PAM 서열은 5'-NNNNRYAC-3' (N은 A, T, G, 또는 C이고, R은 A 또는 G 이고, Y는 C 또는 T임)일 수 있다. A PAM (protospacer associated motif) sequence is required to help the nucleic acid cutting enzyme recognize the target DNA. For Cas12a, which causes collateral cleavage, the PAM sequence is TTTV, and for the Cas9 protein, which does not cause collateral cleavage but is widely utilized in CRISPR gene editing technology, is from Streptococcus pyogenes , the PAM sequence is 5'-NGG-3' (N is A, T, G, or C), and if the Cas9 protein is from Streptococcus thermophilus , the PAM sequence is 5'-NNAGAAW- 3' (N is A, T, G, or C), and if the Cas9 protein is from Staphylococcus aureus , the PAM sequence is 5'-NNGRRT-3' (N is A, T, G, or C and R is A or G), and when the Cas9 protein is from Campylobacter jejuni , the PAM sequence is 5'-NNNNRYAC-3' ( N is A, T, G, or C, R is A or G, and Y is C or T).
본 발명은 다른 관점에서, 상기 조성물을 포함하는 키트에 관한 것이다. In another aspect, the present invention relates to a kit containing the composition.
하나의 실시 예에서, 상기 키트는 샘플을 담는 구획된 캐리어 수단, 시약을 포함하는 용기, 핵산 올리고머를 포함하는 용기를 포함할 수 있다. 경우에 따라서, 유전자 증폭 산물 각각을 검출하기 위한 프로브를 포함하는 용기를 추가로 포함할 수 있다.In one embodiment, the kit may include a compartmentalized carrier means for holding a sample, a container containing a reagent, and a container containing a nucleic acid oligomer. In some cases, a container containing a probe for detecting each gene amplification product may be additionally included.
상기 캐리어 수단은 병, 튜브와 같은 하나 이상의 용기를 함유하기에 적합하고, 각 용기는 본 발명의 방법에 사용되는 독립적 구성요소들을 함유한다. 본 발명의 명세서에서, 당해 분야의 통상의 지식을 가진 자는 용기 중의 필요한 제제를 손쉽게 분배할 수 있다.The carrier means is suitable to contain one or more containers, such as bottles or tubes, each container containing independent components used in the method of the invention. In the context of the present invention, a person skilled in the art can easily dispense the required agent in the container.
본 발명에 따른 키트는 경우에 따라, 효소, 버퍼 등과 같은 등온핵산 증폭 반응을 실시하는데 필요한 시약을 선택적으로 포함할 수 있다. 본 발명에 따른 키트는 또한 다양한 폴리뉴클레오타이드 분자, 다양한 버퍼 및 시약을 추가로 포함할 수 있다.In some cases, the kit according to the present invention may optionally include reagents necessary for carrying out an isothermal nucleic acid amplification reaction, such as enzymes and buffers. The kit according to the present invention may also further include various polynucleotide molecules, various buffers and reagents.
상기 키트에서 특정 반응을 위해 사용되는 시약, 버퍼 또는 반응물의 최적량은 당업자에 의해 결정될 수 있으며, 앞서 언급된 프라이머 또는 프로브 각각을 포함하는 별도의 포장 또는 컴파트먼트(compartment)로 제작될 수 있다.The optimal amount of reagents, buffers or reactants used for a particular reaction in the kit can be determined by a person skilled in the art, and can be manufactured in separate packaging or compartments containing each of the aforementioned primers or probes. .
본 발명은 또한, 바이러스성출혈성패혈증바이러스(VHSV, Viral Hemorrhagic Septicemia Virus)의 핵단백질 (Nucleoprotein) 코딩 유전자 (N 유전자)를 특이적으로 등온증폭할 수 있는 핵산 올리고머를 샘플에 처리하는 단계를 포함하는 VHSV 검출을 위한 정보 제공 방법에 관한 것이다. The present invention also includes the step of treating the sample with a nucleic acid oligomer capable of specifically isothermally amplifying the nucleoprotein coding gene (N gene) of Viral Hemorrhagic Septicemia Virus (VHSV). This relates to a method of providing information for VHSV detection.
하나의 실시예에서, 상기 샘플은 진단 대상 개체 유래의 체액, 세포주, 조직 배양 등에서 얻어지는 폭넓은 범위의 모든 생물학적 체액을 포함할 수 있고, VHSV 유전체를 포함하는 시료라면 제한되는 것은 아니다.In one embodiment, the sample may include a wide range of biological fluids obtained from body fluids, cell lines, tissue cultures, etc. derived from an individual to be diagnosed, and is not limited as long as it is a sample containing the VHSV genome.
상기 샘플로부터 핵산을 추출하는 단계를 추가로 포함할 수 있으며, 핵산의 추출은 예를 들어 상업화 되어 공급되고 있는 다양한 키트 또는 추출 시약을 사용하여 수행될 수 있다. A step of extracting nucleic acids from the sample may be further included, and extraction of nucleic acids may be performed using, for example, various commercially available kits or extraction reagents.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것으로서, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지는 않는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as limited by these examples.
실시예 1: VHSV 검출용 등온증폭 프라이머 설계Example 1: Isothermal amplification primer design for VHSV detection
루프 매개 등온증폭 방법을 사용하여 바이러스성출혈성패혈증 바이러스(VHSV)를 검출하기 위해 프라이머 설계 및 제작을 하였다. Primers were designed and produced to detect viral hemorrhagic sepsis virus (VHSV) using a loop-mediated isothermal amplification method.
먼저, 바이러스성출혈성패혈증 바이러스(VHSV)의 검출을 위한 루프 매개 등온증폭 반응(LAMP)용 프라이머 세트를 제작하기 위하여, Genebank의 레퍼런스 서열(KM926343.1)을 이용하여 뉴클레오단백질 염기서열 부위의 정보를 확인하였다. LAMP에 사용할 프라이머 세트는 PrimerExploler v5를 이용하여 제작하였다(도 1 참조). 도 1은 타겟 서열과 프라이머 서열의 위치를 나타낸 것이다. First, in order to create a primer set for loop-mediated isothermal amplification reaction (LAMP) for detection of viral hemorrhagic sepsis virus (VHSV), information on the nucleoprotein sequence region was obtained using Genebank's reference sequence (KM926343.1). was confirmed. The primer set to be used for LAMP was created using PrimerExplorer v5 (see Figure 1). Figure 1 shows the positions of the target sequence and primer sequence.
실시예 2: 프라이머 세트의 반응 시간 확인Example 2: Confirmation of reaction time of primer set
상기 제작된 뉴클레오단백질 유전자의 염기서열 부위를 표적으로 하는 프라이머 세트가 비특이적 반응으로 보이지 않는 반응 시간을 확인하였다. 본 실시예는 프라이머 세트 간의 비특이적 반응 여부를 확인하는 것이므로 표적 RNA가 없이 프라이머 세트와 등온반응 시약류 만을 넣고 일반적인 루프매개 등온증폭 반응 온도인 65도에서 시간에 따른 증폭 산물 생성 여부를 확인하였다.The reaction time at which the primer set targeting the base sequence region of the prepared nucleoprotein gene did not appear to be a non-specific reaction was confirmed. Since this example is to check for non-specific reactions between primer sets, only the primer set and isothermal reaction reagents without target RNA were added and the generation of amplification products over time was checked at 65 degrees, the general loop-mediated isothermal amplification reaction temperature.
2.5 ㎕의 10배 농도의 등온증폭 반응 완충액 (20 mM Tris-HCl, 10 mM (NH4)2SO4, 50 mM KCl, 2 mM MgSO4, 0,1 % Tween® 20, pH 8.8@25 ℃), 100 mM MgSO4 1.5 ㎕ 10 mM dNTP mix 3.5 ㎕, 프라이머 믹스 5 ㎕ (실제 반응농도 F3/B3 프라이머 1.6 μM, FIP/BIP 0.2 μM, LF/LB 0.4 μM), 등온증폭 효소 1 ㎕ (8 unit), 역전사 효소 0.5 ㎕ (7.5 unit), 8 M betaine 2.5 ㎕, RNAse free water 8.5 ㎕를 섞어 최종 부피 25 ㎕ 의 등온증폭 반응을 위한 혼합액을 제조하였다. 2.5 ㎕ of 10-fold concentration isothermal amplification reaction buffer (20mM Tris-HCl, 10mM ( NH4 ) 2SO4 , 50mMKCl , 2mMMgSO4 , 0,1% Tween® 20, pH 8.8@25°C ), 100 mM MgSO 4 1.5 μl, 10 mM dNTP mix 3.5 μl, primer mix 5 μl (actual reaction concentration F3/B3 primer 1.6 μM, FIP/BIP 0.2 μM, LF/LB 0.4 μM), isothermal amplification enzyme 1 μl (8 unit), 0.5 ㎕ (7.5 unit) of reverse transcriptase, 2.5 ㎕ of 8 M betaine, and 8.5 ㎕ of RNAse free water were mixed to prepare a mixture for isothermal amplification reaction with a final volume of 25 ㎕.
상기 혼합액을 65℃에서 25분, 30분, 35분, 40분, 45분 동안 반응시킨 후, 최종 LAMP 산물을 수득하였다. 상기 수득된 LAMP 산물을 8% 폴리 아크릴아마이드 겔 전기영동을 통해 확인한 결과를 도 2에 나타내었다. 도 2에서 확인되는 바와 같이 반응 시간이 45분 미만에서는 비특이적 등온 증폭 산물이 나타나지 않음을 확인하였고, 이후의 실험에서는 반응시간을 30분 등 45분 미만으로 지정하였다. The mixture was reacted at 65°C for 25 minutes, 30 minutes, 35 minutes, 40 minutes, and 45 minutes, and then the final LAMP product was obtained. The results of confirming the obtained LAMP product through 8% polyacrylamide gel electrophoresis are shown in Figure 2. As seen in Figure 2, it was confirmed that non-specific isothermal amplification products did not appear when the reaction time was less than 45 minutes, and in subsequent experiments, the reaction time was set to less than 45 minutes, such as 30 minutes.
실시예 3: 루프 매개 등온증폭 반응온도의 최적화Example 3: Optimization of loop-mediated isothermal amplification reaction temperature
검체로부터 추출 및 정제를 통해 준비된 바이러스 RNA를 주형으로 사용하였다. Viral RNA prepared through extraction and purification from the specimen was used as a template.
통상적인 방법을 이용하여 검체로부터 추출 및 정제하여 준비한 RNA 주형 1㎕, 2.5 ㎕의 10배 농도의 등온증폭 반응 완충액 (20 mM Tris-HCl, 10 mM (NH4)2SO4, 50 mM KCl, 2 mM MgSO4, 0,1 % Tween® 20, pH 8.8@25 ℃), 100 mM MgSO4 1.5 ㎕ 10 mM dNTP mix 3.5 ㎕, 프라이머 믹스 5 ㎕ (실제 반응농도 F3/B3 프라이머 1.6 μM, FIP/BIP 0.2 μM, LF/LB 0.4 μM), 등온증폭 효소 1 ㎕ (8 unit), 역전사 효소 0.5 ㎕ (7.5 unit), 8 M betaine 2.5 ㎕, RNAse free water 7.5 ㎕ 를 섞어 최종 부피 25 ㎕의 등온증폭 반응을 위한 혼합액을 제조하였다. 1㎕ of RNA template prepared by extraction and purification from the specimen using a conventional method, 2.5㎕ of 10-fold concentration isothermal amplification reaction buffer (20mM Tris-HCl, 10mM ( NH4 ) 2SO4 , 50mM KCl, 2 mM MgSO 4 , 0,1 % Tween® 20, pH 8.8@25 ℃), 100 mM MgSO 4 1.5 ㎕, 10 mM dNTP mix 3.5 ㎕, primer mix 5 ㎕ (actual reaction concentration F3/B3 primer 1.6 M, FIP/ Mix BIP 0.2 μM, LF/LB 0.4 μM), isothermal amplification enzyme 1 μl (8 units), reverse transcriptase 0.5 μl (7.5 units), 8 M betaine 2.5 μl, and RNAse free water 7.5 μl for isothermal amplification in a final volume of 25 μl. A mixed solution for reaction was prepared.
상기 혼합액을 56℃, 59℃, 62℃, 65℃, 68℃, 71℃, 및 74℃ 의 다양한 온도에서 30분 동안 반응시킨 후, 최종 LAMP 산물을 수득하였다. The mixture was reacted for 30 minutes at various temperatures of 56°C, 59°C, 62°C, 65°C, 68°C, 71°C, and 74°C, and then the final LAMP product was obtained.
상기 수득된 LAMP 산물을 8% 폴리아크릴아마이드 겔 전기영동 및 형 광발색을 한 결과를 도 3에 나타내었다. 이 때, 상기 형광발색은 발색제로 100X의 SybrGreen I을 사용하였다. The results of 8% polyacrylamide gel electrophoresis and fluorescence development of the obtained LAMP product are shown in Figure 3. At this time, 100X SybrGreen I was used as a coloring agent for the fluorescence.
*도 3에서 보는 바와 같이, 59℃, 62℃, 65℃, 68℃ 및 71℃ 의 온도에서 30분 동 안 반응 시 등온증폭의 특성인 래더형 진행을 나타냄을 알 수 있다. 이 중 형광 발색의 세기는 68 ℃에서 가장 강한 것으로 확인되었다. *As shown in Figure 3, it can be seen that when reacted at temperatures of 59°C, 62°C, 65°C, 68°C, and 71°C for 30 minutes, it shows ladder-type progression, which is a characteristic of isothermal amplification. Among these, the intensity of fluorescence was confirmed to be strongest at 68°C.
실시예 4: 색도 분석을 통한 VHSV의 검출 한계Example 4: Detection limit of VHSV through colorimetric analysis
바이러스성출혈성패혈증 바이러스(VHSV)의 검출을 위한 루프 매개 등온증폭 반응(LAMP)용 프라이머 세트를 이용하여, 다음과 같이 바이러스성출혈성패혈증 바이러스의 검출한계를 확인하였다.Using a primer set for loop-mediated isothermal amplification reaction (LAMP) for detection of viral hemorrhagic sepsis virus (VHSV), the detection limit of viral hemorrhagic sepsis virus (VHSV) was confirmed as follows.
바이러스성출혈성패혈증 양성 또는 음성 검체 시료로부터 통상적인 방법으로 분리한 RNA를 50 pM 에서 50 aM까지 단계 희석하여 준비하고, 루프 매개 등온 증폭 반응온도 및 반응시간을 68℃ 및 30분으로 하였고, 색도분석을 위해서 하이드록시 나프톨 블루를 첨가하였다. RNA isolated from a positive or negative viral hemorrhagic sepsis sample was prepared by serial dilution from 50 pM to 50 aM, loop-mediated isothermal amplification reaction temperature and reaction time were 68°C and 30 minutes, and colorimetric analysis was performed. For this, hydroxy naphthol blue was added.
하이드록시 나프톨 블루는 루프매개 등온증폭 반응에 활용되는 마그네슘 이온에 결합하는 표지자로서, 마그네슘 이온과 결합해 있을 때에는 보라색을 띄고, 루프매개 등온증폭의 진행에 마그네슘 이온이 사용됨에 따라 마그네슘 이온과의 결합이 감소하게 되면 청색으로 색상이 변하게 된다. 하이드록시 나프톨 블루는 최초 시약을 첨가할 때에 다른 완충용액이나 효소와 함께 첨가하여도 루프매개 등온증폭 과정에 영향을 끼치지 않아 루프매개 등온증폭의 양상을 간단하게 확인할 수 있는 표지자이다. Hydroxynaphthol blue is a marker that binds to magnesium ions used in loop-mediated isothermal amplification reactions. When bound to magnesium ions, it appears purple, and as magnesium ions are used in the loop-mediated isothermal amplification reaction, it binds to magnesium ions. When this decreases, the color changes to blue. Hydroxy naphthol blue is a marker that can easily check the pattern of loop-mediated isothermal amplification because it does not affect the loop-mediated isothermal amplification process even if it is added together with other buffer solutions or enzymes when adding the first reagent.
추출 및 정제하여 준비한 RNA 주형 1㎕, 2.5 ㎕의 10배 농도의 등온증폭 반응 완충액 (20 mM Tris-HCl, 10 mM (NH4)2SO4, 50 mM KCl, 2 mM MgSO4, 0,1 % Tween® 20, pH 8.8@25 ℃), 100 mM MgSO4 1.5 ㎕ 10 mM dNTP mix 3.5 ㎕, 프라이머 믹스 5 ㎕ (실제 반응농도 F3/B3 프라이머 1.6 μM, FIP/BIP 0.2 μM, LF/LB 0.4 μM), 등온증폭 효소 1 ㎕ (8 unit), 역전사 효소 0.5 ㎕ (7.5 unit), 8 M betaine 2.5 ㎕, 1.5 mM 하이드록시 나프톨 블루 2.5 ㎕, RNAse free water 5 ㎕ 를 섞어 최종 부피 25 ㎕의 등온증폭 반응을 위한 혼합액을 제조하였다. 1㎕ of RNA template prepared by extraction and purification, 2.5㎕ of 10-fold concentration isothermal amplification reaction buffer (20mM Tris-HCl, 10mM ( NH4 ) 2SO4 , 50mM KCl , 2mMMgSO4 , 0,1 % Tween® 20, pH 8.8@25 ℃), 100 mM MgSO 4 1.5 μl, 10 mM dNTP mix 3.5 μl, primer mix 5 μl (actual reaction concentration F3/B3 primer 1.6 μM, FIP/BIP 0.2 μM, LF/LB 0.4 µM), 1 ㎕ (8 units) of isothermal amplification enzyme, 0.5 ㎕ (7.5 units) of reverse transcriptase, 2.5 ㎕ of 8 M betaine, 2.5 ㎕ of 1.5 mM hydroxynaphthol blue, and 5 ㎕ of RNAse free water, and mix isothermal in a final volume of 25 ㎕. A mixed solution for amplification reaction was prepared.
수득된 LAMP 산물을 8% 폴리아크릴아마이드 겔 전기영동및 색도분석을 한 결과를 도 4에 나타내었다. The results of 8% polyacrylamide gel electrophoresis and colorimetric analysis of the obtained LAMP product are shown in Figure 4.
도 4에서 보는 바와 같이, 루프매개 등온증폭을 통하여 500 fM 의 검출한계를 확인할 수 있다. As shown in Figure 4, a detection limit of 500 fM can be confirmed through loop-mediated isothermal amplification.
실시예 5: 유전자가위 반응의 신호 증폭을 이용한 VHSV의 검출 한계Example 5: Detection limit of VHSV using signal amplification of gene scissors reaction
바이러스성출혈성패혈증 바이러스(VHSV)의 검출을 위한 루프 매개 등온증폭 반응(LAMP)용 프라이머 세트를 이용하여, 확인한 검출한계는 500 fM 이었다. 이보다 낮은 검출 한계를 얻을 수 있도록 유전자 가위 반응에 필요한 Cas12a, gRNA, 리포터 단일가닥 DNA 를 첨가하여 결과를 다음과 같이 보다 낮은 검출한계를 확인하였다. Using a primer set for loop-mediated isothermal amplification reaction (LAMP) for detection of viral hemorrhagic sepsis virus (VHSV), the detection limit confirmed was 500 fM. To obtain a lower detection limit than this, Cas12a, gRNA, and reporter single-stranded DNA required for the gene editing reaction were added, and the lower detection limit was confirmed as follows.
통상적인 방법으로 분리한 바이러스성출혈성패혈증 바이러스 RNA를 5 fM 에서 50 aM까지 단계 희석하여 준비하고, 루프 매개 등온 증폭 반응온도 및 반응시간을 68℃ 및 30분으로 하였고, 37 ℃ 유전자 가위 반응을 10분 진행하였다. Viral hemorrhagic sepsis virus RNA isolated by a conventional method was prepared by serial dilution from 5 fM to 50 aM, the loop-mediated isothermal amplification reaction temperature and reaction time were 68°C and 30 minutes, and the gene scissors reaction was performed at 37°C for 10 minutes. Minutes progressed.
우선 루프매개 등온증폭을 위하여 추출 및 정제하여 준비한 RNA 주형 1㎕, 2.5 ㎕의 10배 농도의 등온증폭 반응 완충액 (20 mM Tris-HCl, 10 mM (NH4)2SO4, 50 mM KCl, 2 mM MgSO4, 0,1 % Tween® 20, pH 8.8@25 ℃), 100 mM MgSO4 1.5 ㎕, 10 mM dNTP mix 3.5 ㎕, 프라이머 믹스 5 ㎕ (실제 반응농도 F3/B3 프라이머 1.6 μM, FIP/BIP 0.2 μM, LF/LB 0.4 μM), 등온증폭 효소 1 ㎕(8 unit), 역전사 효소 0.5 ㎕ (7.5 unit), 8 M betaine 2.5 ㎕, RNAse free water 7.5 ㎕ 를 섞어 최종 부피 25 ㎕의 등온증폭 반응을 위한 혼합액을 제조하였다. First, 1 ㎕ of RNA template prepared by extraction and purification for loop-mediated isothermal amplification, 2.5 ㎕ of 10-fold concentration isothermal amplification reaction buffer (20 mM Tris-HCl, 10 mM (NH 4 ) 2 SO 4 , 50 mM KCl, 2 mM MgSO 4 , 0,1 % Tween® 20, pH 8.8@25 ℃), 1.5 ㎕ of 100 mM MgSO 4 , 3.5 ㎕ of 10 mM dNTP mix, 5 ㎕ of primer mix (actual reaction concentration F3/B3 primer 1.6 M, FIP/ Mix BIP 0.2 μM, LF/LB 0.4 μM), isothermal amplification enzyme 1 μl (8 units), reverse transcriptase 0.5 μl (7.5 units), 8 M betaine 2.5 μl, and RNAse free water 7.5 μl for isothermal amplification in a final volume of 25 μl. A mixed solution for reaction was prepared.
등온 증폭이 진행되는 동안 1 μM gRNA 5 ㎕, 1 μM Cas12a 5㎕를 37 ℃에서 30분간 반응시켜 Cas 단백질과 gRNA의 중합체를 만들었다. 여기에 수득된 루프매개 등온증폭 산물을 4 ㎕, 리포터 단일가닥 DNA 2 μM 5 ㎕를 넣고 37 ℃에서 10분간 반응시켜 형광신호를 판독한 결과를 도 5에 표기하였다. While isothermal amplification was in progress, 5 μl of 1 μM gRNA and 5 μl of 1 μM Cas12a were reacted at 37°C for 30 minutes to create a polymer of Cas protein and gRNA. 4 μl of the obtained loop-mediated isothermal amplification product and 5 μl of 2 μM reporter single-stranded DNA were added and reacted at 37°C for 10 minutes, and the results of reading the fluorescence signal are shown in Figure 5.
도 5에서 보는 바와 같이, 유전자 가위 반응을 추가하여 검출한계를 50 aM 까지 낮출 수 있었고, 이는 기존의 루프매개 등온증폭만으로 얻은 500 fM의 검출한계 보다 10,000배 낮은 결과임을 알 수 있다. As shown in Figure 5, the detection limit could be lowered to 50 aM by adding the gene scissors reaction, which is 10,000 times lower than the detection limit of 500 fM obtained only with the existing loop-mediated isothermal amplification.
이상으로 본 발명의 내용의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적 기술은 단지 바람직한 실시 양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서, 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다.As the specific parts of the present invention have been described in detail above, those skilled in the art will understand that these specific techniques are merely preferred embodiments and do not limit the scope of the present invention. It will be obvious. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.
본 발명의 유전자 가위 진단 및 등온증폭 기반의 검출방법을 통해 빠른 시간 내에 결과 확인이 가능하여 효과적으로 VHSV를 검출할 수 있음에 따라, 복잡한 열처리 장비 없이 보다 간단하게 VHSV 진단 시스템을 구축할 수 있을 것으로 기대된다. As the gene scissors diagnosis and isothermal amplification-based detection method of the present invention enables quick confirmation of results and effective detection of VHSV, it is expected that a VHSV diagnosis system can be constructed more simply without complex heat treatment equipment. do.
Claims (10)
- 바이러스성출혈성패혈증바이러스(VHSV, Viral Hemorrhagic Septicemia Virus)의 핵단백질 (Nucleoprotein) 코딩 유전자 (N 유전자)를 특이적으로 등온증폭할 수 있는 핵산 올리고머를 포함하는, VHSV 검출용 조성물.A composition for detecting VHSV, comprising a nucleic acid oligomer capable of specifically isothermally amplifying the nucleoprotein coding gene (N gene) of Viral Hemorrhagic Septicemia Virus (VHSV).
- 제1항에 있어서, 상기 VHSV의 N 유전자를 특이적으로 등온증폭할 수 있는 핵산 올리고머는 서열번호 1 내지 서열번호 6으로 구성된 군에서 선택되는 서열을 포함하는 것을 특징으로 하는 조성물.The composition according to claim 1, wherein the nucleic acid oligomer capable of specifically isothermal amplification of the N gene of VHSV comprises a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 6.
- 제1항에 있어서, 상기 핵산 올리고머에 의해 특이적으로 등온증폭된 VHSV의 N 유전자 산물에 상보적으로 혼성화할 수 있는 프로브를 추가로 포함하는 조성물.The composition of claim 1, further comprising a probe capable of hybridizing complementary to the N gene product of VHSV specifically isothermally amplified by the nucleic acid oligomer.
- 제3항에 있어서, 상기 프로브는 서열번호 7의 서열을 포함하는 것을 특징으로 하는 조성물.The composition according to claim 3, wherein the probe comprises the sequence of SEQ ID NO: 7.
- 제1항에 있어서, 상기 VHSV의 N 유전자에 특이적으로 결합하는 가이드 RNA 및 뉴클레아제 또는 이를 코딩하는 핵산을 추가로 포함하는 조성물.The composition of claim 1, further comprising a guide RNA and a nuclease that specifically binds to the N gene of the VHSV or a nucleic acid encoding the same.
- 제5항에 있어서, 상기 gRNA는 서열번호 7의 서열을 포함하는 것을 특징으로 하는 조성물.The composition of claim 5, wherein the gRNA comprises the sequence of SEQ ID NO: 7.
- 제5항에 있어서, 상기 뉴클레아제는 Cas 단백질인 조성물.The composition of claim 5, wherein the nuclease is a Cas protein.
- 제7항에 있어서, 상기 Cas 단백질은 Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas12g, Cas12h, Cas12i, Cas13a, Cas13b, Cas13c, Cas13d, Cas14 Csx17, Csx14, Csx10, Csx16, CsaX인 조성물.The composition of claim 7, wherein the Cas protein is Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas12g, Cas12h, Cas12i, Cas13a, Cas13b, Cas13c, Cas13d, Cas14 Csx17, Csx14, Csx10, Csx16, and CsaX.
- 제1항 내지 제8항 중 어느 한 항에 따른 조성물을 포함하는 VHSV 검출용 키트.A kit for detecting VHSV comprising the composition according to any one of claims 1 to 8.
- 바이러스성출혈성패혈증바이러스(VHSV, Viral Hemorrhagic Septicemia Virus)의 핵단백질 (Nucleoprotein) 코딩 유전자 (N 유전자)를 특이적으로 등온증폭할 수 있는 핵산 올리고머를 샘플에 처리하는 단계를 포함하는 VHSV 검출을 위한 정보 제공 방법.Information for detecting VHSV, including the step of treating a sample with a nucleic acid oligomer capable of specifically isothermally amplifying the nucleoprotein coding gene (N gene) of Viral Hemorrhagic Septicemia Virus (VHSV) How to provide.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020220117305A KR20240038884A (en) | 2022-09-16 | 2022-09-16 | Composition For Detecting Viral Hemorrhagic Septicemia Virus, Kit For Detecting the Same and Method of Detecting VHSV Using the Same based on loop mediated isothermal amplification combined with CRISPR/Cas diagnosis |
| KR10-2022-0117305 | 2022-09-16 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2024058300A1 true WO2024058300A1 (en) | 2024-03-21 |
Family
ID=90275367
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/KR2022/014820 WO2024058300A1 (en) | 2022-09-16 | 2022-09-30 | Vhsv detection composition using gene scissors and loop-mediated isothermal amplification, kit, and vhsv detection method using same |
Country Status (2)
| Country | Link |
|---|---|
| KR (1) | KR20240038884A (en) |
| WO (1) | WO2024058300A1 (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103205508A (en) * | 2013-03-28 | 2013-07-17 | 东莞出入境检验检疫局检验检疫综合技术中心 | RT-LAMP test primer set, kit and test method for viral hemorrhagic septicemia virus |
| CN103215388A (en) * | 2013-05-10 | 2013-07-24 | 国家海洋局第三海洋研究所 | RT-PCR (reverse transcription-polymerase chain reaction) detection kit for viral haemorrhagic septicaemia virus and preparation method thereof |
| KR20220060668A (en) * | 2020-11-05 | 2022-05-12 | 대한민국(국군의무사령부 사령관) | Method for detecting influenza virus using CRISPR-Cas system and multiplex LAMP primer set |
-
2022
- 2022-09-16 KR KR1020220117305A patent/KR20240038884A/en not_active Application Discontinuation
- 2022-09-30 WO PCT/KR2022/014820 patent/WO2024058300A1/en unknown
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103205508A (en) * | 2013-03-28 | 2013-07-17 | 东莞出入境检验检疫局检验检疫综合技术中心 | RT-LAMP test primer set, kit and test method for viral hemorrhagic septicemia virus |
| CN103215388A (en) * | 2013-05-10 | 2013-07-24 | 国家海洋局第三海洋研究所 | RT-PCR (reverse transcription-polymerase chain reaction) detection kit for viral haemorrhagic septicaemia virus and preparation method thereof |
| KR20220060668A (en) * | 2020-11-05 | 2022-05-12 | 대한민국(국군의무사령부 사령관) | Method for detecting influenza virus using CRISPR-Cas system and multiplex LAMP primer set |
Non-Patent Citations (2)
| Title |
|---|
| DATABASE Nucleotide ANONYMOUS : "Viral hemorrhagic septicemia virus isolate BC06-89-4 nucleoprotein gen", XP093150641, retrieved from NCBI * |
| SOLIMAN, H. ; EL-MATBOULI, M.: "Reverse transcription loop-mediated isothermal amplification (RT-LAMP) for rapid detection of viral hemorrhagic septicaemia virus (VHS)", VETERINARY MICROBIOLOGY, ELSEVIER BV, NL, vol. 114, no. 3-4, 31 May 2006 (2006-05-31), NL , pages 205 - 213, XP024935806, ISSN: 0378-1135, DOI: 10.1016/j.vetmic.2005.11.063 * |
Also Published As
| Publication number | Publication date |
|---|---|
| KR20240038884A (en) | 2024-03-26 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| WO2009007438A1 (en) | Method for the simultaneous detection of multiple nucleic acid sequences in a sample | |
| CN117363767B (en) | Probe combination, primer set and kit for real-time fluorescence PCR detection of target genes and application of probe combination and primer set and kit | |
| CN110878381A (en) | Primer composition, kit and method for detecting mycoplasma bovis and infectious bovine rhinotracheitis virus | |
| CN111910017A (en) | Multiplex-time PCR (polymerase chain reaction) kit for detecting respiratory pathogens, method and application | |
| Iovannisci et al. | Ligation amplification and fluorescence detection of Mycobacterium tuberculosis DNA | |
| CN105755134B (en) | Endonuclease-mediated real-time multiple cross-displacement nucleic acid amplification technology and application | |
| KR20220024252A (en) | High sensitive multiplex loop-mediated isothermal amplification primer set for detection of novel coronavirus-19 | |
| WO2022010238A1 (en) | Composition for determining false positives by using specific artificial nucleotide sequence and method for determining false positives by using same | |
| WO2021182850A1 (en) | Method for simultaneous detection of multiple mirnas and mirna detection kit using same | |
| WO2024058300A1 (en) | Vhsv detection composition using gene scissors and loop-mediated isothermal amplification, kit, and vhsv detection method using same | |
| CN113481326B (en) | Isothermal nucleic acid amplification reaction reagent, isothermal nucleic acid amplification method and application thereof | |
| CN110878380B (en) | Primer composition, kit and method for detecting vesicular stomatitis virus Indiana type and new Jersey type | |
| WO2018048021A1 (en) | Method for analyzing snp of mitochondrial dna by using pna probe and melting curve analysis | |
| Saleh et al. | Loop-mediated isothermal amplification (LAMP) for rapid detection of Renibacterium salmoninarum, the causative agent of bacterial kidney disease | |
| KR20190041314A (en) | Oligonucleotide set for detection of chikungunya virus and uses thereof | |
| CN112899385A (en) | Primer group and probe for identifying Brucella S2 vaccine strain and wild strain and application of primer group and probe | |
| CN111004855A (en) | Primer combination and kit for detecting staphylococcus aureus | |
| WO2023096373A1 (en) | Fluorescent labeling material primer set for multiple detection in loop-mediated isothermal amplification method and molecular diagnosis method using same | |
| CN115058493B (en) | DNA probe for multiple nucleic acid detection, CRISPR-reverse dot hybridization nucleic acid detection system and application | |
| CN117987578B (en) | Microfluidic chip for simultaneously detecting pathogenic escherichia coli, salmonella and drug resistance genes and application thereof | |
| Srisutush et al. | Optimization of Colorimetric Assay of Loop Mediated Isothermal Amplification for Detections of Mycobacterium tuberculosis | |
| WO2024034796A1 (en) | Qpcr method for detection of variant through simultaneous analysis of multiple targets | |
| TawheedShafi et al. | Molecular Diagnosis of Infectious Diseases | |
| Poornima et al. | Polymerase Chain Reaction (PCR)–General Principles and Practices | |
| WO2013105775A1 (en) | Method for multiplex-detecting chronic myelogenous leukemia gene using cuttable probe |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| 121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 22958903 Country of ref document: EP Kind code of ref document: A1 |