JP6962707B2 - Detergent composition - Google Patents
Detergent composition Download PDFInfo
- Publication number
- JP6962707B2 JP6962707B2 JP2017099755A JP2017099755A JP6962707B2 JP 6962707 B2 JP6962707 B2 JP 6962707B2 JP 2017099755 A JP2017099755 A JP 2017099755A JP 2017099755 A JP2017099755 A JP 2017099755A JP 6962707 B2 JP6962707 B2 JP 6962707B2
- Authority
- JP
- Japan
- Prior art keywords
- animal
- derived
- tissue
- hard tissue
- dna
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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- 239000000203 mixture Substances 0.000 title claims description 61
- 239000003599 detergent Substances 0.000 title claims description 44
- 210000001519 tissue Anatomy 0.000 claims description 190
- 241001465754 Metazoa Species 0.000 claims description 122
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 117
- 238000000034 method Methods 0.000 claims description 70
- 238000004140 cleaning Methods 0.000 claims description 44
- 108020004707 nucleic acids Proteins 0.000 claims description 34
- 102000039446 nucleic acids Human genes 0.000 claims description 34
- 150000007523 nucleic acids Chemical class 0.000 claims description 34
- 210000000988 bone and bone Anatomy 0.000 claims description 33
- 235000013372 meat Nutrition 0.000 claims description 26
- 238000005406 washing Methods 0.000 claims description 18
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 claims description 16
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 15
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 15
- 235000019333 sodium laurylsulphate Nutrition 0.000 claims description 15
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 claims description 12
- 239000011734 sodium Substances 0.000 claims description 11
- 229910052708 sodium Inorganic materials 0.000 claims description 10
- 239000000126 substance Substances 0.000 claims description 10
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims description 8
- 241000894007 species Species 0.000 claims description 3
- MOTZDAYCYVMXPC-UHFFFAOYSA-N dodecyl hydrogen sulfate Chemical compound CCCCCCCCCCCCOS(O)(=O)=O MOTZDAYCYVMXPC-UHFFFAOYSA-N 0.000 claims description 2
- 229940043264 dodecyl sulfate Drugs 0.000 claims description 2
- 108020004414 DNA Proteins 0.000 description 83
- -1 ammonium hydrogen Chemical class 0.000 description 75
- 239000000047 product Substances 0.000 description 50
- 238000012360 testing method Methods 0.000 description 48
- 230000003321 amplification Effects 0.000 description 46
- 238000003199 nucleic acid amplification method Methods 0.000 description 46
- 241000287828 Gallus gallus Species 0.000 description 38
- 235000013330 chicken meat Nutrition 0.000 description 38
- 239000012459 cleaning agent Substances 0.000 description 38
- 235000013305 food Nutrition 0.000 description 26
- 235000014113 dietary fatty acids Nutrition 0.000 description 25
- 239000000194 fatty acid Substances 0.000 description 25
- 229930195729 fatty acid Natural products 0.000 description 25
- 229920003171 Poly (ethylene oxide) Polymers 0.000 description 18
- 239000000243 solution Substances 0.000 description 17
- 239000007864 aqueous solution Substances 0.000 description 16
- 239000003795 chemical substances by application Substances 0.000 description 15
- 239000002280 amphoteric surfactant Substances 0.000 description 14
- 238000010438 heat treatment Methods 0.000 description 14
- 239000000523 sample Substances 0.000 description 14
- 150000004665 fatty acids Chemical class 0.000 description 13
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 11
- 241000282898 Sus scrofa Species 0.000 description 11
- 239000012634 fragment Substances 0.000 description 11
- 239000004094 surface-active agent Substances 0.000 description 11
- 239000000284 extract Substances 0.000 description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 10
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 8
- LYCAIKOWRPUZTN-UHFFFAOYSA-N ethylene glycol Natural products OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 8
- 238000003753 real-time PCR Methods 0.000 description 8
- KWIUHFFTVRNATP-UHFFFAOYSA-N Betaine Natural products C[N+](C)(C)CC([O-])=O KWIUHFFTVRNATP-UHFFFAOYSA-N 0.000 description 7
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 7
- 239000002253 acid Substances 0.000 description 7
- 229960003237 betaine Drugs 0.000 description 7
- 241000251468 Actinopterygii Species 0.000 description 6
- ROSDSFDQCJNGOL-UHFFFAOYSA-N Dimethylamine Chemical compound CNC ROSDSFDQCJNGOL-UHFFFAOYSA-N 0.000 description 6
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 150000002148 esters Chemical class 0.000 description 6
- RTZKZFJDLAIYFH-UHFFFAOYSA-N ether Substances CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 6
- 150000003839 salts Chemical class 0.000 description 6
- 229910000029 sodium carbonate Inorganic materials 0.000 description 6
- KWIUHFFTVRNATP-UHFFFAOYSA-O N,N,N-trimethylglycinium Chemical compound C[N+](C)(C)CC(O)=O KWIUHFFTVRNATP-UHFFFAOYSA-O 0.000 description 5
- 229910019142 PO4 Inorganic materials 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- 244000144972 livestock Species 0.000 description 5
- 239000002736 nonionic surfactant Substances 0.000 description 5
- 235000021317 phosphate Nutrition 0.000 description 5
- 241000272517 Anseriformes Species 0.000 description 4
- 206010034203 Pectus Carinatum Diseases 0.000 description 4
- 239000004743 Polypropylene Substances 0.000 description 4
- 150000005215 alkyl ethers Chemical class 0.000 description 4
- 239000003945 anionic surfactant Substances 0.000 description 4
- 238000009835 boiling Methods 0.000 description 4
- 239000002540 palm oil Substances 0.000 description 4
- 229920001155 polypropylene Polymers 0.000 description 4
- 229920001451 polypropylene glycol Polymers 0.000 description 4
- 238000005070 sampling Methods 0.000 description 4
- 229910001220 stainless steel Inorganic materials 0.000 description 4
- 239000010935 stainless steel Substances 0.000 description 4
- 238000003756 stirring Methods 0.000 description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- DNIAPMSPPWPWGF-UHFFFAOYSA-N Propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 description 3
- 239000005708 Sodium hypochlorite Substances 0.000 description 3
- 241000282887 Suidae Species 0.000 description 3
- 239000012670 alkaline solution Substances 0.000 description 3
- 125000000217 alkyl group Chemical group 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 239000003093 cationic surfactant Substances 0.000 description 3
- 239000003240 coconut oil Substances 0.000 description 3
- 235000019864 coconut oil Nutrition 0.000 description 3
- 230000002068 genetic effect Effects 0.000 description 3
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 description 3
- 238000013508 migration Methods 0.000 description 3
- 230000005012 migration Effects 0.000 description 3
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 3
- HLERILKGMXJNBU-UHFFFAOYSA-N norvaline betaine Chemical compound CCCC(C([O-])=O)[N+](C)(C)C HLERILKGMXJNBU-UHFFFAOYSA-N 0.000 description 3
- 244000144977 poultry Species 0.000 description 3
- 235000013594 poultry meat Nutrition 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 238000011002 quantification Methods 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 description 3
- 229910021653 sulphate ion Inorganic materials 0.000 description 3
- JNYAEWCLZODPBN-JGWLITMVSA-N (2r,3r,4s)-2-[(1r)-1,2-dihydroxyethyl]oxolane-3,4-diol Chemical compound OC[C@@H](O)[C@H]1OC[C@H](O)[C@H]1O JNYAEWCLZODPBN-JGWLITMVSA-N 0.000 description 2
- HFDVRLIODXPAHB-UHFFFAOYSA-N 1-tetradecene Chemical compound CCCCCCCCCCCCC=C HFDVRLIODXPAHB-UHFFFAOYSA-N 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- QXNVGIXVLWOKEQ-UHFFFAOYSA-N Disodium Chemical compound [Na][Na] QXNVGIXVLWOKEQ-UHFFFAOYSA-N 0.000 description 2
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 2
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- 241000209094 Oryza Species 0.000 description 2
- 235000007164 Oryza sativa Nutrition 0.000 description 2
- 229920001214 Polysorbate 60 Polymers 0.000 description 2
- 241000288906 Primates Species 0.000 description 2
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 2
- 239000000654 additive Substances 0.000 description 2
- 150000003973 alkyl amines Chemical class 0.000 description 2
- AXCZMVOFGPJBDE-UHFFFAOYSA-L calcium dihydroxide Chemical compound [OH-].[OH-].[Ca+2] AXCZMVOFGPJBDE-UHFFFAOYSA-L 0.000 description 2
- 239000000920 calcium hydroxide Substances 0.000 description 2
- 229910001861 calcium hydroxide Inorganic materials 0.000 description 2
- 238000011088 calibration curve Methods 0.000 description 2
- 239000004359 castor oil Substances 0.000 description 2
- 235000019438 castor oil Nutrition 0.000 description 2
- 150000001768 cations Chemical class 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
- ZBCBWPMODOFKDW-UHFFFAOYSA-N diethanolamine Chemical compound OCCNCCO ZBCBWPMODOFKDW-UHFFFAOYSA-N 0.000 description 2
- 238000007598 dipping method Methods 0.000 description 2
- ZEMPKEQAKRGZGQ-XOQCFJPHSA-N glycerol triricinoleate Natural products CCCCCC[C@@H](O)CC=CCCCCCCCC(=O)OC[C@@H](COC(=O)CCCCCCCC=CC[C@@H](O)CCCCCC)OC(=O)CCCCCCCC=CC[C@H](O)CCCCCC ZEMPKEQAKRGZGQ-XOQCFJPHSA-N 0.000 description 2
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 2
- 239000010931 gold Substances 0.000 description 2
- 229910052737 gold Inorganic materials 0.000 description 2
- 235000020993 ground meat Nutrition 0.000 description 2
- MTNDZQHUAFNZQY-UHFFFAOYSA-N imidazoline Chemical class C1CN=CN1 MTNDZQHUAFNZQY-UHFFFAOYSA-N 0.000 description 2
- VTHJTEIRLNZDEV-UHFFFAOYSA-L magnesium dihydroxide Chemical compound [OH-].[OH-].[Mg+2] VTHJTEIRLNZDEV-UHFFFAOYSA-L 0.000 description 2
- 239000000347 magnesium hydroxide Substances 0.000 description 2
- 229910001862 magnesium hydroxide Inorganic materials 0.000 description 2
- HOVAGTYPODGVJG-UHFFFAOYSA-N methyl beta-galactoside Natural products COC1OC(CO)C(O)C(O)C1O HOVAGTYPODGVJG-UHFFFAOYSA-N 0.000 description 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 150000003242 quaternary ammonium salts Chemical class 0.000 description 2
- 235000021067 refined food Nutrition 0.000 description 2
- 235000009566 rice Nutrition 0.000 description 2
- CVHZOJJKTDOEJC-UHFFFAOYSA-N saccharin Chemical compound C1=CC=C2C(=O)NS(=O)(=O)C2=C1 CVHZOJJKTDOEJC-UHFFFAOYSA-N 0.000 description 2
- 229940081974 saccharin Drugs 0.000 description 2
- 235000019204 saccharin Nutrition 0.000 description 2
- 239000000901 saccharin and its Na,K and Ca salt Substances 0.000 description 2
- 235000013580 sausages Nutrition 0.000 description 2
- 150000005846 sugar alcohols Polymers 0.000 description 2
- BDHFUVZGWQCTTF-UHFFFAOYSA-M sulfonate Chemical compound [O-]S(=O)=O BDHFUVZGWQCTTF-UHFFFAOYSA-M 0.000 description 2
- 150000003871 sulfonates Chemical class 0.000 description 2
- 150000003467 sulfuric acid derivatives Chemical class 0.000 description 2
- PDSVZUAJOIQXRK-UHFFFAOYSA-N trimethyl(octadecyl)azanium Chemical compound CCCCCCCCCCCCCCCCCC[N+](C)(C)C PDSVZUAJOIQXRK-UHFFFAOYSA-N 0.000 description 2
- QYIXCDOBOSTCEI-QCYZZNICSA-N (5alpha)-cholestan-3beta-ol Chemical compound C([C@@H]1CC2)[C@@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@H](C)CCCC(C)C)[C@@]2(C)CC1 QYIXCDOBOSTCEI-QCYZZNICSA-N 0.000 description 1
- CMCBDXRRFKYBDG-UHFFFAOYSA-N 1-dodecoxydodecane Chemical compound CCCCCCCCCCCCOCCCCCCCCCCCC CMCBDXRRFKYBDG-UHFFFAOYSA-N 0.000 description 1
- FDCJDKXCCYFOCV-UHFFFAOYSA-N 1-hexadecoxyhexadecane Chemical compound CCCCCCCCCCCCCCCCOCCCCCCCCCCCCCCCC FDCJDKXCCYFOCV-UHFFFAOYSA-N 0.000 description 1
- XXQCYWFEQYXWEL-UHFFFAOYSA-N 2-(1-undecyl-4,5-dihydroimidazol-1-ium-1-yl)ethanol Chemical compound CCCCCCCCCCC[N+]1(CCO)CCN=C1 XXQCYWFEQYXWEL-UHFFFAOYSA-N 0.000 description 1
- HZAXFHJVJLSVMW-UHFFFAOYSA-N 2-Aminoethan-1-ol Chemical compound NCCO HZAXFHJVJLSVMW-UHFFFAOYSA-N 0.000 description 1
- YPDCDBRKWQYBPV-UHFFFAOYSA-N 2-[2-(dodecanoylamino)ethyl-(2-hydroxyethyl)amino]acetic acid;sodium Chemical compound [Na].CCCCCCCCCCCC(=O)NCCN(CCO)CC(O)=O YPDCDBRKWQYBPV-UHFFFAOYSA-N 0.000 description 1
- FXKZPKBFTQUJBA-UHFFFAOYSA-N 2-[2-[bis(carboxymethyl)amino]ethyl-(carboxymethyl)amino]acetic acid;sodium;dihydrate Chemical compound O.O.[Na].[Na].OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O FXKZPKBFTQUJBA-UHFFFAOYSA-N 0.000 description 1
- DUMAFWZFOOOEPH-UHFFFAOYSA-N 2-[bis(2-hydroxyethyl)amino]ethanol;dodecyl benzenesulfonate Chemical compound OCCN(CCO)CCO.CCCCCCCCCCCCOS(=O)(=O)C1=CC=CC=C1 DUMAFWZFOOOEPH-UHFFFAOYSA-N 0.000 description 1
- HVYJSOSGTDINLW-UHFFFAOYSA-N 2-[dimethyl(octadecyl)azaniumyl]acetate Chemical compound CCCCCCCCCCCCCCCCCC[N+](C)(C)CC([O-])=O HVYJSOSGTDINLW-UHFFFAOYSA-N 0.000 description 1
- MPNXSZJPSVBLHP-UHFFFAOYSA-N 2-chloro-n-phenylpyridine-3-carboxamide Chemical compound ClC1=NC=CC=C1C(=O)NC1=CC=CC=C1 MPNXSZJPSVBLHP-UHFFFAOYSA-N 0.000 description 1
- KTCCLEVCNRHWQL-UHFFFAOYSA-N 3-(2-oxotridecylamino)propanoic acid;sodium Chemical compound [Na].CCCCCCCCCCCC(=O)CNCCC(O)=O KTCCLEVCNRHWQL-UHFFFAOYSA-N 0.000 description 1
- CVZYWFZBNHBFDS-UHFFFAOYSA-N 3-(dodecylamino)propanoate;tris(2-hydroxyethyl)azanium Chemical compound OCCN(CCO)CCO.CCCCCCCCCCCCNCCC(O)=O CVZYWFZBNHBFDS-UHFFFAOYSA-N 0.000 description 1
- DDGPBVIAYDDWDH-UHFFFAOYSA-N 3-[dodecyl(dimethyl)azaniumyl]-2-hydroxypropane-1-sulfonate Chemical compound CCCCCCCCCCCC[N+](C)(C)CC(O)CS([O-])(=O)=O DDGPBVIAYDDWDH-UHFFFAOYSA-N 0.000 description 1
- LIFHMKCDDVTICL-UHFFFAOYSA-N 6-(chloromethyl)phenanthridine Chemical compound C1=CC=C2C(CCl)=NC3=CC=CC=C3C2=C1 LIFHMKCDDVTICL-UHFFFAOYSA-N 0.000 description 1
- XZIIFPSPUDAGJM-UHFFFAOYSA-N 6-chloro-2-n,2-n-diethylpyrimidine-2,4-diamine Chemical compound CCN(CC)C1=NC(N)=CC(Cl)=N1 XZIIFPSPUDAGJM-UHFFFAOYSA-N 0.000 description 1
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- ATRRKUHOCOJYRX-UHFFFAOYSA-N Ammonium bicarbonate Chemical compound [NH4+].OC([O-])=O ATRRKUHOCOJYRX-UHFFFAOYSA-N 0.000 description 1
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- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 1
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- VOGVEUAIIKVAOE-VEECZQFMSA-N [(8r,9s,10r,13s,14s,17r)-17-acetyl-6-chloro-10,13-dimethyl-3-oxo-2,8,9,11,12,14,15,16-octahydro-1h-cyclopenta[a]phenanthren-17-yl] acetate;(8r,9s,13s,14s,17r)-17-ethynyl-3-methoxy-13-methyl-7,8,9,11,12,14,15,16-octahydro-6h-cyclopenta[a]phenanthren-17-ol Chemical compound C1C[C@]2(C)[C@@](C#C)(O)CC[C@H]2[C@@H]2CCC3=CC(OC)=CC=C3[C@H]21.C1=C(Cl)C2=CC(=O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@@](C(C)=O)(OC(=O)C)[C@@]1(C)CC2 VOGVEUAIIKVAOE-VEECZQFMSA-N 0.000 description 1
- NPTLAYTZMHJJDP-KTKRTIGZSA-N [3-[3-[3-[3-[3-[3-[3-[3-[3-(2,3-dihydroxypropoxy)-2-hydroxypropoxy]-2-hydroxypropoxy]-2-hydroxypropoxy]-2-hydroxypropoxy]-2-hydroxypropoxy]-2-hydroxypropoxy]-2-hydroxypropoxy]-2-hydroxypropoxy]-2-hydroxypropyl] (z)-octadec-9-enoate Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OCC(O)COCC(O)COCC(O)COCC(O)COCC(O)COCC(O)COCC(O)COCC(O)COCC(O)COCC(O)CO NPTLAYTZMHJJDP-KTKRTIGZSA-N 0.000 description 1
- MHZDPHLNZSQJMO-UHFFFAOYSA-N [Na].CCCCCCCCCCCCN(CC(N)N)CC(O)=O Chemical compound [Na].CCCCCCCCCCCCN(CC(N)N)CC(O)=O MHZDPHLNZSQJMO-UHFFFAOYSA-N 0.000 description 1
- 125000002252 acyl group Chemical group 0.000 description 1
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- 239000011543 agarose gel Substances 0.000 description 1
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- QYIXCDOBOSTCEI-UHFFFAOYSA-N alpha-cholestanol Natural products C1CC2CC(O)CCC2(C)C2C1C1CCC(C(C)CCCC(C)C)C1(C)CC2 QYIXCDOBOSTCEI-UHFFFAOYSA-N 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
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- 239000001099 ammonium carbonate Substances 0.000 description 1
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- BTBJBAZGXNKLQC-UHFFFAOYSA-N ammonium lauryl sulfate Chemical compound [NH4+].CCCCCCCCCCCCOS([O-])(=O)=O BTBJBAZGXNKLQC-UHFFFAOYSA-N 0.000 description 1
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- 238000012850 discrimination method Methods 0.000 description 1
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- DDXLVDQZPFLQMZ-UHFFFAOYSA-M dodecyl(trimethyl)azanium;chloride Chemical compound [Cl-].CCCCCCCCCCCC[N+](C)(C)C DDXLVDQZPFLQMZ-UHFFFAOYSA-M 0.000 description 1
- FLHZRVCNBDNLAL-UHFFFAOYSA-M dodecyl-dimethyl-(16-methylheptadecyl)azanium;chloride Chemical compound [Cl-].CCCCCCCCCCCC[N+](C)(C)CCCCCCCCCCCCCCCC(C)C FLHZRVCNBDNLAL-UHFFFAOYSA-M 0.000 description 1
- XJWSAJYUBXQQDR-UHFFFAOYSA-M dodecyltrimethylammonium bromide Chemical compound [Br-].CCCCCCCCCCCC[N+](C)(C)C XJWSAJYUBXQQDR-UHFFFAOYSA-M 0.000 description 1
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- XLYOFNOQVPJJNP-UHFFFAOYSA-M hydroxide Chemical compound [OH-] XLYOFNOQVPJJNP-UHFFFAOYSA-M 0.000 description 1
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- 239000004615 ingredient Substances 0.000 description 1
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- ILRSCQWREDREME-UHFFFAOYSA-N lauric acid amide propyl betaine Natural products CCCCCCCCCCCC(N)=O ILRSCQWREDREME-UHFFFAOYSA-N 0.000 description 1
- ZLNQQNXFFQJAID-UHFFFAOYSA-L magnesium carbonate Chemical compound [Mg+2].[O-]C([O-])=O ZLNQQNXFFQJAID-UHFFFAOYSA-L 0.000 description 1
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- GNVRJGIVDSQCOP-UHFFFAOYSA-N n-ethyl-n-methylethanamine Chemical compound CCN(C)CC GNVRJGIVDSQCOP-UHFFFAOYSA-N 0.000 description 1
- PSZYNBSKGUBXEH-UHFFFAOYSA-M naphthalene-1-sulfonate Chemical compound C1=CC=C2C(S(=O)(=O)[O-])=CC=CC2=C1 PSZYNBSKGUBXEH-UHFFFAOYSA-M 0.000 description 1
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- GSGDTSDELPUTKU-UHFFFAOYSA-N nonoxybenzene Chemical compound CCCCCCCCCOC1=CC=CC=C1 GSGDTSDELPUTKU-UHFFFAOYSA-N 0.000 description 1
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- RRHXZLALVWBDKH-UHFFFAOYSA-M trimethyl-[2-(2-methylprop-2-enoyloxy)ethyl]azanium;chloride Chemical compound [Cl-].CC(=C)C(=O)OCC[N+](C)(C)C RRHXZLALVWBDKH-UHFFFAOYSA-M 0.000 description 1
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Images
Landscapes
- Detergent Compositions (AREA)
- Sampling And Sample Adjustment (AREA)
Description
本発明は、動物由来の組織の付着した硬組織から動物由来の組織を除去するための洗浄剤組成物及び洗浄方法に関するものである。より詳細には、本発明は、動物由来の組織の付着した硬組織から動物由来の組織を除去し、硬組織由来の核酸を採取するために使用する洗浄剤組成物および洗浄方法に関するものである。 The present invention relates to a detergent composition and a cleaning method for removing animal-derived tissue from hard tissue to which animal-derived tissue is attached. More specifically, the present invention relates to a cleaning agent composition and a cleaning method used for removing animal-derived tissue from hard tissue to which animal-derived tissue is attached and collecting nucleic acid derived from hard tissue. ..
従来、法医学的なＤＮＡ鑑定や、生物学的な遺伝子研究の際に、骨や歯牙等の硬組織からＤＮＡが採取されている。例えば、特許文献１には、ＤＮＡを簡便かつ効率よく採取する方法として、エチレンジアミン四酢酸を含有する水溶液、界面活性剤を含有する可溶化剤緩衝液、タンパク質分解酵素液等の複数の液で処理する方法が開示されている。この方法によれば、ＤＮＡ鑑定を阻害するタンパク質等の不純物を混入させることなく、ＤＮＡを採取することができる。 Conventionally, DNA has been collected from hard tissues such as bones and teeth during forensic DNA analysis and biological genetic research. For example, in Patent Document 1, as a method for collecting DNA easily and efficiently, it is treated with a plurality of solutions such as an aqueous solution containing ethylenediamine tetraacetic acid, a solubilizer buffer solution containing a surfactant, and a proteolytic enzyme solution. The method of doing so is disclosed. According to this method, DNA can be collected without mixing impurities such as proteins that inhibit DNA testing.
挽肉等の食品では骨片が混入する場合があり、品質管理の一環としてこの骨片が挽肉と同種の動物由来のものか、外部からの混入物であるのかを同定する必要がある。また、複数の畜種の肉が混合されている合挽肉等の場合では、再発防止のために、混入した骨片がどの畜種に由来するものであるのかを同定する必要がある。 Minced meat and other foods may contain bone fragments, and as part of quality control, it is necessary to identify whether these bone fragments are derived from animals of the same type as minced meat or are external contaminants. Further, in the case of minced meat or the like in which meats of a plurality of livestock species are mixed, it is necessary to identify which livestock species the mixed bone fragments are derived from in order to prevent recurrence.
そこで、従来の法医学的なＤＮＡの採取方法を利用して、挽肉等の食品中から骨片を取り出し、骨片由来のＤＮＡから畜種の同定を試みたところ、肉由来のＤＮＡを除去できないという問題があった。通常、法医学の分野におけるＤＮＡ採取では、骨片とこれに付着する肉片が同種のものを取り扱うことから、この問題は注視されるものではなかったと推察される。 Therefore, when bone fragments were taken out from foods such as minced meat using the conventional forensic DNA collection method and an attempt was made to identify the livestock species from the DNA derived from the bone fragments, there was a problem that the DNA derived from meat could not be removed. was there. Usually, in DNA collection in the field of forensic medicine, since bone fragments and meat fragments attached to them are usually treated as the same type, it is presumed that this problem was not closely watched.
本発明では、上記のような問題を解決するべく、肉片等の動物由来の組織が付着した骨片等の硬組織から、動物由来の組織を除去し、硬組織の動物種を正確に同定することを目的とするものである。 In the present invention, in order to solve the above problems, animal-derived tissue is removed from hard tissue such as bone fragments to which animal-derived tissue such as meat pieces are attached, and the animal species of the hard tissue is accurately identified. The purpose is to do that.
本発明者らは、上記課題を解決するために鋭意検討した結果、アルカリ剤及び界面活性剤を含有する洗浄剤組成物を用いて硬組織を洗浄することにより、硬組織に付着した動物由来の組織が十分に除去され、硬組織の動物種を正確に同定できることを見出して、本発明を完成した。
すなわち本発明は、以下の洗浄剤組成物及び洗浄方法、核酸採取キット及び核酸採取方法である。
As a result of diligent studies to solve the above problems, the present inventors have washed the hard tissue with a cleaning agent composition containing an alkaline agent and a surfactant, and as a result, the hard tissue is derived from an animal attached to the hard tissue. The present invention was completed by finding that the tissue was sufficiently removed and the hard tissue animal species could be accurately identified.
That is, the present invention is the following detergent composition and cleaning method, nucleic acid collection kit and nucleic acid collection method.
上記課題を解決するための本発明の洗浄剤組成物は、動物由来の組織の付着した硬組織から前記動物由来の組織を除去するための洗浄剤組成物であって、（Ａ）アルカリ剤、及び、（Ｂ）界面活性剤、を含有することを特徴とする。
この洗浄剤組成物によれば、動物由来の組織の付着した硬組織から、動物由来の組織が十分に除去されるため、硬組織の動物種を正確に同定することができる。
The cleaning agent composition of the present invention for solving the above-mentioned problems is a cleaning agent composition for removing the animal-derived tissue from the hard tissue to which the animal-derived tissue is attached, and is (A) an alkaline agent. It is characterized by containing (B) a surfactant.
According to this cleaning agent composition, the animal-derived tissue is sufficiently removed from the hard tissue to which the animal-derived tissue is attached, so that the animal species of the hard tissue can be accurately identified.
更に、本発明の洗浄剤組成物の一実施態様によれば、（Ａ）アルカリ剤の含有量は、０．０１〜０．１質量％であることを特徴とするものである。
この特徴によれば、動物由来の組織の付着した硬組織から、動物由来の組織を除去するという本発明の効果をより発揮することができる。
Further, according to one embodiment of the detergent composition of the present invention, the content of the (A) alkaline agent is 0.01 to 0.1% by mass.
According to this feature, the effect of the present invention of removing the animal-derived tissue from the hard tissue to which the animal-derived tissue is attached can be more exerted.
更に、本発明の洗浄剤組成物の一実施態様によれば、（Ｂ）界面活性剤の含有量は、０．０１〜１質量％であることを特徴とする。
この特徴によれば、動物由来の組織の付着した硬組織から、動物由来の組織を除去するという本発明の効果をより発揮することができる。
Further, according to one embodiment of the detergent composition of the present invention, the content of the (B) surfactant is 0.01 to 1% by mass.
According to this feature, the effect of the present invention of removing the animal-derived tissue from the hard tissue to which the animal-derived tissue is attached can be more exerted.
更に、本発明の洗浄剤組成物の一実施態様によれば、硬組織は、骨片であることを特徴とする。
骨片は、挽肉等の食品等に混入しやすい硬組織であるため、この特徴によれば、本発明の洗浄剤組成物を好適に利用することができる。
Furthermore, according to one embodiment of the detergent composition of the present invention, the hard tissue is characterized by being bone fragments.
Since the bone fragment is a hard tissue that is easily mixed with foods such as minced meat, the cleaning agent composition of the present invention can be preferably used according to this feature.
更に、本発明の洗浄剤組成物の一実施態様によれば、硬組織と動物由来の組織は、種の異なるものであることを特徴とする。
本発明の洗浄剤組成物によれば、硬組織に付着した動物由来の組織を十分に除去することができるため、硬組織と動物由来の組織がそれぞれ種の異なるものである場合に、本発明の効果がより発揮される。
Furthermore, according to one embodiment of the detergent composition of the present invention, the hard tissue and the animal-derived tissue are of different species.
According to the cleaning agent composition of the present invention, the animal-derived tissue adhering to the hard tissue can be sufficiently removed. Therefore, when the hard tissue and the animal-derived tissue are of different species, the present invention is used. The effect of is more exerted.
また、上記課題を解決するための本発明の核酸採取キットは、硬組織からＤＮＡ又はＲＮＡを採取するための核酸採取キットであって、本発明の洗浄剤組成物を含むことを特徴とする。
この核酸採取キットによれば、硬組織由来の核酸を高純度で精製することができるため、硬組織の動物種を正確に同定することができる。
Further, the nucleic acid collection kit of the present invention for solving the above-mentioned problems is a nucleic acid collection kit for collecting DNA or RNA from hard tissue, and is characterized by containing the cleaning agent composition of the present invention.
According to this nucleic acid collection kit, nucleic acid derived from hard tissue can be purified with high purity, so that the animal species of hard tissue can be accurately identified.
また、上記課題を解決するための本発明の洗浄方法は、動物由来の組織の付着した硬組織から動物由来の組織を除去するための洗浄方法であって、本発明の洗浄剤組成物を用いて、動物由来の組織の付着した硬組織を洗浄する工程、を備えたことを特徴とする。
この洗浄方法によれば、動物由来の組織の付着した硬組織から、動物由来の組織が十分に除去されるため、硬組織の動物種を正確に同定することができる。
Further, the cleaning method of the present invention for solving the above-mentioned problems is a cleaning method for removing animal-derived tissue from hard tissue to which animal-derived tissue is attached, and the cleaning agent composition of the present invention is used. It is characterized by including a step of cleaning hard tissue to which animal-derived tissue is attached.
According to this washing method, the animal-derived tissue is sufficiently removed from the hard tissue to which the animal-derived tissue is attached, so that the animal species of the hard tissue can be accurately identified.
また、上記課題を解決するための本発明の核酸採取方法は、動物由来の組織の付着した硬組織からＤＮＡ又はＲＮＡを採取するための核酸採取方法であって、本発明の洗浄剤組成物を用いて、動物由来の組織の付着した硬組織を洗浄する工程、洗浄した硬組織からＤＮＡ又はＲＮＡを採取する工程、を備えたことを特徴とする。
この核酸採取方法によれば、硬組織由来の核酸を高純度で精製することができるため、硬組織の動物種を正確に同定することができる。
Further, the nucleic acid collection method of the present invention for solving the above-mentioned problems is a nucleic acid collection method for collecting DNA or RNA from hard tissue to which animal-derived tissue is attached, and the cleaning agent composition of the present invention can be used. It is characterized by comprising a step of washing a hard tissue to which an animal-derived tissue is attached and a step of collecting DNA or RNA from the washed hard tissue.
According to this nucleic acid collection method, the nucleic acid derived from hard tissue can be purified with high purity, so that the animal species of hard tissue can be accurately identified.
本発明によれば、肉片等の動物由来の組織が付着した骨片等の硬組織から、動物由来の組織を除去し、硬組織の動物種を正確に同定することができる。 According to the present invention, animal-derived tissue can be removed from hard tissue such as bone fragments to which animal-derived tissue such as meat pieces are attached, and the animal species of the hard tissue can be accurately identified.
［洗浄剤組成物］
本発明の洗浄剤組成物は、動物由来の組織の付着した硬組織から動物由来の組織を除去するための洗浄剤組成物であって、（Ａ）アルカリ剤、及び、（Ｂ）界面活性剤、を含有することを特徴とするものである。この洗浄剤組成物によれば、動物由来の組織を硬組織から十分に除去することができる。
[Detergent composition]
The detergent composition of the present invention is a detergent composition for removing animal-derived tissue from hard tissue to which animal-derived tissue is attached, and is (A) an alkaline agent and (B) a surfactant. , Is contained. According to this detergent composition, animal-derived tissue can be sufficiently removed from the hard tissue.
硬組織とは、動物の体を支えるために所定の硬度を有する組織であり、例えば、肉畜や、野生獣、霊長類、家禽、魚類等の歯、爪、骨等が挙げられ、肉畜としてはウシ、ブタ、ヤギ、ヒツジ、ウマ、ウサギ等、野生獣としてはイノシシやシカ等、霊長類としてはヒトやチンパンジー等、家禽としてはニワトリ、ウズラ、シチメンチョウ、アヒル、ガチョウ等、魚類としてはスケトウダラ、マグロ、アジ等が挙げられる。食品等に混入しやすいという観点から、硬組織が骨である場合に、本発明の洗浄剤組成物の利用性が向上する。 The hard tissue is a tissue having a predetermined hardness to support the body of an animal, and examples thereof include meat and livestock, teeth, claws, bones of wild animals, primates, poultry, fish and the like, and meat and livestock. As cattle, pigs, goats, sheep, horses, rabbits, etc., wild boars and deer as wild animals, humans and chimpanzees as primates, chickens, quail, poultry, ducks, geese, etc. as poultry, and fish as fish. Examples include skeletons, geese, and ducks. From the viewpoint that it is easily mixed with foods and the like, the usability of the detergent composition of the present invention is improved when the hard tissue is bone.
動物由来の組織（以下、「動物由来組織」という。）としては、例えば、筋細胞等により構成される筋組織、コラーゲンや線維芽細胞から構成される結合組織、上皮細胞等により構成される上皮組織、神経細胞等により構成される神経組織、脂肪細胞等により構成される脂肪組織等が挙げられる。また、これらの動物由来組織からなる肉片等でもよい。 Examples of animal-derived tissue (hereinafter referred to as “animal-derived tissue”) include muscle tissue composed of muscle cells, connective tissue composed of collagen and fibroblasts, and epithelium composed of epithelial cells. Examples thereof include tissues, nerve tissues composed of nerve cells and the like, and adipose tissues composed of fat cells and the like. Further, a piece of meat or the like made of these animal-derived tissues may be used.
動物由来組織における動物種は、硬組織と同一種でも、異種でもよい。本発明の洗浄剤組成物は、硬組織から動物由来組織を除去する性能に優れることから、動物由来組織と硬組織が異種である場合に、本発明の効果をより発揮することができる。 The animal species in the animal-derived tissue may be the same as or different from the hard tissue. Since the cleaning agent composition of the present invention is excellent in the ability to remove animal-derived tissue from hard tissue, the effect of the present invention can be more exerted when the animal-derived tissue and the hard tissue are different from each other.
動物由来組織の付着した硬組織とは、動物由来組織が硬組織に付着した状態のものであれば、特に制限されないが、例えば、動物由来組織が硬組織に癒着した状態のもの、動物由来組織と硬組織を混練することにより動物由来組織が硬組織に付着した状態のもの等が挙げられる。 The hard tissue to which the animal-derived tissue is attached is not particularly limited as long as it is in a state where the animal-derived tissue is attached to the hard tissue. And the one in which the animal-derived tissue is attached to the hard tissue by kneading the hard tissue and the like.
本発明の洗浄剤組成物は、動物由来組織の付着した硬組織から動物由来組織を除去する用途に利用されるものであれば、特に制限されず、例えば、挽肉等の食品に混入した硬組織の動物種の同定方法や、法医学的なＤＮＡ鑑定や、生物学的な遺伝子研究におけるＤＮＡの採取や、動物の骨の標本作製等に利用することができる。 The cleaning composition of the present invention is not particularly limited as long as it is used for removing animal-derived tissue from hard tissue to which animal-derived tissue is attached, and for example, hard tissue mixed in food such as ground meat. It can be used for identification methods of animal species, forensic DNA analysis, DNA collection in biological genetic research, preparation of animal bone specimens, and the like.
食品に混入した硬組織の動物種の同定方法では、動物由来組織と硬組織の動物種が異なるものかどうかを判別することが求められるため、動物由来の組織を硬組織から十分に除去するという本発明の効果をより発揮することができる。
前記食品の具体例としては、例えば、挽肉、合挽肉、食肉の練肉、魚の練肉、ネギトロ等の未加熱食品や、ソーセージ、ハンバーグ、ナゲット等の食肉を含む加工食品、魚肉ソーセージ、はんぺん、さつま揚げ等の魚肉を含む加工食品等の加熱食品が挙げられる。
In the method of identifying a hard tissue animal species mixed in food, it is required to determine whether the animal-derived tissue and the hard tissue animal species are different, so that the animal-derived tissue is sufficiently removed from the hard tissue. The effect of the present invention can be more exerted.
Specific examples of the foods include unheated foods such as minced meat, minced meat, meat paste, fish paste, and negitoro, processed foods including meat such as sausage, hamburger, and nugget, fish sausage, and hampen. Examples thereof include cooked foods such as processed foods containing fish meat such as fried minced meat.
次に、本発明の洗浄剤組成物に含有する成分について、詳細に説明する。
＜（Ａ）アルカリ剤＞
本発明に用いるアルカリ剤は、脂肪やタンパク質を分解するための成分であり、例えば、無機アルカリ剤および有機アルカリ剤を用いることができる。無機アルカリ剤としては、具体的には、水酸化ナトリウム、水酸化カリウム、水酸化カルシウム、水酸化マグネシウム等の水酸化物、炭酸ナトリウム、炭酸アンモニウム、炭酸マグネシウム等の炭酸塩、炭酸水素ナトリウム、炭酸水素アンモニウム等の炭酸水素塩、アンモニア等が挙げられる。有機アルカリ剤としては、具体的には、モノエタノールアミン、ジエタノールアミン、トリエタノールアミン等のアルカノールアミン、アルギニン、リジン等の塩基性アミノ酸等が挙げられる。
汎用性に優れるという観点から、好ましくは、無機アルカリ剤であり、より好ましくは、水酸化物であり、特に好ましくは、水酸化ナトリウム、水酸化カリウム、水酸化カルシウム、水酸化マグネシウムである。
Next, the components contained in the detergent composition of the present invention will be described in detail.
<(A) Alkaline agent>
The alkaline agent used in the present invention is a component for decomposing fat and protein, and for example, an inorganic alkaline agent and an organic alkaline agent can be used. Specific examples of the inorganic alkaline agent include hydroxides such as sodium hydroxide, potassium hydroxide, calcium hydroxide and magnesium hydroxide, carbonates such as sodium carbonate, ammonium carbonate and magnesium carbonate, sodium hydrogencarbonate and carbonate. Examples thereof include hydrogen carbonates such as ammonium hydrogen, ammonia and the like. Specific examples of the organic alkaline agent include alkanolamines such as monoethanolamine, diethanolamine and triethanolamine, and basic amino acids such as arginine and lysine.
From the viewpoint of excellent versatility, it is preferably an inorganic alkaline agent, more preferably a hydroxide, and particularly preferably sodium hydroxide, potassium hydroxide, calcium hydroxide, or magnesium hydroxide.
アルカリ剤の含有量は、特に制限されないが、好ましくは０．００１〜１質量％である。下限値としては、より好ましくは０．００５質量％以上であり、特に好ましくは０．０１質量％以上である。上限値としては、より好ましくは０．５質量％以下であり、更に好ましくは０．３質量％以下であり、特に好ましくは０．１質量％以下である。 The content of the alkaline agent is not particularly limited, but is preferably 0.001 to 1% by mass. The lower limit is more preferably 0.005% by mass or more, and particularly preferably 0.01% by mass or more. The upper limit value is more preferably 0.5% by mass or less, further preferably 0.3% by mass or less, and particularly preferably 0.1% by mass or less.
＜（Ｂ）界面活性剤＞
本発明に用いる界面活性剤は、親水性部分と疎水性部分を有する化合物であり、親水性部分の性質により、陰イオン界面活性剤、陽イオン界面活性剤、両性界面活性剤、非イオン界面活性剤に大別される。界面活性剤は、硬組織表面の微細な孔に埋まっている動物由来組織を除去する作用を有すると推察される。
<(B) Surfactant>
The surfactant used in the present invention is a compound having a hydrophilic portion and a hydrophobic portion, and depending on the nature of the hydrophilic portion, an anionic surfactant, a cationic surfactant, an amphoteric surfactant, and a nonionic surfactant. It is roughly divided into agents. It is presumed that the surfactant has an action of removing the animal-derived tissue buried in the fine pores on the surface of the hard tissue.
（陰イオン界面活性剤）
陰イオン界面活性剤としては、例えば、脂肪酸セッケン、アルキルエーテルカルボン酸塩、脂肪酸アミドエーテルカルボン酸塩、アシル乳酸塩、Ｎ−アシルグルタミン酸塩、Ｎ−アシルメチルアラニン塩、Ｎ−アシルサルコシン塩、Ｎ−アシルアミノ酸塩等のカルボン酸塩；アルカンスルホン酸塩、α−オレフィンスルホン酸塩、α−スルホ脂肪酸メチルエステル塩、アシルイセチオン酸塩、アルキルグリシジルエーテルスルホン酸塩、アルキルスルホコハク酸塩、アルキルスルホ酢酸塩、アルキルベンゼンスルホン酸塩、アルキルナフタレンスルホン酸塩、Ｎ−アシルメチルタウリン塩、ナフタレンスルホン酸塩ホルマリン縮合物等のスルホン酸塩；アルキル硫酸塩、アルキルエーテル硫酸塩、アルキルアリールエーテル硫酸塩、脂肪酸アルカノールアミド硫酸塩、脂肪酸モノグリセリド硫酸塩等の硫酸塩；アルキルリン酸塩、ポリオキシエチレンアルキルエーテルリン酸塩、アルキルアリルエーテルリン酸塩、脂肪酸アミドエーテルリン酸塩等のリン酸塩等が挙げられる。
(Anionic surfactant)
Examples of anionic surfactants include fatty acid sequens, alkyl ether carboxylates, fatty acid amide ether carboxylates, acyl lactates, N-acylglutamates, N-acylmethylalanine salts, N-acylsulfosin salts, and N. Carboates such as −acylamino acid salts; alkane sulfonates, α-olefin sulfonates, α-sulfo fatty acid methyl ester salts, acyl citeionates, alkyl glycidyl ether sulfonates, alkyl sulfosuccinates, alkyl sulfoacetates. , Alkylbenzene sulfonate, alkylnaphthalene sulfonate, N-acylmethyl taurine salt, naphthalene sulfonate formalin condensate and other sulfonates; alkyl sulphate, alkyl ether sulphate, alkylaryl ether sulphate, fatty acid alkanolamide Sulfates such as sulfates and fatty acid monoglyceride sulfates; phosphates such as alkyl phosphates, polyoxyethylene alkyl ether phosphates, alkyl allyl ether phosphates, and fatty acid amide ether phosphates can be mentioned.
より具体的には、ラウリル硫酸ナトリウム（ドデシル硫酸ナトリウム（ＳＤＳ））、ミリスチル硫酸ナトリウム、ラウリル硫酸カリウム、ラウリル硫酸アンモニウム、ラウリル硫酸トリエタノールアミン、セチル硫酸ナトリウム、ステアリル硫酸ナトリウム、ポリオキシエチレンラウリルエーテル硫酸ナトリウム、ポリオキシエチレンラウリルエーテル硫酸トリエタノールアミン、ポリオキシエチレンラウリルエーテル硫酸アンモニウム、ポリオキシエチレンステアリルエーテル硫酸ナトリウム、ステアロイルメチルタウリンナトリウム、ドデシルベンゼンスルホン酸トリエタノールアミン、テトラデセンスルホン酸ナトリウム、ラウリルリン酸ナトリウム、ポリオキシエチレンラウリルエーテルリン酸ナトリウム、ラウロイルグルタミン酸ナトリウム、Ｎ−ラウロイルメチル−β−アラニンナトリウム、ラウリン酸ナトリウム、ミリスチン酸ナトリウム等が例示される。 More specifically, sodium lauryl sulfate (sodium dodecyl sulfate (SDS)), sodium myristyl sulfate, potassium lauryl sulfate, ammonium lauryl sulfate, triethanolamine lauryl sulfate, sodium cetyl sulfate, sodium stearyl sulfate, sodium polyoxyethylene lauryl ether sulfate. , Polyoxyethylene lauryl ether sulfate triethanolamine, polyoxyethylene lauryl ether sulfate ammonium, polyoxyethylene stearyl ether sulfate sodium, stearoyl methyl taurine sodium, dodecylbenzene sulfonate triethanolamine, tetradecene sulfonate sodium, sodium lauryl phosphate, Examples thereof include sodium polyoxyethylene lauryl ether phosphate, sodium lauroyl glutamate, sodium N-lauroylmethyl-β-alanine, sodium laurate, and sodium myristate.
（陽イオン界面活性剤）
陽イオン界面活性剤としては、例えば、１級アミン塩、２級アミン塩、３級アミン塩、脂肪酸アミドアミン塩等の脂肪族アミン塩；アルキル４級アンモニウム塩、アルキルトリアルキレングリコールアンモニウム塩、アルキルエーテルアンモニウム塩、ベンザルコニウム塩、ベンゼトニウム塩、ピリジニウム塩等の４級アンモニウム塩等が挙げられる。
(Cationic surfactant)
Examples of the cationic surfactant include aliphatic amine salts such as primary amine salts, secondary amine salts, tertiary amine salts, and fatty acid amidamine salts; alkyl quaternary ammonium salts, alkyltrialkylene glycol ammonium salts, and alkyl ethers. Examples thereof include quaternary ammonium salts such as ammonium salt, benzalconium salt, benzethonium salt and pyridinium salt.
より具体的には、塩化ラウリルトリメチルアンモニウム、臭化ラウリルトリメチルアンモニウム、塩化アルキル（１６，１８）トリメチルアンモニウム、塩化セチルトリメチルアンモニウム、臭化セチルトリメチルアンモニウム、セチルトリメチルアンモニウムサッカリン、塩化ステアリルトリメチルアンモニウム、臭化ステアリルトリメチルアンモニウム、塩化ベヘニルトリメチルアンモニウム、ステアリルトリメチルアンモニウムサッカリン、塩化アルキル（２８）トリメチルアンモニウム、塩化ジポリオキシエチレン（２）オレイルメチルアンモニウム、塩化ジポリオキシエチレンステアリルメチルアンモニウム、塩化ポリオキシエチレン（１）ポリオキシプロピレン（２５）ジエチルメチルアンモニウム、塩化ポリオキシプロピレンメチルジエチルアンモニウム、塩化メタクリロイルオキシエチルトリメチルアンモニウム、メチル硫酸ベヘニルトリメチルアンモニウム、塩化ジアルキル（１２〜１５）ジメチルアンモニウム、塩化ジアルキル（１２〜１８）ジメチルアンモニウム、塩化ジアルキル（１４〜１８）ジメチルアンモニウム、塩化ジココイルジメチルアンモニウム、塩化ジセチルジメチルアンモニウム、塩化ジステアリルジメチルアンモニウム、塩化イソステアリルラウリルジメチルアンモニウム等が挙げられる。なお、「アルキル」に続くカッコ内の数字は炭素数、「ポリオキシエチレン」及び「ポリオキシプロピレン」に続くカッコ内の数字は付加モル数を表す。 More specifically, lauryltrimethylammonium chloride, lauryltrimethylammonium bromide, alkyl (16,18) trimethylammonium chloride, cetyltrimethylammonium chloride, cetyltrimethylammonium bromide, cetyltrimethylammonium saccharin, stearyltrimethylammonium chloride, bromide. Stearyltrimethylammonium, behenyltrimethylammonium chloride, stearyltrimethylammonium saccharin, alkyl (28) trimethylammonium, dipolyoxyethylene chloride (2) oleylmethylammonium, dipolyoxyethylene chloride stearylmethylammonium, polyoxyethylene chloride (1) Polyoxypropylene (25) diethylmethylammonium, polyoxypropylenemethyldiammonium chloride, methacryloyloxyethyltrimethylammonium chloride, behenyltrimethylammonium methylsulfate, dialkyl (12-15) dimethylammonium, dialkyl chloride (12-18) dimethylammonium , Dialkyl (14-18) dimethylammonium, dicocoyl dimethylammonium chloride, disetyldimethylammonium chloride, distearyldimethylammonium chloride, isostearyllauryldimethylammonium chloride and the like. The number in parentheses following "alkyl" indicates the number of carbon atoms, and the number in parentheses following "polyoxyethylene" and "polyoxypropylene" indicates the number of added moles.
（両性界面活性剤）
両性界面活性剤としては、例えば、グリシン型、アミノプロピオン酸型、カルボキシベタイン型、イミダゾリニウム塩等のカルボン酸塩型両性界面活性剤；スルホベタイン型両性界面活性剤；スルホン酸型両性界面活性剤；硫酸型両性界面活性剤；リン酸型両性界面活性剤等が挙げられる。また、カルボン酸塩型両性界面活性剤は、アミン塩のカチオン部をもつアミノ酸型両性界面活性剤（グリシン型、アミノプロピオン酸型）と、４級アンモニウム塩のカチオン部をもつベタイン型両性界面活性剤に大別される。
(Amphoteric surfactant)
Examples of amphoteric surfactants include carboxylate-type amphoteric surfactants such as glycine-type, aminopropionic acid-type, carboxybetaine-type, and imidazolinium salts; sulfobetaine-type amphoteric surfactants; and sulfonic acid-type amphoteric surfactants. Agents; sulfuric acid-type amphoteric surfactants; phosphoric acid-type amphoteric surfactants and the like. The carboxylate-type amphoteric tenside is an amino acid-type amphoteric tenside having a cation portion of an amine salt (glycine type, aminopropionic acid type) and a betaine-type amphoteric tenside having a cation portion of a quaternary ammonium salt. It is roughly divided into agents.
アミノ酸型両性界面活性剤の具体例としては、例えば、Ｎ−ラウロイル−Ｎ’−カルボキシメチル−Ｎ’−ヒドロキシエチルエチレンジアミンナトリウム（ラウロアンホ酢酸Ｎａ）、２−アルキル−Ｎ−カルボキシメチル−Ｎ−ヒドロキシエチルイミダゾリニウムベタイン、ウンデシルヒドロキシエチルイミダゾリニウムベタインナトリウム、塩酸アルキルジアミノエチルグリシン、Ｎ−ヤシ油脂肪酸アシル−Ｎ’−カルボキシエチル−Ｎ’−ヒドロキシエチルエチレンジアミンナトリウム、Ｎ−ヤシ油脂肪酸アシル−Ｎ’−カルボキシエトキシエチル−Ｎ’−カルボキシエチルエチレンジアミン二ナトリウム、Ｎ−ヤシ油脂肪酸アシル−Ｎ’−カルボキシメトキシエチル−Ｎ’−カルボキシメチルエチレンジアミン二ナトリウム、ラウリルジアミノエチルグリシンナトリウム、パーム油脂肪酸アシル−Ｎ−カルボキシエチル−Ｎ−ヒドロキシエチルエチレンジアミンナトリウム等のグリシン型両性界面活性剤；ラウリルアミノプロピオン酸ナトリウム、ラウリルアミノジプロピオン酸ナトリウム、ラウリルアミノプロピオン酸トリエタノールアミン等のアミノプロピオン酸型両性界面活性剤等が挙げられる。
ベタイン型両性界面活性剤の具体例としては、例えば、ヤシ油アルキルベタイン、ラウリルジメチルアミノ酢酸ベタイン、ミリスチルジメチルアミノ酢酸ベタイン、ステアリルジメチルアミノ酢酸ベタイン、ステアリルジメチルベタインナトリウム、ヤシ油脂肪酸アミドプロピルベタイン、パーム油脂肪酸アミドプロピルベタイン、ラウリン酸アミドプロピルベタイン、リシノレイン酸アミドプロピルベタイン、ステアリルジヒドロキシエチルベタイン等のアミノ酢酸ベタイン型両性界面活性剤；ラウリルヒドロキシスルホベタイン等のスルホベタイン型両性界面活性剤等が挙げられる。
Specific examples of the amino acid-type amphoteric surfactant include N-lauroyl-N'-carboxymethyl-N'-hydroxyethylethylenediamine sodium (Na lauroamphoacetate) and 2-alkyl-N-carboxymethyl-N-hydroxyethyl. Imidazolinium betaine, undecyl hydroxyethyl imidazolinium betaine sodium, alkyldiaminoethylglycine hydrochloride, N-palm oil fatty acid acyl-N'-carboxyethyl-N'-hydroxyethyl ethylenediamine sodium, N-palm oil fatty acid acyl-N '-Carboxyethoxyethyl-N'-carboxyethylethylenediamine disodium, N-palm oil fatty acid acyl-N'-carboxymethoxyethyl-N'-carboxymethylethylenediamine disodium, lauryldiaminoethylglycine sodium, palm oil fatty acid acyl-N Glycine-type amphoteric surfactants such as -carboxyethyl-N-hydroxyethylethylenediamine sodium; aminopropionic acid-type amphoteric surfactants such as sodium laurylaminopropionate, sodium laurylaminodipropionate, triethanolamine laurylaminopropionate, etc. Can be mentioned.
Specific examples of the betaine-type amphoteric surfactant include coconut oil alkyl betaine, lauryl dimethylamino acetate betaine, myristyl dimethyl amino acetate betaine, stearyl dimethyl amino acetate betaine, stearyl dimethyl betaine sodium, coconut oil fatty acid amide propyl betaine, and palm. Examples thereof include aminoacetate betaine-type amphoteric surfactants such as oil fatty acid amide propyl betaine, lauric acid amide propyl betaine, ricinoleic acid amide propyl betaine, and stearyldihydroxyethyl betaine; and sulfobetaine-type amphoteric surfactants such as lauryl hydroxysulfobetaine. ..
（非イオン界面活性剤）
非イオン界面活性剤としては、例えば、プロピレングリコールモノ脂肪酸エステル、エチレングリコールモノ脂肪酸エステル、グリセリンモノ脂肪酸エステル、ポリグリセリン脂肪酸エステル、ソルビタン脂肪酸エステル、ショ糖脂肪酸エステル、メチルグルコシド脂肪酸エステル、アルキルポリグルコシド等の多価アルコール脂肪酸エステル及び多価アルコールアルキルエーテル；ポリオキエチレンアルキルエーテル、ポリオキエチレンアルキルフェニルエーテル、ポリオキエチレンフィトステロール、ポリオキエチレンフィトスタノール、ポリオキエチレンコレステロール、ポリオキエチレンコレスタノール、ポリオキエチレンポリオキシプロピレンアルキルエーテル等のポリオキシエチレンエーテル；ポリオキエチレンモノ脂肪酸エステル、ポリオキエチレングリコールジ脂肪酸エステル、ポリオキエチレングリセリン脂肪酸エステル、ポリオキエチレンソルビタン脂肪酸エステル、ポリオキエチレンソルビトール脂肪酸エステル、ポリオキエチレンメチルグルコシド脂肪酸エステル、ポリオキエチレンヒマシ油・硬化ヒマシ油、ポリオキエチレン動植物油、ポリオキエチレンアルキルエーテル脂肪酸エステル、ポリオキエチレンポリオキシプロピレングリコール等のエーテルエステル；ポリオキエチレンアルキルアミン、ポリオキエチレン脂肪酸アミド、脂肪酸ジエタノールアミド、アルキルアミンオキシド等の含窒素誘導体等が挙げられる。
(Nonionic surfactant)
Examples of the nonionic surfactant include propylene glycol monofatty acid ester, ethylene glycol monofatty acid ester, glycerin monofatty acid ester, polyglycerin fatty acid ester, sorbitan fatty acid ester, sucrose fatty acid ester, methylglucoside fatty acid ester, alkylpolyglucoside and the like. Polyhydric Alcohol Fatty Acid Ethers and Polyhydric Alcohol Alkyl Ethers; Polyokiethylene Alkyl Ethers, Polyokiethylene Alkylphenyl Ethers, Polyokiethylene Phytosterol, Polyokiethylene Phytostanol, Polyokiethylene Cholesterol, Polyokiethylene Cholestanol, Polyoki Polyoxyethylene ethers such as ethylene polyoxypropylene alkyl ethers; polyoxyethylene monofatty acid esters, polyoxyethylene glycol difatty acid esters, polyoxyethylene glycerin fatty acid esters, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene sorbitol fatty acid esters, polyo Ether esters such as chiethylene methyl glucoside fatty acid ester, polyoxyethylene castor oil / hardened castor oil, polyoxyethylene animal and vegetable oil, polyoxyethylene alkyl ether fatty acid ester, polyoxyethylene polyoxypropylene glycol; polyoxyethylene alkylamine, polyo Examples thereof include nitrogen-containing derivatives such as chiethylene fatty acid amide, fatty acid diethanolamide, and alkylamine oxide.
より具体的には、ポリオキシエチレン（２０）ソルビタンモノラウレート（Ｔｗｅｅｎ２０）、ポリオキシエチレン（２０）ソルビタンモノオレート、ポリオキシエチレン（５〜１０）ヤシ油アルコールエーテル、ポリオキシエチレン（５〜１０）合成アルコールエーテル、ポリオキシエチレン（５〜７）ラウリルエーテル、ポリオキシエチレン（７．５〜１５）ノニルフェニルエーテル、ポリオキシエチレン（５）ポリオキシプロピレン（８）セチルエーテル、ポリオキシエチレンポリオキシプロピレンデシルテトラデシルエーテル、ポリオキシエチレン（１０〜５５）モノステアリン酸エステル、モノカプリン酸ソルビタン、モノミリスチン酸デカグリセリル、モノオレイン酸デカグリセリル、モノラウリン酸デカグリセリル、モノカプリン酸ヘキサグリセリル等が挙げられる。 More specifically, polyoxyethylene (20) sorbitan monolaurate (Tween 20), polyoxyethylene (20) sorbitan monoolate, polyoxyethylene (5-10) coconut oil alcohol ether, polyoxyethylene (5-10). ) Synthetic alcohol ether, polyoxyethylene (5-7) lauryl ether, polyoxyethylene (7.5-15) nonylphenyl ether, polyoxyethylene (5) polyoxypropylene (8) cetyl ether, polyoxyethylene polyoxy Examples thereof include propylene decyltetradecyl ether, polyoxyethylene (10-55) monostearic acid ester, sorbitan monocaprate, decaglyceryl monomyristate, decaglyceryl monooleate, decaglyceryl monolaurate, hexaglyceryl monocaprate and the like. ..
界面活性剤として、好ましくは、陰イオン界面活性剤、非イオン界面活性剤であり、より好ましくは、アルキル硫酸塩、ポリオキエチレンソルビタン脂肪酸エステルであり、特に好ましくは、ラウリル硫酸ナトリウム、ポリオキシエチレン（２０）ソルビタンモノラウレートである。界面活性剤は、１種又は２種以上を混合して使用することができる。 The surfactant is preferably an anionic surfactant or nonionic surfactant, more preferably alkyl sulfate or polyoxyethylene sorbitan fatty acid ester, and particularly preferably sodium lauryl sulfate or polyoxyethylene. (20) Solbitan monolaurate. The surfactant may be used alone or in admixture of two or more.
界面活性剤の含有量は、特に制限されないが、好ましくは０．００１〜５質量％である。下限値としては、より好ましくは０．０１質量％以上であり、更に好ましくは０．０２質量％以上であり、特に好ましくは０．０５質量％以上である。上限値としては、より好ましくは１質量％以下であり、更に好ましくは０．９質量％以下であり、特に好ましくは０．５質量％以下である。
また、イオン性界面活性剤を使用する場合には、好ましくは０．０１質量％超であり、非イオン界面活性剤を使用する場合には、好ましくは１質量％未満である。
The content of the surfactant is not particularly limited, but is preferably 0.001 to 5% by mass. The lower limit value is more preferably 0.01% by mass or more, further preferably 0.02% by mass or more, and particularly preferably 0.05% by mass or more. The upper limit value is more preferably 1% by mass or less, further preferably 0.9% by mass or less, and particularly preferably 0.5% by mass or less.
Further, when an ionic surfactant is used, it is preferably more than 0.01% by mass, and when a nonionic surfactant is used, it is preferably less than 1% by mass.
＜その他の成分＞
本発明の洗浄剤組成物には上記の各成分以外にも、各種の添加剤を配合することができる。例えば、ＥＤＴＡ（エチレンジアミン四酢酸）等のキレート剤や、次亜塩素酸ナトリウム等の各種添加剤を加えることができる。
<Other ingredients>
In addition to the above-mentioned components, various additives can be added to the detergent composition of the present invention. For example, a chelating agent such as EDTA (ethylenediaminetetraacetic acid) and various additives such as sodium hypochlorite can be added.
また、本発明の洗浄剤組成物において、以上の各成分を液体に溶解又は分散して使用してもよい。液体としては、水のほか、エタノール、メタノール、イソプロパノール等のアルコール、クロロホルム、ヘキサン等を使用することができる。また、２種以上の液体を混合して使用することもできる。好ましい液体は、水である。 Further, in the detergent composition of the present invention, each of the above components may be dissolved or dispersed in a liquid for use. As the liquid, in addition to water, alcohols such as ethanol, methanol and isopropanol, chloroform, hexane and the like can be used. Further, two or more kinds of liquids can be mixed and used. The preferred liquid is water.
［洗浄方法］
本発明の洗浄方法は、動物由来の組織の付着した硬組織から動物由来の組織を除去するための洗浄方法であって、本発明の洗浄剤組成物を用いて、動物由来の組織の付着した硬組織を洗浄する工程（以下、「洗浄工程」という。）、を備えたことを特徴とする。
[Washing method]
The cleaning method of the present invention is a cleaning method for removing animal-derived tissue from hard tissue to which animal-derived tissue is attached, and the cleaning agent composition of the present invention is used to attach animal-derived tissue. It is characterized by including a step of cleaning hard tissue (hereinafter referred to as "cleaning step").
＜洗浄工程＞
洗浄工程は、動物由来組織の付着した硬組織（以下、「被洗浄物」という。）を、本発明の洗浄剤組成物と接触させる手段であれば、どのような手段を用いてもよい。例えば、被洗浄物を洗浄剤組成物に浸漬洗浄する手段や、被洗浄物に洗浄剤組成物を掛け流して洗浄する手段や、洗浄剤組成物を付したブラシ等の洗浄具を用いて動物由来組織をこすり落とす洗浄手段等が挙げられる。硬組織が小さい片である場合には、被洗浄物を洗浄剤組成物に浸漬洗浄する手段により洗浄処理することが好ましい。
<Washing process>
In the cleaning step, any means may be used as long as the hard tissue to which the animal-derived tissue is attached (hereinafter, referred to as “object to be cleaned”) is brought into contact with the cleaning agent composition of the present invention. For example, an animal using a means for immersing and cleaning the object to be cleaned in the cleaning agent composition, a means for flushing the cleaning agent composition over the object to be cleaned, and a cleaning tool such as a brush to which the cleaning agent composition is attached. Examples include a cleaning means for scraping off the derived tissue. When the hard tissue is a small piece, it is preferable to wash the object to be cleaned by a means for dipping and cleaning the cleaning agent composition.
被洗浄物を洗浄剤組成物に浸漬洗浄する手段においては、ボルテックスミキサー等の撹拌装置を用いて撹拌することが好ましい。撹拌することにより、動物由来組織を除去する作用をより高めることができる。 In the means for dipping and cleaning the object to be cleaned in the detergent composition, it is preferable to stir using a stirring device such as a vortex mixer. By stirring, the action of removing animal-derived tissue can be further enhanced.
＜前処理工程＞
洗浄工程の前に、被洗浄物をアルカリ溶液により処理する前処理工程を行うことが好ましい。前処理工程により、洗浄工程における動物由来組織の除去効果を高めることができる。
<Pretreatment process>
Prior to the cleaning step, it is preferable to perform a pretreatment step of treating the object to be cleaned with an alkaline solution. The pretreatment step can enhance the effect of removing animal-derived tissue in the washing step.
前処理工程としては、被洗浄物をアルカリ溶液に接触させる手段であれば、どのような手段を用いてもよく、例えば、被洗浄物を５０〜１００℃に加温したアルカリ溶液に、１〜３０分間浸漬した後、該アルカリ溶液から硬組織を摘出する工程等が挙げられる。この前処理工程は、２回以上行うことが好ましい。 As the pretreatment step, any means may be used as long as it is a means for bringing the object to be cleaned into contact with the alkaline solution. Examples thereof include a step of removing hard tissue from the alkaline solution after soaking for 30 minutes. This pretreatment step is preferably performed twice or more.
＜加熱工程＞
洗浄工程の前に、被洗浄物を加熱処理する加熱工程を行ってもよい。加熱工程としては、動物由来組織を変性する程度に加熱する手段であれば、どのような手段を用いてもよく、例えば、被洗浄物を真空状態で密封し、沸騰水中にて５〜６０分間加熱する工程や、ステンレス製容器に入れた状態でオートクレーブを用いて１２１℃にて１５分間加熱する工程等が挙げられる。
<Heating process>
Before the cleaning step, a heating step of heat-treating the object to be cleaned may be performed. As the heating step, any means may be used as long as it is a means for heating the animal-derived tissue to the extent that it is denatured. For example, the object to be cleaned is sealed in a vacuum state and placed in boiling water for 5 to 60 minutes. Examples thereof include a step of heating and a step of heating in a stainless steel container at 121 ° C. for 15 minutes using an autoclave.
以下、本発明の洗浄剤組成物を用いて、動物由来組織の付着した硬組織から前記動物由来組織を除去するための洗浄方法、洗浄した硬組織から核酸を採取するための核酸採取方法、及び、硬組織から採取した核酸を用いて動物種を判別する方法の実施形態を、図面を参照して詳細に説明する。 Hereinafter, using the cleaning agent composition of the present invention, a cleaning method for removing the animal-derived tissue from the hard tissue to which the animal-derived tissue is attached, a nucleic acid collection method for collecting nucleic acid from the washed hard tissue, and a nucleic acid collection method. , An embodiment of a method for discriminating an animal species using nucleic acid collected from hard tissue will be described in detail with reference to the drawings.
図１に示すように、動物由来組織の付着した硬組織から前記動物由来組織を除去するための洗浄方法は、（１）前処理工程と（２）洗浄工程を備えており、洗浄した硬組織から核酸を採取するための核酸採取方法は、（３）脱灰工程と（４）核酸採取工程を備えている。 As shown in FIG. 1, the washing method for removing the animal-derived tissue from the hard tissue to which the animal-derived tissue is attached includes (1) a pretreatment step and (2) a washing step, and the washed hard tissue. The nucleic acid collection method for collecting nucleic acid from the tissue includes (3) decalcification step and (4) nucleic acid collection step.
（１）前処理工程
本実施形態における前処理工程では、動物由来組織に混入した硬組織を、前処理液に浸漬、摘出等の処理を施して清浄し、硬組織の表面に付着した動物由来組織を除去した。前処理液としては、炭酸ナトリウム水溶液を用いた。
(1) Pretreatment Step In the pretreatment step in the present embodiment, the hard tissue mixed in the animal-derived tissue is cleaned by immersing it in a pretreatment solution, removing it, or the like, and the animal-derived tissue adheres to the surface of the hard tissue. Tissue was removed. As the pretreatment liquid, an aqueous sodium carbonate solution was used.
具体的には、動物由来組織に混入した硬組織を、７５℃に加温した１質量％炭酸ナトリウム水溶液に１０分間浸漬後、ステンレス製ピンセットを用いて硬組織を摘出した。次いで、摘出した硬組織を新たな１質量％炭酸ナトリウム水溶液を用いて再度浸漬し、ステンレス製ピンセットを用いて硬組織表面に付着した動物由来組織を除去した。さらに、新たな１質量％炭酸ナトリウム水溶液で硬組織表面のすすぎを行った後、風乾させた。 Specifically, the hard tissue mixed in the animal-derived tissue was immersed in a 1% by mass sodium carbonate aqueous solution heated to 75 ° C. for 10 minutes, and then the hard tissue was removed using a stainless steel tweezers. Then, the removed hard tissue was immersed again with a new 1% by mass sodium carbonate aqueous solution, and the animal-derived tissue adhering to the hard tissue surface was removed using stainless steel tweezers. Further, the surface of the hard tissue was rinsed with a new 1 mass% sodium carbonate aqueous solution, and then air-dried.
（２）洗浄工程
本実施形態における洗浄工程では、前処理工程において動物由来組織を除去した硬組織を、更に洗浄剤組成物を用いて浸漬洗浄した。これにより、硬組織に残存した動物由来組織を除去した。
(2) Cleaning Step In the cleaning step in the present embodiment, the hard tissue from which the animal-derived tissue was removed in the pretreatment step was further immersed and washed with a cleaning agent composition. As a result, the animal-derived tissue remaining in the hard tissue was removed.
具体的には、ポリプロピレン製２ｍＬサンプリングチューブに１ｍＬの洗浄剤組成物を投入し、これに前処理工程において動物由来組織を除去した硬組織を浸漬させ、ボルテックスミキサーを用いて攪拌を行った。 Specifically, 1 mL of the detergent composition was put into a 2 mL sampling tube made of polypropylene, and the hard tissue from which the animal-derived tissue had been removed in the pretreatment step was immersed therein, and the mixture was stirred using a vortex mixer.
次いで、硬組織をポリプロピレン製２ｍＬサンプリングチューブに加えた１ｍＬの滅菌水に浸漬させ、ボルテックスミキサーを用いて攪拌を行った。 Next, the hard tissue was immersed in 1 mL of sterile water added to a 2 mL sampling tube made of polypropylene, and the mixture was stirred using a vortex mixer.
次いで、硬組織をポリプロピレン製２ｍＬサンプリングチューブに加えた１ｍＬのエタノール（質量分率９９．５＋％）に浸漬させ、ボルテックスミキサーを用いて攪拌を行った後、風乾させた。 Next, the hard tissue was immersed in 1 mL of ethanol (mass fraction 99.5 +%) added to a polypropylene 2 mL sampling tube, stirred using a vortex mixer, and then air-dried.
（３）脱灰工程
実施形態における硬組織の脱灰工程では、ＥＤＴＡ水溶液に浸漬させることで、硬組織に含まれるカルシウム成分を除去した。
(3) Decalcification Step In the hard tissue decalcification step of the embodiment, the calcium component contained in the hard tissue was removed by immersing it in an EDTA aqueous solution.
具体的には、洗浄工程において動物由来組織を除去した硬組織を、ポリプロピレン製２ｍＬサンプリングチューブに加えた１．５ｍＬの５０ｍＭ ＥＤＴＡ水溶液（ｐＨ８．０）に浸漬させ、ボルテックスミキサーを用いて攪拌を行った。次に、上清を除去し、１．５ｍＬの５０ｍＭ ＥＤＴＡ水溶液（ｐＨ８．０）を再度加えてボルテックスミキサーを用いて攪拌を行った。本工程をさらに２回繰り返した後、風乾させた。 Specifically, the hard tissue from which the animal-derived tissue was removed in the washing step was immersed in 1.5 mL of a 50 mM EDTA aqueous solution (pH 8.0) added to a polypropylene 2 mL sampling tube, and stirred using a vortex mixer. rice field. Next, the supernatant was removed, 1.5 mL of a 50 mM EDTA aqueous solution (pH 8.0) was added again, and the mixture was stirred using a vortex mixer. After repeating this step two more times, it was air-dried.
上記（２）洗浄工程および（３）脱灰工程において、ボルテックスミキサーを用いて攪拌を行う際の温度、強度および時間は、硬組織の容量や形状に応じ、適宜調整することができる。温度としては１０〜３０℃であると一層好ましく、強度および時間は１，５００〜３，０００ｒｐｍで１分間以上であると一層好ましい。 In the above (2) cleaning step and (3) decalcification step, the temperature, strength and time when stirring with a vortex mixer can be appropriately adjusted according to the capacity and shape of the hard tissue. The temperature is more preferably 10 to 30 ° C., and the intensity and time are more preferably 1,500 to 3,000 rpm for 1 minute or more.
（４）核酸採取工程
実施形態における硬組織からの核酸採取工程では、核酸採取キットを用いて、脱灰した硬組織から核酸を採取した。
(4) Nucleic Acid Collection Step In the nucleic acid collection step from the hard tissue in the embodiment, nucleic acid was collected from the decalcified hard tissue using a nucleic acid collection kit.
具体的には、核酸採取キット、例えばＨｉｇｈ Ｐｕｒｅ ＰＣＲ Ｔｅｍｐｌａｔｅ Ｐｒｅｐａｒａｔｉｏｎ Ｋｉｔ（ロッシュ社製；商品名）を用いて、脱灰した硬組織から核酸を採取した。核酸の採取はキット指定の方法に従って行った。
なお、核酸採取キットとしては、ＩＳＯＨＡＩＲ（ニッポンジーン社製；商品名）を用いてもよい。
Specifically, nucleic acids were collected from decalcified hard tissue using a nucleic acid collection kit, for example, High Pure PCR Template Preparation Kit (manufactured by Roche; trade name). Nucleic acid was collected according to the method specified in the kit.
As the nucleic acid collection kit, ISOHAIR (manufactured by Nippon Gene Co., Ltd .; trade name) may be used.
（５）動物種判別工程
実施形態における動物種判別工程では、核酸採取工程で得たＤＮＡ抽出液を用いてＰＣＲ法または定量ＰＣＲ法により硬組織の動物種を判別した。
(5) Animal Species Discrimination Step In the animal species discrimination step of the embodiment, a hard tissue animal species was discriminated by a PCR method or a quantitative PCR method using the DNA extract obtained in the nucleic acid collection step.
＜ＰＣＲ法による動物種判別＞
具体的には、松永ら（日本食品科学工学会誌，４６（３），１８７−１９４，１９９９）の方法でＰＣＲ法による動物種判別を行った。ＰＣＲ反応液は、ＡｍｐｌｉＴａｑ Ｇｏｌｄ ３６０ Ｍａｓｔｅｒ Ｍｉｘ（サーモフィッシャーサイエンティフィック社製；商品名）１２．５μＬ、プライマー、ＤＮＡ抽出液１μＬに滅菌水を加えて合計２５μＬとした。使用するプライマーおよび反応条件は松永ら（１９９９）の方法に従い、９４℃で５分反応後、９４℃・３０秒、６０℃・３０秒、７２℃・３０秒を１サイクルとして繰り返し、７２℃で７分反応させた。ＰＣＲはＧｅｎｅａｍｐ ＰＣＲ Ｓｙｓｔｅｍ ９７００（サーモフィッシャーサイエンティフィック社製；商品名）を用いて行い、ＰＣＲ産物５μＬを４質量％アガロースゲル電気泳動で分析した。泳動条件は１００Ｖで３５分間とし、泳動後アガロースゲルをエチジウムブロマイド溶液で染色し、泳動図をＡＥ−６９３３ＦＸＥＳプリントグラフ（アトー社製；商品名）で画像化のうえ、ＣＳ Ａｎａｌｙｚｅｒ ３（アトー社製；商品名）で増幅産物のバンドを定量した。９４℃・３０秒、６０℃・３０秒、７２℃・３０秒の繰り返しはＤＮＡ抽出液の濃度や精製度に応じ、適宜調整することが好ましく、２５〜３５サイクルであると一層好ましい。
<Animal species discrimination by PCR method>
Specifically, animal species were discriminated by the PCR method by the method of Matsunaga et al. (Journal of the Japan Society for Food Science and Technology, 46 (3), 187-194, 1999). The PCR reaction solution was made up to 25 μL in total by adding 12.5 μL of AmpliTaq Gold 360 Master Mix (manufactured by Thermo Fisher Scientific Co., Ltd .; trade name), 1 μL of primer and DNA extract, and sterilized water. The primers and reaction conditions used were the method of Matsunaga et al. (1999), and after reacting at 94 ° C. for 5 minutes, repeating 94 ° C. for 30 seconds, 60 ° C. for 30 seconds, and 72 ° C. for 30 seconds as one cycle, and at 72 ° C. The reaction was carried out for 7 minutes. PCR was performed using Geneamp PCR System 9700 (manufactured by Thermo Fisher Scientific Co., Ltd .; trade name), and 5 μL of PCR product was analyzed by 4 mass% agarose gel electrophoresis. The migration conditions were 100 V for 35 minutes, and after the migration, the agarose gel was stained with an ethidium bromide solution, and the migration map was imaged with an AE-6933FXES print graph (manufactured by Atto Co., Ltd .; trade name), and then CS Analyzer 3 (manufactured by Atto Co., Ltd.). The band of the amplified product was quantified by (trade name). The repetition of 94 ° C. for 30 seconds, 60 ° C. for 30 seconds, and 72 ° C. for 30 seconds is preferably adjusted as appropriate according to the concentration of the DNA extract and the degree of purification, and more preferably 25 to 35 cycles.
＜定量ＰＣＲ法による動物種判別＞
具体的には、Ｔａｎａｂｅ ｅｔ ａｌ.（Ｂｉｏｓｃｉ． Ｂｉｏｔｅｃｈｎｏｌ． Ｂｉｏｃｈｅｍ．，７１（１２），３１３１−３１３５，２００７）の方法に従い、定量ＰＣＲ法による動物種判別を行った。定量ＰＣＲ反応液は、ＡｍｐｌｉＴａｑ Ｇｏｌｄ ３６０ Ｍａｓｔｅｒ Ｍｉｘ（サーモフィッシャーサイエンティフィック社製；商品名）１２．５μＬ、プライマー、プローブ、ＤＮＡ抽出液２．５μＬに滅菌水を加えて合計２５μＬとした。使用するプライマー、プローブおよび反応条件はＴａｎａｂｅ ｅｔ ａｌ.（２００７）の方法に従い、５０℃で２分保持後、９５℃・１０秒、９５℃・１５秒、６０℃・１分を１サイクルとして繰り返した。なお、ＤＮＡ抽出液は、ＱｕａｎｔｉＦｌｕｏｒ Ｏｎｅ ｄｓＤＮＡ ｓｙｓｔｅｍ（プロメガ社製；商品名）を用いて濃度測定を行い、脱イオン蒸留水にて１ｎｇ／μＬに調製したものを用いた。また、定量ＰＣＲは、Ｔａｋａｒａ ｔｈｅｒｍａｌ ｃｙｃｌｅｒ ｄｉｃｅ（タカラバイオ社製；商品名）を用いて行った。
<Animal species discrimination by quantitative PCR method>
Specifically, animal species were discriminated by the quantitative PCR method according to the method of Tanabe et al. (Bioscii. Biotechnol. Biochem., 71 (12), 3131-3135, 2007). The quantitative PCR reaction solution was made up to 25 μL in total by adding 12.5 μL of AmpliTaq Gold 360 Master Mix (manufactured by Thermo Fisher Scientific Co., Ltd .; trade name), 2.5 μL of primer, probe, and DNA extract to 2.5 μL of DNA extract. The primers, probes and reaction conditions used were kept at 50 ° C. for 2 minutes according to the method of Tanabe et al. (2007), and then 95 ° C. for 10 seconds, 95 ° C. for 15 seconds, and 60 ° C. for 1 minute were repeated as one cycle. rice field. As the DNA extract, the concentration was measured using QuantiFluor One dsDNA system (manufactured by Promega Co., Ltd .; trade name), and the DNA extract was prepared to 1 ng / μL with deionized distilled water. In addition, quantitative PCR was performed using Takara thermal cycler dice (manufactured by Takara Bio Inc .; trade name).
以下、本発明の実施例を詳細に説明するが、本発明の技術的範囲はこれらの実施例に限定されるものではない。 Hereinafter, examples of the present invention will be described in detail, but the technical scope of the present invention is not limited to these examples.
［実施例１］未加熱食品の洗浄工程に各種洗浄剤を用いた硬組織の動物種判別（ＰＣＲ法）
表１−１に示す試験区の各種洗浄剤組成物について、硬組織の洗浄試験を行った。なお、各洗浄剤組成物において、表に示す成分１、２以外の残部は、精製水である。また、表中の「％」は「質量％」を示す（以下同様。）。被験試料には、硬組織として０．２〜８．０ｍｇに細切したブタ肋骨を、該ブタ肋骨の１，０００倍重量のニワトリむね挽肉に混入したものを使用した。
洗浄試験では、上記実施態様に従って硬組織の洗浄および核酸の採取を行い、得られたＤＮＡ抽出液について、ブタ及びニワトリプライマーを使用して、ＰＣＲ法による硬組織の動物種の判別を行った。
[Example 1] Identification of hard tissue animal species using various detergents in the washing process of unheated food (PCR method)
Hard tissue cleaning tests were conducted on various detergent compositions in the test groups shown in Table 1-1. In each detergent composition, the rest other than the components 1 and 2 shown in the table is purified water. In addition, "%" in the table indicates "mass%" (the same shall apply hereinafter). As the test sample, a pig rib shredded to 0.2 to 8.0 mg as a hard tissue was mixed with minced chicken ribs having a weight 1,000 times the weight of the pig rib.
In the washing test, the hard tissue was washed and the nucleic acid was collected according to the above embodiment, and the obtained DNA extract was used to discriminate the animal species of the hard tissue by the PCR method using pig and chicken primers.
各試験区におけるブタおよびニワトリＤＮＡ由来の増幅産物のバンドの定量値を、「（２）洗浄工程」を実施しない試験区１におけるブタおよびニワトリＤＮＡ由来の増幅産物のバンドの定量値でそれぞれ除した値が、０．５以上のものを「Ａ；バンドを検出」、０．１以上０．５未満のものを「Ｂ；バンドを僅かに検出」、０．１未満のものを「Ｃ；バンドを不検出」とし、結果を表１−１に示した。 The quantitative value of the band of the amplification product derived from the pig and chicken DNA in each test group was divided by the quantitative value of the band of the amplification product derived from the pig and chicken DNA in the test group 1 in which "(2) washing step" was not performed. A value of 0.5 or more is "A; band detected", a value of 0.1 or more and less than 0.5 is "B; a slight band is detected", and a value of less than 0.1 is "C; band". Was not detected, and the results are shown in Table 1-1.
上記表１−１から明らかなとおり、洗浄剤を使用しない試験区１では、ブタＤＮＡ由来の増幅産物と、ニワトリＤＮＡ由来の増幅産物がともに検出された。また、特許文献１に記載の洗浄剤を使用した試験区２、次亜塩素酸ナトリウム、ラウリル硫酸ナトリウムもしくはポリオキシエチレン（２０）ソルビタンモノラウレートを洗浄剤として使用した試験区３〜５においても同様に、ブタＤＮＡ由来の増幅産物と、ニワトリＤＮＡ由来の増幅産物がともに検出された。一方で、０．０３質量％の水酸化ナトリウムを洗浄剤として使用した試験区６では、ブタＤＮＡ由来の増幅産物は検出されたが、ニワトリＤＮＡ由来の増幅産物のバンドは前記試験区と比較して薄く、僅かに検出されるのみであり、未加熱食品に混入した骨異物の動物種判別における硬組織の洗浄処理として、水酸化ナトリウム水溶液を使用する方法が好ましいことが確認された。 As is clear from Table 1-1 above, both the porcine DNA-derived amplification product and the chicken DNA-derived amplification product were detected in Test Group 1 in which no detergent was used. Further, also in Test Group 2 using the cleaning agent described in Patent Document 1, and Test Group 3 to 5 using sodium hypochlorite, sodium lauryl sulfate or polyoxyethylene (20) sorbitan monolaurate as the cleaning agent. Similarly, both porcine DNA-derived amplification products and chicken DNA-derived amplification products were detected. On the other hand, in the test group 6 in which 0.03% by mass of sodium hydroxide was used as a cleaning agent, the amplification product derived from porcine DNA was detected, but the band of the amplification product derived from chicken DNA was compared with that in the test group. It was thin and only slightly detected, and it was confirmed that a method using an aqueous sodium hydroxide solution was preferable as a treatment for cleaning hard tissues in determining the animal species of bone foreign substances mixed in unheated foods.
次いで、未加熱食品に混入した骨異物の動物種判別における硬組織の洗浄処理として、水酸化ナトリウム水溶液を使用する方法が好ましいことが確認されたことから、各濃度の水酸化ナトリウムを洗浄工程に用いて、実施形態に従ってＰＣＲ法による硬組織の動物種判別の検討を行った。 Next, since it was confirmed that a method using an aqueous sodium hydroxide solution was preferable as a treatment for cleaning hard tissues in determining the animal species of bone foreign substances mixed in unheated foods, sodium hydroxide at each concentration was used in the cleaning step. Using, the animal species discrimination of hard tissue by the PCR method was examined according to the embodiment.
［実施例２］未加熱食品の洗浄工程に各濃度の水酸化ナトリウム水溶液を用いた硬組織の動物種判別（ＰＣＲ法）
表１−２に示す試験区の各種洗浄剤組成物について、硬組織の洗浄試験を行った。被験試料として、３．５〜２４．４ｍｇに細切したブタ肋骨を、該ブタ肋骨の１，０００倍重量のニワトリむね挽肉に混入したものを使用した以外は、実施例１と同様に評価した。
[Example 2] Identification of hard tissue animal species using an aqueous solution of sodium hydroxide at each concentration in the washing step of unheated food (PCR method)
Hard tissue cleaning tests were conducted on various detergent compositions in the test groups shown in Table 1-2. As a test sample, porcine ribs chopped to 3.5 to 24.4 mg were evaluated in the same manner as in Example 1 except that a mixture of 1,000 times the weight of the porcine ribs was mixed with minced chicken breast. ..
上記表１−２から明らかなとおり、洗浄剤を使用しない試験区１では、ブタＤＮＡ由来の増幅産物と、ニワトリＤＮＡ由来の増幅産物がともに検出された。また、０．００１質量％および１質量％の水酸化ナトリウムを洗浄剤として使用した試験区２および６においても同様に、ブタＤＮＡ由来の増幅産物と、ニワトリＤＮＡ由来の増幅産物がともに検出された。一方で、０．０１質量％、０．０３質量％および０．１質量％の水酸化ナトリウムを洗浄剤として使用した試験区３〜５では、ブタＤＮＡ由来の増幅産物は検出されたが、ニワトリＤＮＡ由来の増幅産物のバンドは前記試験区と比較して薄く、僅かに検出されるのみであり、未加熱食品に混入した骨異物の動物種判別における硬組織の洗浄処理として、０．０１〜０．１質量％の水酸化ナトリウムを使用する方法が好ましいことが確認された。 As is clear from Table 1-2 above, both the porcine DNA-derived amplification product and the chicken DNA-derived amplification product were detected in Test Group 1 in which no detergent was used. Similarly, in Test Groups 2 and 6 using 0.001% by mass and 1% by mass of sodium hydroxide as a cleaning agent, both porcine DNA-derived amplification products and chicken DNA-derived amplification products were detected. .. On the other hand, in Test Groups 3 to 5 in which 0.01% by mass, 0.03% by mass and 0.1% by mass of sodium hydroxide were used as a cleaning agent, amplification products derived from porcine DNA were detected, but chickens. The band of the amplification product derived from DNA is thinner than that of the test group and is only slightly detected. As a treatment for washing hard tissue in determining the animal species of bone foreign matter mixed in unheated food, 0.01 to It was confirmed that the method using 0.1% by mass of sodium hydroxide is preferable.
次いで、未加熱食品に混入した骨異物の動物種判別における硬組織の洗浄処理として、０．０１〜０．１質量％の水酸化ナトリウムを使用する方法が好ましいことが確認されたことから、０．０１〜０．１質量％の水酸化ナトリウムに各種試薬を添加した洗浄剤を洗浄工程に用いて、実施形態に従ってＰＣＲ法による硬組織の動物種判別の検討を行った。 Next, it was confirmed that a method using 0.01 to 0.1% by mass of sodium hydroxide is preferable as a treatment for cleaning hard tissues in determining the animal species of bone foreign substances mixed in unheated foods. Using a cleaning agent prepared by adding various reagents to 0.01 to 0.1% by mass of sodium hydroxide in the cleaning step, the animal species of hard tissue was examined by the PCR method according to the embodiment.
［実施例３］未加熱食品の洗浄工程に水酸化ナトリウム水溶液に各種試薬を添加した洗浄剤を用いた硬組織の動物種判別（ＰＣＲ法）
表１−３に示す試験区の各種洗浄剤組成物について、硬組織の洗浄試験を行った。被験試料として、０．５〜７．４ｍｇに細切したブタ肋骨を、該ブタ肋骨の１，０００倍重量のニワトリむね挽肉に混入したものを使用した以外は、実施例１と同様に評価した。
[Example 3] Identification of hard tissue animal species using a detergent obtained by adding various reagents to an aqueous sodium hydroxide solution in the washing step of unheated food (PCR method)
Hard tissue cleaning tests were conducted on various detergent compositions in the test groups shown in Table 1-3. As a test sample, a pig rib shredded to 0.5 to 7.4 mg was evaluated in the same manner as in Example 1 except that a mixture of 1,000 times the weight of the pig rib was mixed with minced chicken breast. ..
上記表１−３から明らかなとおり、洗浄剤を使用しない試験区１では、ブタＤＮＡ由来の増幅産物と、ニワトリＤＮＡ由来の増幅産物がともに検出された。次いで、０．０３質量％の水酸化ナトリウム水溶液を洗浄剤として使用した試験区２では、Ｎ＝２で実施したいずれの試料でもブタＤＮＡ由来の増幅産物が検出され、Ｎ＝２で実施した一方ではニワトリＤＮＡ由来の増幅産物のバンドは薄く、僅かに検出されるのみであり、もう一方ではニワトリＤＮＡ由来の増幅産物のバンドは検出されなかった。次いで、エチレンジアミン四酢酸二ナトリウム二水和物を添加した０．０３質量％の水酸化ナトリウムを洗浄剤として使用した試験区３では、Ｎ＝２で実施したいずれの試料でもブタＤＮＡ由来の増幅産物が検出され、Ｎ＝２で実施した一方ではニワトリＤＮＡ由来の増幅産物が検出されたが、もう一方ではニワトリＤＮＡ由来の増幅産物のバンドは検出されなかった。次いで、次亜塩素酸ナトリウムを添加した０．０３質量％の水酸化ナトリウムを洗浄剤として使用した試験区４では、Ｎ＝２で実施したいずれの試料でもブタＤＮＡ由来の増幅産物が検出され、Ｎ＝２で実施した一方ではニワトリＤＮＡ由来の増幅産物が検出されたが、もう一方ではニワトリＤＮＡ由来の増幅産物のバンドは薄く、僅かに検出されるのみであった。次いで、０．１質量％のラウリル硫酸ナトリウムを添加した０．０３質量％の水酸化ナトリウムを洗浄剤として使用した試験区５では、Ｎ＝２で実施したいずれの試料でもブタＤＮＡ由来の増幅産物が検出されたが、ニワトリＤＮＡ由来の増幅産物は検出されなかった。次いで、０．１質量％のポリオキシエチレン（２０）ソルビタンモノラウレートを添加した０．０３質量％の水酸化ナトリウムを洗浄剤として使用した試験区６では、Ｎ＝２で実施したいずれの試料でもブタＤＮＡ由来の増幅産物が検出されたが、Ｎ＝２で実施した一方ではニワトリＤＮＡ由来の増幅産物のバンドは薄く、僅かに検出されるのみであり、もう一方ではニワトリＤＮＡ由来の増幅産物のバンドは検出されなかった。よって、未加熱食品に混入した骨異物の動物種判別における硬組織の洗浄処理として、水酸化ナトリウム水溶液を使用する方法が好ましく、水酸化ナトリウム水溶液に加えて０．１質量％のラウリル硫酸ナトリウムもしくはポリオキシエチレン（２０）ソルビタンモノラウレートを添加した水溶液を使用する方法が一層好ましいことが確認された。 As is clear from Table 1-3 above, both the porcine DNA-derived amplification product and the chicken DNA-derived amplification product were detected in Test Group 1 in which no detergent was used. Next, in Test Group 2 in which 0.03 mass% sodium hydroxide aqueous solution was used as a cleaning agent, an amplification product derived from porcine DNA was detected in all the samples carried out at N = 2, while it was carried out at N = 2. The band of the amplification product derived from chicken DNA was thin and only slightly detected, while the band of the amplification product derived from chicken DNA was not detected. Next, in Test Group 3 in which 0.03% by mass of sodium hydroxide to which ethylenediaminetetraacetic acid disodium dihydrate was added was used as a cleaning agent, amplification products derived from porcine DNA were obtained in all the samples carried out at N = 2. Was detected, and on the one hand, the amplification product derived from chicken DNA was detected, but on the other hand, the band of the amplification product derived from chicken DNA was not detected. Next, in Test Group 4 in which 0.03% by mass of sodium hydroxide to which sodium hypochlorite was added was used as a cleaning agent, an amplification product derived from porcine DNA was detected in all the samples carried out at N = 2. On the one hand, the amplification product derived from chicken DNA was detected at N = 2, but on the other hand, the band of the amplification product derived from chicken DNA was thin and only slightly detected. Next, in Test Group 5 in which 0.03% by mass of sodium hydroxide to which 0.1% by mass of sodium lauryl sulfate was added was used as a detergent, the amplification products derived from porcine DNA were obtained in all the samples carried out at N = 2. Was detected, but no amplification product derived from chicken DNA was detected. Next, in Test Group 6 in which 0.03% by mass of sodium hydroxide to which 0.1% by mass of polyoxyethylene (20) sorbitan monolaurate was added was used as a cleaning agent, any sample carried out at N = 2 was used. However, the amplification product derived from porcine DNA was detected, but on the one hand, the band of the amplification product derived from chicken DNA was thin and only slightly detected, and on the other hand, the amplification product derived from chicken DNA was detected. Band was not detected. Therefore, a method of using an aqueous sodium hydroxide solution is preferable as a treatment for cleaning hard tissues in determining the animal species of bone foreign substances mixed in unheated foods, and in addition to the aqueous sodium hydroxide solution, 0.1% by mass of sodium lauryl sulfate or It was confirmed that the method of using an aqueous solution to which polyoxyethylene (20) sorbitan monolaurate was added was more preferable.
次いで、未加熱食品に混入した骨異物の動物種判別における硬組織の洗浄処理として、水酸化ナトリウム水溶液を使用する方法が好ましく、水酸化ナトリウム水溶液に加えて０．１質量％のラウリル硫酸ナトリウムもしくはポリオキシエチレン（２０）ソルビタンモノラウレートを添加した水溶液を使用する方法が一層好ましいことが確認されたことから、水酸化ナトリウム水溶液に各濃度のラウリル硫酸ナトリウムもしくはポリオキシエチレン（２０）ソルビタンモノラウレートを加えた洗浄剤を洗浄工程に用いて、実施形態に従ってＰＣＲ法による硬組織の動物種判別の検討を行った。 Next, as a treatment for cleaning the hard tissue in determining the animal species of bone foreign substances mixed in unheated food, a method of using an aqueous sodium hydroxide solution is preferable, and in addition to the aqueous sodium hydroxide solution, 0.1% by mass of sodium lauryl sulfate or Since it was confirmed that the method of using an aqueous solution containing polyoxyethylene (20) sorbitan monolaurate was more preferable, each concentration of sodium lauryl sulfate or polyoxyethylene (20) sorbitan monolaurate in the sodium hydroxide aqueous solution was confirmed. Using the cleaning agent to which the rate was added in the cleaning step, the animal species discrimination of hard tissue by the PCR method was examined according to the embodiment.
［実施例４］未加熱食品の洗浄工程に各濃度のラウリル硫酸ナトリウムもしくはポリオキシエチレン（２０）ソルビタンモノラウレートを加えた水酸化ナトリウム水溶液を用いた硬組織の動物種判別（ＰＣＲ法）
表１−４に示す試験区の各種洗浄剤組成物について、硬組織の洗浄試験を行った。被験試料として、８．９〜３０．６ｍｇに細切したブタ肋骨を、ブタ肋骨の１，０００倍重量のニワトリむね挽肉に混入したものを使用した以外は、実施例１と同様に評価した。
[Example 4] Identification of hard tissue animal species using an aqueous solution of sodium hydroxide obtained by adding sodium lauryl sulfate or polyoxyethylene (20) sorbitan monolaurate at each concentration to the washing step of unheated food (PCR method).
Hard tissue cleaning tests were conducted on various detergent compositions in the test groups shown in Table 1-4. As a test sample, porcine ribs chopped to 8.9 to 30.6 mg were evaluated in the same manner as in Example 1 except that a mixture of 1,000 times the weight of minced pork ribs was mixed with minced chicken breast.
上記表１−４から明らかなとおり、洗浄剤を使用しない試験区１では、ブタＤＮＡ由来の増幅産物と、ニワトリＤＮＡ由来の増幅産物がともに検出された。一方で、０．１質量％もしくは１質量％のラウリル硫酸ナトリウムを添加した０．０３質量％の水酸化ナトリウムを洗浄剤として使用した試験区２、３では、ブタＤＮＡ由来の増幅産物は検出されたが、ニワトリＤＮＡ由来の増幅産物のバンドは薄く、僅かに検出されるのみであった。また、０．０１質量％もしくは０．１質量％のポリオキシエチレン（２０）ソルビタンモノラウレートを添加した０．０３質量％の水酸化ナトリウムを洗浄剤として使用した試験区４、５では、ブタＤＮＡ由来の増幅産物は検出されたが、ニワトリＤＮＡ由来の増幅産物のバンドは薄く、僅かに検出されるのみであった。よって、未加熱食品に混入した骨異物の動物種判別における硬組織の洗浄処理として、０．１質量％もしくは１質量％のラウリル硫酸ナトリウムを添加した０．０３質量％の水酸化ナトリウム、０．０１質量％もしくは０．１質量％のポリオキシエチレン（２０）ソルビタンモノラウレートを添加した０．０３質量％の水酸化ナトリウムを使用する方法が好ましいことが確認された。 As is clear from Table 1-4 above, both the porcine DNA-derived amplification product and the chicken DNA-derived amplification product were detected in Test Group 1 in which no detergent was used. On the other hand, amplification products derived from porcine DNA were detected in Test Groups 2 and 3 in which 0.03% by mass of sodium hydroxide added with 0.1% by mass or 1% by mass of sodium lauryl sulfate was used as a detergent. However, the band of the amplification product derived from chicken DNA was thin and only slightly detected. Further, in Test Groups 4 and 5 in which 0.03% by mass of sodium hydroxide supplemented with 0.01% by mass or 0.1% by mass of polyoxyethylene (20) sorbitan monolaurate was used as a cleaning agent, pigs were used. DNA-derived amplification products were detected, but the band of chicken DNA-derived amplification products was thin and only slightly detected. Therefore, as a treatment for cleaning the hard tissue in determining the animal species of bone foreign matter mixed in the unheated food, 0.03% by mass or 1% by mass of sodium lauryl sulfate was added to 0.03% by mass of sodium hydroxide, 0. It was confirmed that the method using 0.03% by mass of sodium hydroxide supplemented with 01% by mass or 0.1% by mass of polyoxyethylene (20) sorbitan monolaurate was preferable.
［実施例５］加熱食品の洗浄工程に各種洗浄剤を用いた硬組織の動物種判別（ＰＣＲ法）
次に、加熱処理した被験試料について検討した。被験試料には、硬組織として１．３〜１２．７ｍｇに細切したブタ肋骨を、該ブタ肋骨の１，０００倍重量のニワトリむね挽肉に混入させ、冷蔵条件にて一晩静置後、加熱処理したものを使用した。加熱処理条件としては、真空状態で沸騰水中にて３０分もしくはステンレス製容器に入れた状態でオートクレーブを用いて１２１℃にて１５分の加熱処理を施した。これらの加熱処理した被験試料について、表２に示す試験区の各種洗浄剤組成物を用いて洗浄試験を行い、実施例１と同様に評価した。
[Example 5] Animal species discrimination of hard tissue using various detergents in the washing step of heated food (PCR method)
Next, the heat-treated test sample was examined. In the test sample, pig ribs chopped into 1.3 to 12.7 mg as hard tissue were mixed with minced chicken ribs 1,000 times as heavy as the pig ribs, and allowed to stand overnight under refrigerated conditions. The heat-treated one was used. As the heat treatment conditions, heat treatment was performed at 121 ° C. for 15 minutes using an autoclave in a vacuum state in boiling water for 30 minutes or in a stainless steel container. These heat-treated test samples were subjected to a cleaning test using various detergent compositions in the test group shown in Table 2 and evaluated in the same manner as in Example 1.
上記表２から明らかなとおり、洗浄剤を使用しない試験区１、特許文献１に記載の洗浄剤を使用した試験区２では、いずれの加熱条件で作製した試料でもブタＤＮＡ由来の増幅産物と、ニワトリＤＮＡ由来の増幅産物がともに検出された。一方で、０．０３質量％の水酸化ナトリウムを洗浄剤として使用した試験区３では、いずれの加熱条件で作製した試料でもブタＤＮＡ由来の増幅産物が検出され、沸騰水中にて３０分間の加熱処理を施したものではニワトリＤＮＡ由来の増幅産物が検出されたが、オートクレーブを用いて１２１℃にて１５分の加熱処理を施したものではニワトリＤＮＡ由来の増幅産物は検出されなかった。また、０．１質量％のラウリル硫酸ナトリウムを添加した０．０３質量％の水酸化ナトリウムを洗浄剤として使用した試験区４では、いずれの加熱条件で作製した試料でもブタＤＮＡ由来の増幅産物が検出されたが、ニワトリＤＮＡ由来の増幅産物のバンドは薄く、僅かに検出されるのみであった。さらに、０．１質量％のポリオキシエチレン（２０）ソルビタンモノラウレートを添加した０．０３質量％の水酸化ナトリウムを洗浄剤として使用した試験区５では、いずれの加熱条件で作製した試料でもブタＤＮＡ由来の増幅産物が検出され、沸騰水中にて３０分間の加熱処理を施したものではニワトリＤＮＡ由来の増幅産物が検出されず、オートクレーブを用いて１２１℃にて１５分の加熱処理を施したものではニワトリＤＮＡ由来の増幅産物のバンドは薄く、僅かに検出されるのみであった。よって、加熱食品に混入した骨異物の動物種判別における硬組織の洗浄処理として、水酸化ナトリウム水溶液を使用する方法が好ましく、水酸化ナトリウム水溶液に加えて０．１質量％のラウリル硫酸ナトリウムもしくはポリオキシエチレン（２０）ソルビタンモノラウレートを添加した水溶液を使用する方法が一層好ましいことが確認された。 As is clear from Table 2 above, in the test group 1 in which no cleaning agent was used and in the test group 2 in which the cleaning agent described in Patent Document 1 was used, the sample prepared under any of the heating conditions contained the amplification product derived from porcine DNA. Amplification products derived from chicken DNA were both detected. On the other hand, in Test Group 3 in which 0.03% by mass of sodium hydroxide was used as a cleaning agent, an amplification product derived from porcine DNA was detected in the sample prepared under any heating condition, and the sample was heated in boiling water for 30 minutes. Amplification products derived from chicken DNA were detected in the treated products, but no amplification products derived from chicken DNA were detected in the products subjected to heat treatment at 121 ° C. for 15 minutes using an autoclave. Further, in Test Group 4 in which 0.03% by mass of sodium hydroxide to which 0.1% by mass of sodium lauryl sulfate was added was used as a detergent, amplification products derived from porcine DNA were found in the samples prepared under any of the heating conditions. Although it was detected, the band of the amplification product derived from chicken DNA was thin and only slightly detected. Further, in Test Group 5 in which 0.03% by mass of sodium hydroxide to which 0.1% by mass of polyoxyethylene (20) sorbitan monolaurate was added was used as a cleaning agent, the sample prepared under any heating condition was used. Amplification products derived from porcine DNA were detected, and those subjected to heat treatment in boiling water for 30 minutes did not detect amplification products derived from chicken DNA, and were heat-treated at 121 ° C. for 15 minutes using an autoclave. The band of the amplification product derived from chicken DNA was thin and only slightly detected. Therefore, a method of using an aqueous sodium hydroxide solution is preferable as a treatment for cleaning hard tissues in determining the animal species of bone foreign substances mixed in a heated food, and 0.1% by mass of sodium lauryl sulfate or poly is added to the aqueous sodium hydroxide solution. It was confirmed that the method of using an aqueous solution containing oxyethylene (20) sorbitan monolaurate was more preferable.
［実施例６］未加熱食品の洗浄工程に各種洗浄剤を用いて硬組織より採取した核酸の定量（定量ＰＣＲ法）
実施例１および実施例３で採取したＤＮＡ抽出液を用いて、ブタおよびニワトリプライマー、ブタおよびニワトリプローブを使用して、実施形態に従って定量ＰＣＲ法による硬組織の動物種判別の検討を行った。ＤＮＡの定量には、実施形態における核酸採取方法によりブタもも肉及びニワトリむね肉から採取したＤＮＡ抽出液を、それぞれブタ由来及びニワトリ由来の標準品ＤＮＡとして作成した検量線を用いた。これらの検量線から算出したブタＤＮＡの定量値及びニワトリＤＮＡの定量値を表３に示す。また、ブタＤＮＡおよびニワトリＤＮＡの定量値から算出したＤＮＡ定量値比（ニワトリＤＮＡ／ブタＤＮＡ）を表３に示す。
[Example 6] Quantification of nucleic acid collected from hard tissue using various detergents in the washing step of unheated food (quantitative PCR method)
Using the DNA extracts collected in Examples 1 and 3, the animal species of hard tissue was examined by quantitative PCR using porcine and chicken primers, porcine and chicken probes, according to the embodiment. For the quantification of DNA, calibration curves prepared as standard DNAs derived from pigs and chickens from DNA extracts collected from pig thighs and chicken breasts by the nucleic acid collection method in the embodiment were used. Table 3 shows the quantitative values of porcine DNA and chicken DNA calculated from these calibration curves. Table 3 shows the DNA quantification value ratio (chicken DNA / porcine DNA) calculated from the quantified values of porcine DNA and chicken DNA.
上記表３から明らかなとおり、洗浄剤を使用しない試験区１、特許文献１に記載の洗浄剤を使用した試験区２と比較し、０．０３質量％の水酸化ナトリウムを洗浄剤として使用した試験区３、０．０３質量％の水酸化ナトリウムに加えて０．１質量％のラウリル硫酸ナトリウムもしくはポリオキシエチレン（２０）ソルビタンモノラウレートを添加した試験区４、５では、ニワトリＤＮＡの定量値が小さく、未加熱食品に混入した骨異物の動物種判別における硬組織の洗浄処理として、水酸化ナトリウム水溶液を使用する方法が好ましく、水酸化ナトリウム水溶液に加えて０．１質量％のラウリル硫酸ナトリウムもしくはポリオキシエチレン（２０）ソルビタンモノラウレートを添加した水溶液を使用する方法が一層好ましいことが定量ＰＣＲでも確認された。 As is clear from Table 3 above, 0.03% by mass of sodium hydroxide was used as the cleaning agent as compared with the test group 1 in which no cleaning agent was used and the test group 2 in which the cleaning agent described in Patent Document 1 was used. Test group 3, quantification of chicken DNA in test groups 4 and 5 in which 0.1% by mass of sodium lauryl sulfate or polyoxyethylene (20) sorbitan monolaurate was added in addition to 0.03% by mass of sodium hydroxide. A method of using an aqueous sodium hydroxide solution is preferable as a treatment for cleaning hard tissue in determining the animal species of bone foreign substances mixed in unheated foods because the value is small. In addition to the sodium hydroxide aqueous solution, 0.1% by mass of lauryl sulfate is used. Quantitative PCR also confirmed that the method of using an aqueous solution containing sodium or polyoxyethylene (20) sorbitan monolaurate was more preferable.
本発明の洗浄剤組成物及び洗浄方法は、動物由来組織が付着した硬組織から、動物由来組織を除去する方法に利用することができる。例えば、挽肉等の食品に混入した硬組織の動物種の同定方法や、法医学的なＤＮＡ鑑定や、生物学的な遺伝子研究におけるＤＮＡの採取や、動物の骨の標本作製等に利用することができる。 The detergent composition and cleaning method of the present invention can be used as a method for removing animal-derived tissue from hard tissue to which animal-derived tissue is attached. For example, it can be used for identification of hard tissue animal species mixed in foods such as ground meat, forensic DNA analysis, DNA collection in biological genetic research, and animal bone specimen preparation. can.
また、本発明の洗浄剤組成物及び洗浄方法は、硬組織からＤＮＡやＲＮＡを採取する方法に好適に利用することができる。
In addition, the detergent composition and cleaning method of the present invention can be suitably used for a method of collecting DNA or RNA from hard tissue.
Claims (5)
（Ａ）水酸化ナトリウム又は水酸化カリウム、及び、（Ｂ）ラウリル硫酸ナトリウム又はポリオキシエチレン（２０）ソルビタンモノラウレート、を含有し、
洗浄剤組成物中における前記（Ａ）水酸化ナトリウム又は水酸化カリウムの含有量は、０．００１〜１質量％であり、
洗浄剤組成物中における前記（Ｂ）ラウリル硫酸ナトリウム又はポリオキシエチレン（２０）ソルビタンモノラウレートの含有量は、０．００１〜５質量％であることを特徴とする、洗浄剤組成物。 When collecting DNA from bone foreign matters mixed into meat piece consisting of animal tissue, a piece of meat comprising the animal-derived tissue a detergent composition used for removal from the bone foreign body,
It contains (A) sodium hydroxide or potassium hydroxide, and (B) sodium lauryl sulfate or polyoxyethylene (20) sorbitan monolaurate .
The content of the sodium hydroxide or potassium hydroxide (A) in the detergent composition is 0.001 to 1% by mass.
The detergent composition, wherein the content of the sodium (B) lauryl sulfate or polyoxyethylene (20) sorbitan monolaurate in the detergent composition is 0.001 to 5% by mass.
前記請求項１又は２に記載の洗浄剤組成物を含むことを特徴とする、核酸採取キット。 A nucleic acid collection kit for collecting DNA from the bone foreign body.
A nucleic acid collection kit comprising the detergent composition according to claim 1 or 2.
前記請求項１又は２に記載の洗浄剤組成物を用いて、前記動物由来組織からなる肉片の付着した骨異物を洗浄する工程、
を備えたことを特徴とする、洗浄方法。 A washing method for removing the meat pieces made of animal-derived tissue from bone foreign substances mixed in the meat pieces made of animal-derived tissue.
A step of cleaning a bone foreign substance to which a piece of meat made of an animal-derived tissue is attached, using the detergent composition according to claim 1 or 2.
A cleaning method characterized by being equipped with.
請求項１又は２に記載の洗浄剤組成物を用いて、前記動物由来組織からなる肉片の付着した骨異物を洗浄する工程、
洗浄した骨異物からＤＮＡを採取する工程、
を備えたことを特徴とする、核酸採取方法。
A nucleic acid collection method for collecting DNA from bone foreign bodies mixed in meat pieces made of animal-derived tissue.
A step of cleaning a bone foreign substance to which a piece of meat made of an animal-derived tissue is attached, using the detergent composition according to claim 1 or 2.
The process of collecting DNA from washed bone foreign bodies,
A nucleic acid collection method, which comprises the above.
Priority Applications (1)
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