CN107083382B - A kind of blood preseration agent and its application for protecting dissociative DNA - Google Patents
A kind of blood preseration agent and its application for protecting dissociative DNA Download PDFInfo
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- CN107083382B CN107083382B CN201710380209.9A CN201710380209A CN107083382B CN 107083382 B CN107083382 B CN 107083382B CN 201710380209 A CN201710380209 A CN 201710380209A CN 107083382 B CN107083382 B CN 107083382B
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- 239000008280 blood Substances 0.000 title claims abstract description 161
- 210000004369 blood Anatomy 0.000 title claims abstract description 161
- 230000002633 protecting effect Effects 0.000 title claims abstract description 12
- 239000003755 preservative agent Substances 0.000 claims abstract description 72
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 45
- 239000007853 buffer solution Substances 0.000 claims abstract description 13
- KDCGOANMDULRCW-UHFFFAOYSA-N 7H-purine Chemical compound N1=CNC2=NC=NC2=C1 KDCGOANMDULRCW-UHFFFAOYSA-N 0.000 claims abstract description 12
- 239000003146 anticoagulant agent Substances 0.000 claims abstract description 11
- 229940127219 anticoagulant drug Drugs 0.000 claims abstract description 11
- 239000004599 antimicrobial Substances 0.000 claims abstract description 10
- 230000002587 anti-hemolytic effect Effects 0.000 claims abstract description 5
- 239000003112 inhibitor Substances 0.000 claims abstract description 5
- 108010039918 Polylysine Proteins 0.000 claims description 20
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 14
- QLBHNVFOQLIYTH-UHFFFAOYSA-L dipotassium;2-[2-[bis(carboxymethyl)amino]ethyl-(carboxylatomethyl)amino]acetate Chemical compound [K+].[K+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O QLBHNVFOQLIYTH-UHFFFAOYSA-L 0.000 claims description 14
- 229930024421 Adenine Natural products 0.000 claims description 7
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 claims description 7
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 claims description 7
- 229930195725 Mannitol Natural products 0.000 claims description 7
- 229960000643 adenine Drugs 0.000 claims description 7
- 239000004202 carbamide Substances 0.000 claims description 7
- 239000000594 mannitol Substances 0.000 claims description 7
- 235000010355 mannitol Nutrition 0.000 claims description 7
- 150000008442 polyphenolic compounds Chemical class 0.000 claims description 7
- 235000013824 polyphenols Nutrition 0.000 claims description 7
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 6
- 239000004311 natamycin Substances 0.000 claims description 6
- NCXMLFZGDNKEPB-FFPOYIOWSA-N natamycin Chemical compound O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1/C=C/C=C/C=C/C=C/C[C@@H](C)OC(=O)/C=C/[C@H]2O[C@@H]2C[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 NCXMLFZGDNKEPB-FFPOYIOWSA-N 0.000 claims description 6
- 229960003255 natamycin Drugs 0.000 claims description 6
- 235000010298 natamycin Nutrition 0.000 claims description 6
- 239000000872 buffer Substances 0.000 claims description 5
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 claims description 4
- RCEAADKTGXTDOA-UHFFFAOYSA-N OS(O)(=O)=O.CCCCCCCCCCCC[Na] Chemical compound OS(O)(=O)=O.CCCCCCCCCCCC[Na] RCEAADKTGXTDOA-UHFFFAOYSA-N 0.000 claims description 2
- 229910019142 PO4 Inorganic materials 0.000 claims description 2
- 229930006000 Sucrose Natural products 0.000 claims description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 2
- MOXADUNKHOQYSJ-UHFFFAOYSA-N [K].[K].[K].CC(O)=O.CC(O)=O.CC(O)=O.CC(O)=O.NCCN Chemical compound [K].[K].[K].CC(O)=O.CC(O)=O.CC(O)=O.CC(O)=O.NCCN MOXADUNKHOQYSJ-UHFFFAOYSA-N 0.000 claims description 2
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 2
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 2
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 2
- -1 disodium hydrogen Chemical class 0.000 claims description 2
- ZJYYHGLJYGJLLN-UHFFFAOYSA-N guanidinium thiocyanate Chemical compound SC#N.NC(N)=N ZJYYHGLJYGJLLN-UHFFFAOYSA-N 0.000 claims description 2
- 239000010452 phosphate Substances 0.000 claims description 2
- 239000005720 sucrose Substances 0.000 claims description 2
- 241001122767 Theaceae Species 0.000 claims 2
- 239000003109 Disodium ethylene diamine tetraacetate Substances 0.000 claims 1
- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 claims 1
- 235000019301 disodium ethylene diamine tetraacetate Nutrition 0.000 claims 1
- 239000001257 hydrogen Substances 0.000 claims 1
- 229910052739 hydrogen Inorganic materials 0.000 claims 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims 1
- NQMRYBIKMRVZLB-UHFFFAOYSA-N methylamine hydrochloride Chemical compound [Cl-].[NH3+]C NQMRYBIKMRVZLB-UHFFFAOYSA-N 0.000 claims 1
- XONPDZSGENTBNJ-UHFFFAOYSA-N molecular hydrogen;sodium Chemical compound [Na].[H][H] XONPDZSGENTBNJ-UHFFFAOYSA-N 0.000 claims 1
- 238000004321 preservation Methods 0.000 abstract description 27
- 238000012360 testing method Methods 0.000 abstract description 10
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 abstract description 7
- 239000008103 glucose Substances 0.000 abstract description 7
- 210000000601 blood cell Anatomy 0.000 abstract description 3
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 3
- 230000000593 degrading effect Effects 0.000 abstract 1
- 210000002381 plasma Anatomy 0.000 description 63
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 18
- 238000001514 detection method Methods 0.000 description 18
- 230000032258 transport Effects 0.000 description 17
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 14
- 229960002897 heparin Drugs 0.000 description 14
- 229920000669 heparin Polymers 0.000 description 14
- 238000001190 Q-PCR Methods 0.000 description 13
- 230000000694 effects Effects 0.000 description 13
- 239000000126 substance Substances 0.000 description 12
- 230000015556 catabolic process Effects 0.000 description 10
- 238000006731 degradation reaction Methods 0.000 description 10
- 239000000284 extract Substances 0.000 description 10
- 238000000034 method Methods 0.000 description 8
- 238000013467 fragmentation Methods 0.000 description 7
- 238000006062 fragmentation reaction Methods 0.000 description 7
- 238000002156 mixing Methods 0.000 description 7
- 230000002335 preservative effect Effects 0.000 description 7
- 230000002255 enzymatic effect Effects 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 6
- 108090000623 proteins and genes Proteins 0.000 description 6
- 231100000111 LD50 Toxicity 0.000 description 5
- 239000000463 material Substances 0.000 description 5
- 239000003223 protective agent Substances 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 4
- 238000000605 extraction Methods 0.000 description 4
- 230000036541 health Effects 0.000 description 4
- 231100000252 nontoxic Toxicity 0.000 description 4
- 230000003000 nontoxic effect Effects 0.000 description 4
- 231100000614 poison Toxicity 0.000 description 4
- 239000002574 poison Substances 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 239000003440 toxic substance Substances 0.000 description 4
- 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 description 3
- 244000269722 Thea sinensis Species 0.000 description 3
- 230000007059 acute toxicity Effects 0.000 description 3
- 231100000403 acute toxicity Toxicity 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 231100000481 chemical toxicant Toxicity 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- 210000003743 erythrocyte Anatomy 0.000 description 3
- 210000000265 leukocyte Anatomy 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 239000011734 sodium Substances 0.000 description 3
- 229910052708 sodium Inorganic materials 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- KEQGZUUPPQEDPF-UHFFFAOYSA-N 1,3-dichloro-5,5-dimethylimidazolidine-2,4-dione Chemical compound CC1(C)N(Cl)C(=O)N(Cl)C1=O KEQGZUUPPQEDPF-UHFFFAOYSA-N 0.000 description 2
- 108010085238 Actins Proteins 0.000 description 2
- 235000005979 Citrus limon Nutrition 0.000 description 2
- 244000131522 Citrus pyriformis Species 0.000 description 2
- 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 2
- PIICEJLVQHRZGT-UHFFFAOYSA-N Ethylenediamine Chemical compound NCCN PIICEJLVQHRZGT-UHFFFAOYSA-N 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 238000010241 blood sampling Methods 0.000 description 2
- KCIDZIIHRGYJAE-YGFYJFDDSA-L dipotassium;[(2r,3r,4s,5r,6r)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl] phosphate Chemical compound [K+].[K+].OC[C@H]1O[C@H](OP([O-])([O-])=O)[C@H](O)[C@@H](O)[C@H]1O KCIDZIIHRGYJAE-YGFYJFDDSA-L 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 231100000053 low toxicity Toxicity 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 108700019599 monomethylolglycine Proteins 0.000 description 2
- 230000001473 noxious effect Effects 0.000 description 2
- 210000005259 peripheral blood Anatomy 0.000 description 2
- 239000011886 peripheral blood Substances 0.000 description 2
- 229920001184 polypeptide Polymers 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 230000002441 reversible effect Effects 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 235000015170 shellfish Nutrition 0.000 description 2
- 229940101011 sodium hydroxymethylglycinate Drugs 0.000 description 2
- CITBNDNUEPMTFC-UHFFFAOYSA-M sodium;2-(hydroxymethylamino)acetate Chemical compound [Na+].OCNCC([O-])=O CITBNDNUEPMTFC-UHFFFAOYSA-M 0.000 description 2
- 231100000331 toxic Toxicity 0.000 description 2
- 230000002588 toxic effect Effects 0.000 description 2
- 231100000419 toxicity Toxicity 0.000 description 2
- 230000001988 toxicity Effects 0.000 description 2
- 231100000027 toxicology Toxicity 0.000 description 2
- WBTIFBJEYFLFFW-UHFFFAOYSA-N 2-(hydroxymethylazaniumyl)acetate Chemical compound OCNCC(O)=O WBTIFBJEYFLFFW-UHFFFAOYSA-N 0.000 description 1
- 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 1
- YTIXGBHAPNMOKU-UHFFFAOYSA-N 2-nitropropane-1,3-diol Chemical compound OCC(CO)[N+]([O-])=O YTIXGBHAPNMOKU-UHFFFAOYSA-N 0.000 description 1
- 230000035502 ADME Effects 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 206010064571 Gene mutation Diseases 0.000 description 1
- 206010018910 Haemolysis Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- WYNCHZVNFNFDNH-UHFFFAOYSA-N Oxazolidine Chemical compound C1COCN1 WYNCHZVNFNFDNH-UHFFFAOYSA-N 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 150000001299 aldehydes Chemical class 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 230000001194 anti-hemostatic effect Effects 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 230000023555 blood coagulation Effects 0.000 description 1
- 230000000711 cancerogenic effect Effects 0.000 description 1
- 231100000315 carcinogenic Toxicity 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 238000005345 coagulation Methods 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- SOROIESOUPGGFO-UHFFFAOYSA-N diazolidinylurea Chemical compound OCNC(=O)N(CO)C1N(CO)C(=O)N(CO)C1=O SOROIESOUPGGFO-UHFFFAOYSA-N 0.000 description 1
- 229960001083 diazolidinylurea Drugs 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229960001484 edetic acid Drugs 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000007515 enzymatic degradation Effects 0.000 description 1
- NUVBSKCKDOMJSU-UHFFFAOYSA-N ethylparaben Chemical compound CCOC(=O)C1=CC=C(O)C=C1 NUVBSKCKDOMJSU-UHFFFAOYSA-N 0.000 description 1
- 230000029142 excretion Effects 0.000 description 1
- 210000004700 fetal blood Anatomy 0.000 description 1
- 210000004907 gland Anatomy 0.000 description 1
- 230000008588 hemolysis Effects 0.000 description 1
- 229940071206 hydroxymethylglycinate Drugs 0.000 description 1
- ZCTXEAQXZGPWFG-UHFFFAOYSA-N imidurea Chemical compound O=C1NC(=O)N(CO)C1NC(=O)NCNC(=O)NC1C(=O)NC(=O)N1CO ZCTXEAQXZGPWFG-UHFFFAOYSA-N 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 231100000636 lethal dose Toxicity 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 230000005291 magnetic effect Effects 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 238000011017 operating method Methods 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- QUBQYFYWUJJAAK-UHFFFAOYSA-N oxymethurea Chemical compound OCNC(=O)NCO QUBQYFYWUJJAAK-UHFFFAOYSA-N 0.000 description 1
- 229950005308 oxymethurea Drugs 0.000 description 1
- 230000005298 paramagnetic effect Effects 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- QELSKZZBTMNZEB-UHFFFAOYSA-N propylparaben Chemical compound CCCOC(=O)C1=CC=C(O)C=C1 QELSKZZBTMNZEB-UHFFFAOYSA-N 0.000 description 1
- 229960003415 propylparaben Drugs 0.000 description 1
- YGSDEFSMJLZEOE-UHFFFAOYSA-M salicylate Chemical compound OC1=CC=CC=C1C([O-])=O YGSDEFSMJLZEOE-UHFFFAOYSA-M 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 231100000004 severe toxicity Toxicity 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 231100000167 toxic agent Toxicity 0.000 description 1
- 230000002110 toxicologic effect Effects 0.000 description 1
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 1
Classifications
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- 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
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
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- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Zoology (AREA)
- General Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- Wood Science & Technology (AREA)
- Biomedical Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Crystallography & Structural Chemistry (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention belongs to the preservation reagents of blood sample, and in particular to a kind of blood preseration agent for protecting dissociative DNA.The preservative agent, every 100 milliliters of volumes include following components：Anti-coagulants：6.0~8.8g, natural antiseptic agent：0.1~2.0g, dnase inhibitor：4.8~11.7g, glucose：5.0~10.0g, antihemolytic：5.0~8.0g, purine：0.1~0.5g, buffer solution are mended to 100ml.Preservative agent of the present invention can effectively prevent blood cell breakage release genomic DNA to pollute, prevent plasma DNA from degrading, stable environment is provided for plasma DNA, more importantly safety, to human body and environment nonhazardous, is conducive to more accurately test and analyze dissociative DNA.
Description
Technical field
The invention belongs to the preservation reagents of blood sample, and in particular to a kind of blood preseration agent for protecting dissociative DNA
And its application.
Background technology
Currently, plasma DNA clinically apply it is more and more, such as noninvasive prenatal gene detection and diagnosing tumor all
Be with plasma DNA be detection target.In detection process, the number of the amount of plasma DNA is an important detection
Index.Human plasma dissociative DNA content is few, is easily degraded by the DNA enzymatic in blood, and is easy to be cracked by leucocyte and discharge
Contaminating genomic DNA, seriously affect testing result.
In order to solve these problems, the measure mainly taken at this stage has：(1) band anti-coagulants (EDTA, heparin, lemon are selected
Lemon hydrochlorate) heparin tube acquire blood sample, to prevent whole blood cells blood coagulation, reduce leucocyte and discharge genomic DNA；(2) 6
Blood plasma, 4 DEG C of Cord blood transports are isolated in hour, and the holding time cannot be long, prevent DNA enzymatic degradation plasma free
DNA。
By the above technical finesse, certain improvement can be obtained to the accurate detection of plasma DNA.But 4 DEG C of low temperature
It preserves under traffic condition, still there is DNA enzymatic certain activity, long-term preservation plasma DNA can be made largely to lose, be unfavorable for blood plasma
The accurate detection of dissociative DNA.Simultaneously under existence conditions, the storage and transport of blood all carry out at low temperature, bring many not
Just, make plasma DNA detection economic cost higher.
Therefore, it is necessary to provide a kind of blood preseration agent for protecting dissociative DNA, it is intended to solve to use existing blood
Dissociative DNA store method is starched, plasma DNA is vulnerable to contaminating genomic DNA, is easily degraded by DNA enzymatic, and the holding time is short, protects
Deposit the problems such as temperature is low.
WS-10001- (HD-0230) -2002 discloses a kind of alserver's solution, glucose therein, adenine, sweet
Dew alcohol, phosphate are conducive to the preservation of red blood cell, but make the whole blood sample with karyocyte in 7 to 14 days periods of ambient-temp-stable
Effect it is still undesirable.
The patent of application number 201510226039.X discloses a kind of preservative agent preserving dissociative DNA in peripheral blood, protects
Deposit in agent component contain chemical preservative, including DIAZOLIDINYL UREA, imidazolidinyl urea, 1,3-, bis- chloro- 5,5- dimethyl hydantoins,
The bromo- 2- nitros -1,3- propylene glycol of sodium hydroxymethylglycinate, dimethylol urea, 2-, oxazolidine, sodium hydroxymethylglycinate and Buddha's warrior attendant
One or more combinations of alkane quaternary ammonium.
The patent of application number 201510132774.4 disclose it is a kind of for protect dissociative DNA blood anticoagulant and its answer
With blood anticoagulant component contains formaldehyde releaser, including 1,3- dihydroxymethyls -5,5- Dimethyl Hydan, hydroxymethylglycinate
The one or more of sodium, soluble metyl hydroxybenzoate, nipagin A sodium, soluble propylhydroxybenzoate.
It is well known that many chemical preservatives are toxic, it is harmful, and easily pollute the environment；Formaldehyde is
A kind of carcinogenic toxicant is also harmful to human body.
Epsilon-polylysine is a kind of polypeptide with bacteriostasis efficacy, safe and nontoxic.
The toxicity that table 1 enumerates several chemical substances compares, and wherein epsilon-polylysine data source is in NeDaK's et al.
《NedaK,SakuraiT,etal.Two-generation reproductionstudywithteratologytestofε-
poly-L-lysineby dietaryadministrationinrats[J]》.JppharmcolTher,1999,27:1139-
1159.), the data source of other chemical substances is in the competing Chemicals Database (http of object://www.basechem.org/).
The toxicology data of 1 several chemical substances of table
| Chemical substance | Rat oral LD50(mg/kg) |
| Epsilon-polylysine | ＞ 20000 |
| 1,3 dichloro 5,5 dimethyl hydantoin | 542 |
| The bromo- 2- nitros -1,3- propylene glycol of 2- | 180~400 |
| Formaldehyde | 800 |
| Tea polyphenols (＞ 95%) | 3940 |
| Natamycin | 4000 |
Median lethal dose (lethaldose50%, LD50)：It is the drug agent for referring to cause the death of experimental animal half
Amount, recommends a Pyatyi standard for the compound acute toxicity graded combination World Health Organization of state (WHO), is shown in Table 2.
2 foreign compound acute toxicity of table is classified
| Chemical substance | Rat oral LD50(mg/kg) |
| Severe toxicity | ＜ 1 |
| High poison | 1- |
| Medium poison | 50- |
| Low toxicity | 500- |
| Micro- poison | 5000- |
It can be seen that the patent of application number 201510226039.X discloses a kind of guarantor preserving dissociative DNA in peripheral blood
It is noxious material to deposit 1,3 dichloro 5,5 dimethyl hydantoin and the bromo- 2- nitros -1,3- propylene glycol of 2- in agent component.Application
Numbers 201510132774.4 patent discloses the formaldehyde releaser in a kind of blood anticoagulant component for protecting dissociative DNA
The formaldehyde of generation belongs to noxious material, can be harmful.
It can be seen that there is also larger deficiencies for the prior art.
Invention content
In consideration of it, it is necessary to provide a kind of blood preseration agent for protecting dissociative DNA regarding to the issue above, blood plasma is swum
, safety good from DNA preservation effects, is more advantageous to the accurate detection of plasma DNA.
The object of the invention is realized by following technological means：
A kind of blood preseration agent for protecting dissociative DNA, every 100 milliliters of protective agents include following components：
Further, the anti-coagulants is EDTAP dipotassium ethylene diamine tetraacetate, ethylenediamine tetra-acetic acid tripotassium, ethylenediamine tetra-acetic acid two
Sodium, citric acid one or more of are received.The addition of anti-coagulants, can anti-hemostasis-coagulation, inhibition DNA enzymatic.
Preferably, the anti-coagulants is EDTAP dipotassium ethylene diamine tetraacetate.
Further, the natural antiseptic agent is one kind in epsilon-polylysine, tea polyphenols (＞ 95%), natamycin.
When wherein, using tea polyphenols (＞ 95%) or natamycin, and preservation effect phase of the epsilon-polylysine to sample dissociative DNA is used
When.The addition of natural antiseptic agent is conducive to haemocyte monolithic stability, can anti-Hemostatic Oral Liquid it is rotten.Preferably, the natural antiseptic agent
For epsilon-polylysine.
Further, the dnase inhibitor is one in urea, ammonium sulfate, lauryl sodium sulfate, guanidinium isothiocyanate
Kind is a variety of.The addition of dnase inhibitor, the plasma DNA degradation that can prevent DNA enzymatic from mediating.Preferably, the DNA enzymatic
Inhibitor is urea.
Further, the buffer solution is 0.05mol/l tris-HCI buffers, 0.2mol/l phosphorus
One or more of sour disodium hydrogen-phosphate sodium dihydrogen buffer solution, 1 × PBS buffer solution, the pH value range of buffer solution is 6.8~
8.0.The addition of buffer solution can stablize blood pH, extend the storage life of haemocyte.
Preferably, the buffer solution is 0.05mol/l tris-HCI buffers, pH7.4.
Further, the glucose is the nutritional ingredient maintained needed for haemocyte metabolism, extends the red cell preservation time.
Further, the antihemolytic is mannitol, sorbierite, sucrose.The addition of antihemolytic can reinforce cell
Film alleviates erythrocyte hemolysis.
Preferably, the antihemolytic is mannitol.
Further, the purine is the combination of one or both of adenine, guanine.The addition of purine can maintain
The content of ATP maintains red cell morphology.
Preferably, the purine is adenine.
Further, preservative agent of the present invention is used as acquisition blood sample, transport blood sample or storage blood sample
When sample dissociative DNA preservative agent.For example, preservative agent of the present invention can be located in a special equipment, and before blood sampling
It is added, which is a collection container vacuumized, can be but not only vacuum blood collection tube.Blood sample is inhaled into
In vacuum blood collection tube containing preservative agent of the present invention, 7~14 days, even more than 14 days can be preserved at normal temperatures, haemocyte do not rupture,
Form stable, plasma DNA is completely without degradation.
Further, when the preservative agent is used as the preservative agent of sample dissociative DNA, the body of the preservative agent and blood sample
Accumulating ratio is：1:30~1:45.
Advantageous effect of the present invention：
What is added in preservative agent component of the present invention is natural antiseptic agent.Natural antiseptic agent be by organism secretion or in vivo
In the presence of the substance obtained by modern industry means.Such preservative is natural materials, and safety is harmless to the human body, more favorably
In the accurate detection of plasma DNA.
The anticoagulant for storage of whole blood of the present invention tends to be nontoxic using safe and non-toxic natural antiseptic agent epsilon-polylysine and low toxicity
Tea polyphenols (＞ 95%) and natamycin, without formaldehyde and other toxic chemical preservatives.Epsilon-polylysine is a kind of with suppression
The polypeptide of bacterium effect, NeDaK's et al.《NedaK,SakuraiT,etal.Two-generationreproductionstudy
withteratologytestofε-poly-L-lysinebydietary administrationinrats[J]
.JppharmcolTher,1999,27:1139-1159.》To epsilon-polylysine carry out toxicologic study, by it is acute with it is chronic
Mouse test proves, epsilon-polylysine does not have toxicity, or even epsilon-polylysine is up to the dosage of 20000mg/kg and will not generate
Any unfavorable effect and gene mutation.HirakiJ's et al.《Hiraki J,IchikawaT,NinomiyaS,
etal.UseofADMEstudiestoconfirm thesafetyofpolylysineasapreservativeinfood[J]
.RegulatoryToxicologyandPharmacology,2003,37(2):328-340.》With14The epsilon-polylysine of C flag
Carry out the safety that ADME experiments (absorbability, distributivity, metabolic and the excretion experiment of animal) also indicate that epsilon-polylysine.
As shown in table 1, the half lethal dose of epsilon-polylysine rat oral is more than 20000mg/kg, according to the United Nations's world health group
The Pyatyi standard (table 2) being classified for compound acute toxicity of (WHO) recommendation is knitted it is found that natural anti-in preservative agent of the present invention
Rotten agent epsilon-polylysine is micro- poison or even nontoxic substance, will not be damaged to personnel.
It is of the present invention a kind of for protecting the blood preseration agent of dissociative DNA that blood cell breakage can be effectively prevent to discharge gene
The protective agent that DNA is polluted, plasma DNA is degraded is organized, stable environment is provided for plasma DNA, is conducive to free
DNA is more accurately tested and analyzed.
7~14 days, even more than 14 days, room temperature fortune can be preserved after blood sample and protective agent mixing of the present invention at normal temperatures
It transports to 4 days less, extends the holding time of sample, reduce the condition of storage after sample collection, to save dissociative DNA inspection
Economic cost is surveyed, the promotion and popularization of dissociative DNA detection technique are conducive to.
Description of the drawings
After Fig. 1 is stored at room temperature preservation 0 day for blood sample in 1 vacuum blood collection tube 1 (containing 125 μ l preservative agents 1) of embodiment
Dissociative DNA segment distribution map.
After Fig. 2 is stored at room temperature preservation 4 days for blood sample in 1 vacuum blood collection tube 1 (containing 125 μ l preservative agents 1) of embodiment
Dissociative DNA segment distribution map.
After Fig. 3 is stored at room temperature preservation 7 days for blood sample in 1 vacuum blood collection tube 1 (containing 125 μ l preservative agents 1) of embodiment
Dissociative DNA segment distribution map.
After Fig. 4 is stored at room temperature preservation 14 days for blood sample in 1 vacuum blood collection tube 1 (containing 125 μ l preservative agents 1) of embodiment
Dissociative DNA segment distribution map.
After Fig. 5 is stored at room temperature preservation 0 day for blood sample in 1 vacuum blood collection tube 2 (containing 125 μ l preservative agents 2) of embodiment
Dissociative DNA segment distribution map.
After Fig. 6 is stored at room temperature preservation 4 days for blood sample in 1 vacuum blood collection tube 2 (containing 125 μ l preservative agents 2) of embodiment
Dissociative DNA segment distribution map.
After Fig. 7 is stored at room temperature preservation 7 days for blood sample in 1 vacuum blood collection tube 2 (containing 125 μ l preservative agents 2) of embodiment
Dissociative DNA segment distribution map.
After Fig. 8 is stored at room temperature preservation 14 days for blood sample in 1 vacuum blood collection tube 2 (containing 125 μ l preservative agents 2) of embodiment
Dissociative DNA segment distribution map.
Fig. 9 is that blood sample is stored at room temperature the dissociative DNA segment point preserved in 2 hours in embodiment 1EDTA-2K heparin tubes
Butut.
Figure 10 is that blood sample is stored at room temperature the dissociative DNA segment point after preserving 4 days in embodiment 1EDTA-2K heparin tubes
Butut.
Figure 11 is free after blood sample room temperature transports 0 day in 2 vacuum blood collection tube 1 of embodiment (containing 125 μ l preservative agents 1)
DNA fragmentation distribution map.
Figure 12 is free after blood sample room temperature transports 4 days in 2 vacuum blood collection tube 1 of embodiment (containing 125 μ l preservative agents 1)
DNA fragmentation distribution map.
Figure 13 is free after blood sample room temperature transports 0 day in 2 vacuum blood collection tube 2 of embodiment (containing 125 μ l preservative agents 2)
DNA fragmentation distribution map.
Figure 14 is free after blood sample room temperature transports 4 days in 2 vacuum blood collection tube 2 of embodiment (containing 125 μ l preservative agents 2)
DNA fragmentation distribution map.
To Fig. 1-14 it should be further noted that the peak of 35bp is the peak that DNALadder is generated in figure, other peaks are then
The peak generated for sample.
Fig. 1-9 needs are illustrated further, each figure main peak size meets plasma DNA all in 180bp or so
Clip size；Without apparent miscellaneous peak, illustrate the intact no degradations of DNA；Plasma DNA is relatively stablized.
Figure 10 need to be illustrated further, without 180 or so peak, plasma DNA may degrade.In the presence of
The multiple very high peaks 200bp or more illustrate that there are the segments after lots of genes group DNA degradation.Plasma DNA it is unstable and by
The pollution of genomic DNA is arrived.
Figure 11-14 needs are illustrated further, each figure main peak size meets plasma free all in 180bp or so
DNA fragmentation size；Without apparent miscellaneous peak, illustrate the intact no degradations of DNA；Plasma DNA is relatively stablized.
Specific implementation mode
The problem of solved in order to better illustrate the present invention, used technical solution and the effect reached, are now tied
It closes specific embodiment and related data is expanded on further.It should be noted that the content of present invention is including but not limited to following implementation
Example and combinations thereof embodiment.
Embodiment 1 is stored at room temperature preservation experiment
Blood preseration agent 1 provided in this embodiment, every 100 milliliters of protective agents 1 include following components weight (g)：
| EDTAP dipotassium ethylene diamine tetraacetate | 8.8g |
| Epsilon-polylysine | 1.8g |
| Urea | 8.0g |
| Glucose | 9.6g |
| Mannitol | 5.0g |
| Adenine | 0.3g |
Preparation method：100ml blood preserations agent 1 is prepared, is calculated by said components concentration, material is prepared：Ethylenediamine tetrem
Sour dipotassium：8.8g, epsilon-polylysine：1.8g, urea：8.0g, glucose：9.6g, mannitol：5.0g, adenine：0.3g.
Said mixture is settled to 100ml, 0.05mol/l, pH7.4 tris-HCI buffers, most
1 finished product of blood preseration agent is obtained eventually.
Blood preseration agent 1 obtained by the present embodiment is used as acquisition blood sample, transport blood sample or storage blood sample
When sample dissociative DNA preservative agent when, the volume ratio with blood sample is：1:40,125 μ should be added by such as handling 5ml blood samples
L blood preserations agent 1.
As a contrast, every 100 milliliters of protective agents 2 include following components weight for blood preseration agent 2 provided in this embodiment
(g)：
| EDTAP dipotassium ethylene diamine tetraacetate | 8.8g |
| The bromo- 2- nitros -1,3- propylene glycol of 2- | 3.2g |
| Urea | 8.0g |
| Glucose | 9.6g |
| Mannitol | 5.0g |
| Adenine | 0.3g |
Preparation method：100ml blood preserations agent 2 is prepared, is calculated by said components concentration, material is prepared：Ethylenediamine tetrem
Sour dipotassium：The bromo- 2- nitros -1,3- propylene glycol of 8.8g, 2-：3.2g, urea：8.0g, glucose：9.6g, mannitol：5.0g, gland
Purine：0.3g.
Said mixture is settled to 100ml, 0.05mol/l, pH7.4 tris-HCI buffers, most
2 finished product of blood preseration agent is obtained eventually.
Blood preseration agent 2 obtained by the present embodiment is used as acquisition blood sample, transport blood sample or storage blood sample
When sample dissociative DNA preservative agent when, the volume ratio with blood sample is：1:40,125 μ should be added by such as handling 5ml blood samples
L blood preserations agent 2.
Blood preseration agent 1 can be used as one kind epsilon-polylysine containing natural antiseptic agent of the present invention, without chemical preservative, first
The blood preseration agent of aldehyde releasing agent.
Blood preseration agent 2 can be used as the blood preseration agent of the control of the present embodiment, containing the toxic bromo- 2- of chemical preservative 2-
Nitro -1,3- propylene glycol.
(1) experimental method：
Using vacuum blood collection tube 1 (containing 125 μ l preservative agents 1), to 6 volunteers, everyone extracts 4 pipe 5ml blood samples, up and down
Reverse mixing 10 times makes blood with preservative agent at uniform system.It is again that the blood sample after mixing is quiet at ambient temperature respectively
Preservation 0 day (being no more than 6 hours), 4 days, 7 days, 14 days is set, each time point takes the 1 pipe blood sample of each volunteer to divide
From blood plasma.
Separated plasma method：Blood sample is put into centrifuge, 4 DEG C, 1600g centrifuges 10min, careful absorption supernatant
Into new centrifuge tube, if getting leucocyte or red blood cell, supernatant is drawn after re-starting centrifugation.After first time is centrifuged
The supernatant of acquisition is placed again into centrifuge, 4 DEG C, and 16000g centrifuges 10min, and careful only 600 μ l supernatants of absorption are new to one
Centrifuge tube in, the solution being achieved in that is exactly plasma sample.
The extraction of plasma DNA：The 600 μ l plasma samples isolated to each volunteer extract plasma DNA
(the auspicious plasma DNA extracts kit (magnetic bead with health Biotechnology Ltd. of Beijing shellfish can be selected in extracts kit
Method) or Beijing Genmagbio Biotechnology Co., Ltd. free serum DNA extracts kit etc.), by the blood plasma extracted swim
It is dissolved in 40 μ lTris-HCl buffer solutions (pH8.0) from DNA, obtains plasma DNA sample.
It takes the plasma DNA sample of 3 μ l said extracteds to carry out Q-PCR tests as template, utilizes β-actin genes
CT values come detect in blood sample dissociate amount of DNA variation.
The Q-PCR reaction systems are：
The Q-PCR reaction conditions：
The primer sequence is：
Primer-F：CTGGGAAGGTTACAGGAAGA；
Primer-R：AATTGGCTCAAACAACGTGAAT.
The isometric mixing of plasma DNA sample for preserving the identical 6 volunteer's blood samples extraction of number of days will be stood, takes 1 μ
The plasma DNA sample of l mixings detects the piece of dissociative DNA in blood sample with Agilent 2100DNA high sensitivity chip
Duan great little.
It respectively extracts 4 pipe 5ml blood samples again respectively to above 6 volunteers and is placed in vacuum blood collection tube 2 (containing 125 μ l preservations
Agent 2) in, sample handling characteristics (contain 125 μ l preservative agents with vacuum blood collection tube 1 (containing 125 μ l preservative agents 1) with vacuum blood collection tube 2
2) sample is stored at room temperature the test result of preservation 0 day (being no more than 6 hours), 4 days, 7 days, 14 days as a contrast.
To above 6 volunteers, respectively extracting 2 pipe 5ml blood samples again is placed in the (examination in the pipe of EDTA-2K heparin tubes respectively
Agent is containing only anti-coagulants EDTAP dipotassium ethylene diamine tetraacetate, no other components), sample handling characteristics (are protected with vacuum blood collection tube 1 containing 125 μ l
Deposit agent 1), it is stored at room temperature using EDTA-2K heparin tube samples and preserves the test result of 0 day (be no more than 6 hours), 4 days as blank
Control.
(2) experimental result：
0 day, 4 days, 7 days, 14 days vacuum blood collection tubes 1 (containing 125 μ l preservative agents 1) blood sample are preserved to being stored at room temperature
Dissociative DNA, the CT values detected using Q-PCR, as shown in table 3.
0 day, 4 days, 7 days, 14 days vacuum blood collection tubes 2 (containing 125 μ l preservative agents 2) blood sample are preserved to being stored at room temperature
Dissociative DNA, the CT values detected using Q-PCR, as shown in table 4.
Dissociative DNA to the EDTA-2K heparin tube blood samples for being stored at room temperature preservation 0 day, 4 days uses Q-PCR detections
CT values, as shown in table 5.
After being stored at room temperature preservation 0 day, 4 days, 7 days, 14 days, vacuum blood collection tube 1 (containing 125 μ l preservative agents 1) and vacuum blood collection
Pipe 2 (the containing 125 μ l preservative agents 2) otherness that blood sample preserves compares：(contain 125 μ l using Q-PCR detection vacuum blood collection tubes 1
Preservative agent 1) and vacuum blood collection tube 2 (contain 125 μ l preservative agents 2) blood sample dissociative DNA CT values all relatively, about 30；
Illustrate that the amount of plasma DNA is stablized, does not carry out the pollution of leukocytes release lots of genes group DNA.As a result referring to table 3 and table
4。
It is stored at room temperature preservation 0 day, the free DN of 4 days EDTA-2K heparin tube blood samples and vacuum blood collection tube 1 and (contains 125
μ l preservative agents 1) otherness of Sample storage compares：It is quiet using Q-PCR detections vacuum blood collection tube 1 (containing 125 μ l preservative agents 1) room temperature
Set preserve 0 day, the CT values of 4 days blood sample dissociative DNAs all relatively, about 30；But EDTA-2K heparin tube room temperatures are quiet
(CT values difference is obviously reduced than preserving 0 day (about 30) in the CT values (about 26) for setting the blood sample dissociative DNA of preservation 4 days
4, DNA amount difference 24Times, i.e., 16 times), illustrate that the blood sample for preserving 4 days is stored at room temperature using EDTA-2K heparin tubes to be existed
Carry out the pollution of leukocytes release lots of genes group DNA.
The DNA fragmentation size of each heparin tube sample please refers to Fig. 1 to Figure 10, and (2100 biological analyser of Agilent is big to DNA
Small and concentration analysis).Comparison diagram 1 can be seen that in 14 days to Fig. 8, vacuum blood collection tube 1 (containing 125 μ l preservative agents 1) and very
The plasma DNA testing result of empty heparin tube 2 (containing 125 μ l preservative agents 2)：Main peak size distribution is consistent, all on the left sides 180bp
The right side meets Circulating DNA fragments size, and plasma DNA is completely without degradation；200bp or more illustrates not deposit without apparent miscellaneous peak
Segment after genomic DNA degradation, is not present the pollution of genomic DNA.
Fig. 9 is similar to Fig. 1 to Fig. 8, and plasma DNA is stablized pollution-free.Figure 10 is without 180 or so peak, and plasma free
The peak of DNA is about 180bp, and plasma DNA may degrade；There are the multiple very high peaks 200bp or more, illustrate in the presence of big
Measure the segment after genomic DNA degradation.The plasma DNA that preservation 4 days is stood using 1 temperature of EDTA-2K heparin tubes room is unstable
And receive the pollution of genomic DNA.
In summary：It was stored at room temperature in the holding time at 14 days, plasma DNA is in the vacuum blood collection tube 1 containing preservative agent 1
Middle preservation effect is preferable, consistent with the preservation effect in the vacuum blood collection tube 2 containing preservative agent 2.And common EDTA-2K blood samplings
Pipe cannot be used for preserving plasma DNA for a long time.Room temperature preserve blood sample when using containing natural antiseptic agent blood preseration agent 1 with
The blood preseration agent 2 of the preservative containing toxic chemical is consistent to the protecting effect of plasma DNA, but the blood preseration agent of the present invention
1 is safer, more endangers smaller to the operating personnel and environment of the contact preservative agent in detection process.
Table 3
Table 4
Table 5
2 room temperature trafficking experiments of embodiment：
The component and its ratio of blood preseration agent 1 and blood preseration agent 2 provided in this embodiment are the same as embodiment 1.
(1) experimental method：
Using vacuum blood collection tube (1 contains 125 μ l preservative agents 1), to 6 volunteers, everyone extracts 2 pipe 5ml blood samples, up and down
Reverse mixing 10 times makes blood with preservative agent at uniform system.The blood sample of each volunteer is respectively in sampling (6 hours 0 day
It is interior) and after room temperature transports 4 days (96 hours), 1 pipe is respectively taken to carry out separated plasma, it often manages and only takes 600 μ l blood plasma as plasma sample,
The method of separated plasma can be the same as embodiment 1.
The extraction of plasma DNA：Plasma DNA (extracts reagent is extracted to the 600 μ l plasma samples of each volunteer
The auspicious plasma DNA extracts kit (paramagnetic particle method) or north with health Biotechnology Ltd. of Beijing shellfish can be selected in box
Free serum DNA extracts kit of Jing Jinmaige Bioisystech Co., Ltd etc.), the DNA extracted is dissolved in 40 μ l
In Tris-HCl buffer solutions (pH8.0), plasma DNA sample is obtained.
It takes 3 μ l plasma DNAs samples to carry out Q-PCR tests as template, is detected using the CT values of β-actin genes
The variation of free amount of DNA in blood sample, Q-PCR reaction systems, reaction condition and primer sequence can be the same as embodiments 1.
The isometric mixing of plasma DNA sample of the identical 6 volunteer's blood samples extraction of number of days will be transported, takes 1 μ l mixed
Even plasma DNA sample is big come the segment for detecting dissociative DNA in blood sample with Agilent 2100DNA high sensitivity chip
It is small.
2 pipe 5ml blood samples are extracted again respectively to above 6 volunteers and are placed in vacuum blood collection tube 2 (containing 125 μ l preservative agents
2) in, sample handling characteristics are with vacuum blood collection tube 1 (containing 125 μ l preservative agents 1), with vacuum blood collection tube 2 (containing 125 μ l preservative agents 2)
Room temperature transports the sample of 0 day (being no more than 6 hours) and room temperature transports the sample tests of 4 days (96 hours) as a contrast.
(2) experimental result：
The dissociative DNA that 4 days vacuum blood collection tube 1 (containing 125 μ l preservative agents 1) blood sample is transported to 0 day and room temperature, uses
The CT values of Q-PCR detections, as shown in table 6.
The dissociative DNA that 4 days vacuum blood collection tube 2 (containing 125 μ l preservative agents 2) blood sample is transported to 0 day and room temperature, uses
The CT values of Q-PCR detections, as shown in table 7.
After room temperature transports 4 days, vacuum blood collection tube 1 (containing 125 μ l preservative agents 1) and vacuum blood collection tube 2 (contain 125 μ l preservative agents
2) otherness that blood sample preserves compares：Use Q-PCR detections vacuum blood collection tube 1 (containing 125 μ l preservative agents 1) and vacuum blood collection
The CT values of pipe 2 (contain 125 μ l preservative agents 2) blood sample dissociative DNA all relatively, about 30, illustrate the amount of plasma DNA
Stablize, does not carry out the pollution of leukocytes release lots of genes group DNA.
The DNA fragmentation size of each heparin tube sample please refers to Figure 11 to Figure 14, and (2100 biological analyser of Agilent is big to DNA
Small and concentration analysis).It can be seen from the figure that after room temperature transports 4 days, vacuum blood collection tube 1 (contain 125 μ l preservative agents 1) and
The plasma DNA testing result of vacuum blood collection tube 2 (containing 125 μ l preservative agents 2)：Main peak size distribution is consistent, all on the left sides 180bp
The right side meets Circulating DNA fragments size, and plasma DNA is completely without degradation；200bp or more illustrates not deposit without apparent miscellaneous peak
Segment after genomic DNA degradation, is not present the pollution of genomic DNA.
In summary：In 4 days room temperature haulage times, plasma DNA preserves in the vacuum blood collection tube containing preservative agent 1
Effect is preferable, consistent with the preservation effect in the vacuum blood collection tube containing preservative agent 2.Using containing natural when i.e. room temperature transports blood sample
The blood preseration agent 1 of preservative is with the blood preseration agent 2 of the preservative containing toxic chemical to the protecting effect one of plasma DNA
It causes, but the blood preseration agent 1 of the present invention is safer, more endangers to the operating personnel and environment of the contact preservative agent in detection process
Evil smaller.
Table 6
Table 7
By time-proven, formula, proportioning involved by the specific embodiment of the invention, operating method, test method etc.
It is suitable for tea polyphenols (＞ 95%) and natamycin, and can basically reaches the advantageous effect of epsilon-polylysine, herein no longer
It repeats.
Several embodiments of the invention above described embodiment only expresses, the description thereof is more specific and detailed, but simultaneously
Cannot the limitation to the scope of the claims of the present invention therefore be interpreted as.It should be pointed out that for those of ordinary skill in the art
For, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to the guarantor of the present invention
Protect range.Therefore, the protection domain of patent of the present invention should be determined by the appended claims.
Claims (8)
1. a kind of blood preseration agent for protecting dissociative DNA, which is characterized in that every 100 milliliters of preservative agents include following
Component：
The natural antiseptic agent is one kind in epsilon-polylysine, tea polyphenols, natamycin；The purity of the tea polyphenols is more than
95%.
2. blood preseration agent according to claim 1, which is characterized in that the anti-coagulants be EDTAP dipotassium ethylene diamine tetraacetate,
Ethylenediamine tetra-acetic acid tripotassium, disodium ethylene diamine tetraacetate, citric acid one or more of are received.
3. blood preseration agent according to claim 1, which is characterized in that the dnase inhibitor be urea, ammonium sulfate,
One or more of lauryl sodium sulfate, guanidinium isothiocyanate.
4. blood preseration agent according to claim 1, which is characterized in that the buffer solution is 0.05mol/l trihydroxy methyls
One in aminomethane-hydrochloride buffer, 0.2mol/l disodium hydrogen phosphates-phosphate sodium dihydrogen buffer solution or 1 × PBS buffer solution
Kind, the pH value range of buffer solution is 6.8~8.0.
5. blood preseration agent according to claim 1, which is characterized in that the antihemolytic be mannitol, sorbierite or
Sucrose.
6. blood preseration agent according to claim 1, which is characterized in that the purine is one in adenine, guanine
Kind or two kinds of combinations.
7. the preservative agent described in claim 1-6 any one, which is characterized in that the preservative agent be used as acquisition blood sample,
Blood sample dissociative DNA preservative agent when transporting blood sample or storing blood sample.
8. blood preseration agent according to claim 7, which is characterized in that the volume of the blood preseration agent and blood sample
Than for：1:30~1:45.
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| CN105985904A (en) * | 2015-02-26 | 2016-10-05 | 付士明 | Circulating free DNA vacuum collection tube |
| CN106244535A (en) * | 2016-08-30 | 2016-12-21 | 成都瑞琦科技实业股份有限公司 | The preservative agent of fetal cell-free DNA in maternal plasma and the vacuum test tube of composition thereof |
| CN106666329A (en) * | 2016-12-30 | 2017-05-17 | 江苏诺兴生物科技有限公司 | Preservative for improving quality and fresh keeping effect of food and application of preservative |
| CN106701743A (en) * | 2016-12-26 | 2017-05-24 | 广州和实生物技术有限公司 | Blood collection tube for protecting and stabilizing free DNA |
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| CN102823579A (en) * | 2012-08-27 | 2012-12-19 | 杭州博拓生物技术有限公司 | Blood protective agent, preparation method thereof and application |
| CN105985904A (en) * | 2015-02-26 | 2016-10-05 | 付士明 | Circulating free DNA vacuum collection tube |
| CN104830830A (en) * | 2015-03-25 | 2015-08-12 | 厦门艾德生物医药科技有限公司 | A blood anticoagulant used for protecting free DNA and applications thereof |
| CN105087470A (en) * | 2015-07-31 | 2015-11-25 | 何静 | Method for separating and purifying human embryo trophoblast and placental mesenchymal stem cells |
| CN106244535A (en) * | 2016-08-30 | 2016-12-21 | 成都瑞琦科技实业股份有限公司 | The preservative agent of fetal cell-free DNA in maternal plasma and the vacuum test tube of composition thereof |
| CN106701743A (en) * | 2016-12-26 | 2017-05-24 | 广州和实生物技术有限公司 | Blood collection tube for protecting and stabilizing free DNA |
| CN106666329A (en) * | 2016-12-30 | 2017-05-17 | 江苏诺兴生物科技有限公司 | Preservative for improving quality and fresh keeping effect of food and application of preservative |
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