CN107083382A - A kind of blood preseration agent and its application for being used to protect dissociative DNA - Google Patents
A kind of blood preseration agent and its application for being used to protect dissociative DNA Download PDFInfo
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- CN107083382A CN107083382A CN201710380209.9A CN201710380209A CN107083382A CN 107083382 A CN107083382 A CN 107083382A CN 201710380209 A CN201710380209 A CN 201710380209A CN 107083382 A CN107083382 A CN 107083382A
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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
Abstract
The invention belongs to the preservation reagent of blood sample, and in particular to a kind of blood preseration agent for being used to protect 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 from discharging genomic DNA and polluting, 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 testing and analyzing dissociative DNA.
Description
Technical field
The invention belongs to the preservation reagent of blood sample, and in particular to a kind of blood preseration agent for being used to protect dissociative DNA
And its application.
Background technology
At present, plasma DNA clinically applies more and more, such as noninvasive prenatal gene detection and diagnosing tumor are all
It is using plasma DNA as 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 easily cracked release by leucocyte
Contaminating genomic DNA, have a strong impact on testing result.
In order to solve these problems, the measure mainly taken at this stage has：(1) from band anti-coagulants (EDTA, heparin, lemon
Lemon hydrochlorate) heparin tube collection blood sample, to prevent whole blood cells blood coagulation, reduce leucocyte release genomic DNA；(2) 6
Blood plasma, 4 DEG C of Cord blood transports are isolated in hour, and the holding time can not be long, prevents DNA enzymatic degraded plasma free
DNA。
By above technical finesse, the accurate detection to plasma DNA can obtain certain improvement.But 4 DEG C of low temperature
Preserve under traffic condition, DNA enzymatic still has certain activity, long-term preserve can be such that plasma DNA largely loses, be unfavorable for blood plasma
The accurate detection of dissociative DNA.Simultaneously under existence conditions, the storage and transport of blood are all carried out at low temperature, bring many not
Just, plasma DNA is made to detect that financial cost is higher.
Therefore, it is necessary that a kind of blood preseration agent for being used to protect dissociative DNA is provided, it is intended to solve to use existing blood
Dissociative DNA store method is starched, plasma DNA is vulnerable to contaminating genomic DNA, easily degraded by DNA enzymatic, the holding time is short, protect
Deposit the low problem of temperature.
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.
Application number 201510226039.X patent discloses a kind of preservative agent for preserving dissociative DNA in peripheral blood, and it is protected
Deposit and contain chemical preservative in agent component, including chloro- 5, the 5- dimethyl hydantoins of DIAZOLIDINYL UREA, imidazolidinyl urea, 1,3- bis-,
The bromo- 2- nitros -1,3- propane diols 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 discloses a kind of blood anticoagulant for being used to protect dissociative DNA and its should
With its blood anticoagulant component contains formaldehyde releaser, including 1,3- dihydroxymethyl -5,5- DMH, hydroxymethylglycinate
Sodium, soluble metyl hydroxybenzoate, nipagin A sodium, the one or more of soluble propylhydroxybenzoate.
It is well known that many chemical preservatives are poisonous, it is harmful, and easily environment is polluted；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 thing://www.basechem.org/).
The toxicology data of several chemical substances of table 1
| Chemical substance | Rat oral LD50(mg/kg) |
| Epsilon-polylysine | ＞ 20000 |
| 1,3 dichloro 5,5 dimethyl hydantoin | 542 |
| The bromo- 2- nitros -1,3- propane diols of 2- | 180~400 |
| Formaldehyde | 800 |
| Tea Polyphenols (＞ 95%) | 3940 |
| Natamycin | 4000 |
Median lethal dose (lethaldose50%, LD50)：It is the medicine agent for referring to cause experimental animal half dead
Amount, recommends a Pyatyi standard for the compound acute toxicity graded combination World Health Organization of state (WHO), is shown in Table 2.
The foreign compound acute toxicity of table 2 is classified
| Chemical substance | Rat oral LD50(mg/kg) |
| Severe toxicity | ＜ 1 |
| High poison | 1- |
| Medium poison | 50- |
| Low toxicity | 500- |
| Micro- poison | 5000- |
As can be seen here, application number 201510226039.X patent discloses a kind of guarantor for 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- propane diols of 2- in agent component.Application
Numbers 201510132774.4 patent, which is disclosed, a kind of is used to protect the formaldehyde releaser in the blood anticoagulant component of dissociative DNA
The formaldehyde of generation belongs to noxious material, can be harmful.
As can be seen here, also there is larger deficiency in prior art.
The content of the invention
In consideration of it, being necessary to provide a kind of blood preseration agent for being used to protect dissociative DNA regarding to the issue above, blood plasma is swum
, safety good from DNA preservation effects, is more beneficial for the accurate detection of plasma DNA.
The object of the invention is realized by following technological means：
A kind of blood preseration agent for being used to protect 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 receive in one or more.The addition of anti-coagulants, can anti-hemostasis-coagulation, suppression 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, with using preservation effect phase of the epsilon-polylysine to sample dissociative DNA
When.The addition of natural antiseptic agent, is conducive to haemocyte monolithic stability, can anti-Hemostatic Oral Liquid go bad.Preferably, the natural antiseptic agent
For epsilon-polylysine.
Further, the dnase inhibitor is one in urea, ammonium sulfate, lauryl sodium sulfate, guanidinium isothiocyanate
Plant or a variety of.The addition of dnase inhibitor, the plasma DNA degraded 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 in sour disodium hydrogen-phosphate sodium dihydrogen buffer solution, 1 × PBS, the pH value range of buffer solution for 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 to maintain the nutritional ingredient 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 be maintained
ATP content, maintenance red cell morphology.
Preferably, the purine is adenine.
Further, preservative agent of the present invention is used as collection blood sample, transport blood sample or storage blood sample
When sample dissociative DNA preservative agent.Such as, preservative agent of the present invention can be located in a special equipment, and before blood sampling
Add, the equipment is a collection container vacuumized, can be but not exclusively vacuum blood collection tube.Blood sample is inhaled into
In vacuum blood collection tube containing preservative agent of the present invention, 7~14 days can be preserved at normal temperatures, even more than 14 days, haemocyte do not rupture,
Form stable, plasma DNA is completely without degraded.
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.
Beneficial effect of the present invention：
What is added in preservative agent component of the present invention is natural antiseptic agent.Natural antiseptic agent is by organism secretion or internal
In the presence of the material 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 the natural antiseptic agent epsilon-polylysine and low toxicity of safety non-toxic
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 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 20000mg/kg dosage and will not also produced
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 security 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
Being understood for the Pyatyi standard (table 2) that compound acute toxicity is classified for (WHO) recommendation is knitted, it is natural anti-in preservative agent of the present invention
Rotten agent epsilon-polylysine is micro- malicious or even nontoxic material, and personnel will not be damaged.
A kind of blood preseration agent for being used to protect dissociative DNA of the present invention can effectively prevent blood cell breakage from discharging gene
Group DNA is polluted, the protective agent of plasma DNA degraded, is provided stable environment for plasma DNA, is conducive to free
DNA is more accurately tested and analyzed.
Blood sample and protective agent of the present invention can be preserved 7~14 days at normal temperatures after mixing, even more than 14 days, normal temperature fortune
Transport to 4 days less, extend the holding time of sample, reduce the condition of storage after sample collection, so as to save dissociative DNA inspection
Financial cost is surveyed, is conducive to popularizing for dissociative DNA detection technique.
Brief description of the drawings
After Fig. 1 is stored at room temperature preservation 0 day for blood sample in the vacuum blood collection tube 1 (containing 125 μ l preservative agents 1) of embodiment 1
Dissociative DNA fragment distribution map.
After Fig. 2 is stored at room temperature preservation 4 days for blood sample in the vacuum blood collection tube 1 (containing 125 μ l preservative agents 1) of embodiment 1
Dissociative DNA fragment distribution map.
After Fig. 3 is stored at room temperature preservation 7 days for blood sample in the vacuum blood collection tube 1 (containing 125 μ l preservative agents 1) of embodiment 1
Dissociative DNA fragment distribution map.
After Fig. 4 is stored at room temperature preservation 14 days for blood sample in the vacuum blood collection tube 1 (containing 125 μ l preservative agents 1) of embodiment 1
Dissociative DNA fragment distribution map.
After Fig. 5 is stored at room temperature preservation 0 day for blood sample in the vacuum blood collection tube 2 (containing 125 μ l preservative agents 2) of embodiment 1
Dissociative DNA fragment distribution map.
After Fig. 6 is stored at room temperature preservation 4 days for blood sample in the vacuum blood collection tube 2 (containing 125 μ l preservative agents 2) of embodiment 1
Dissociative DNA fragment distribution map.
After Fig. 7 is stored at room temperature preservation 7 days for blood sample in the vacuum blood collection tube 2 (containing 125 μ l preservative agents 2) of embodiment 1
Dissociative DNA fragment distribution map.
After Fig. 8 is stored at room temperature preservation 14 days for blood sample in the vacuum blood collection tube 2 (containing 125 μ l preservative agents 2) of embodiment 1
Dissociative DNA fragment distribution map.
Fig. 9 is stored at room temperature the dissociative DNA fragment point preserved in 2 hours for blood sample in embodiment 1EDTA-2K heparin tubes
Butut.
Figure 10 is stored at room temperature the dissociative DNA fragment point after preserving 4 days for blood sample in embodiment 1EDTA-2K heparin tubes
Butut.
Figure 11 is free after blood sample normal temperature is transported 0 day in the vacuum blood collection tube 1 of embodiment 2 (containing 125 μ l preservative agents 1)
DNA fragmentation distribution map.
Figure 12 is free after blood sample normal temperature is transported 4 days in the vacuum blood collection tube 1 of embodiment 2 (containing 125 μ l preservative agents 1)
DNA fragmentation distribution map.
Figure 13 is free after blood sample normal temperature is transported 0 day in the vacuum blood collection tube 2 of embodiment 2 (containing 125 μ l preservative agents 2)
DNA fragmentation distribution map.
Figure 14 is free after blood sample normal temperature is transported 4 days in the vacuum blood collection tube 2 of embodiment 2 (containing 125 μ l preservative agents 2)
DNA fragmentation distribution map.
To Fig. 1-14 it should be further stated that, in figure 35bp peak be DNALadder produce peak, other peaks are then
The peak produced for sample.
Fig. 1-9 is needed to illustrate further, each figure main peak size meets plasma DNA all in 180bp or so
Clip size；Without obvious miscellaneous peak, illustrate that DNA is intact without degraded；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 of more than 200bp, illustrate the fragment for having after lots of genes group DNA degradation.Plasma DNA it is unstable and by
The pollution of genomic DNA is arrived.
Figure 11-14 is needed to illustrate further, each figure main peak size meets plasma free all in 180bp or so
DNA fragmentation size；Without obvious miscellaneous peak, illustrate that DNA is intact without degraded；Plasma DNA is relatively stablized.
Embodiment
In order to which problem solved by the invention, the technical scheme used and the effect reached is better described, now tie
Close specific embodiment and related data is expanded on further.It should be noted that present invention is implemented including but not limited to following
Example and combinations thereof embodiment.
Embodiment 1 is stored at room temperature preservation experiment
The blood preseration agent 1 that the present embodiment is provided, 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 |
Compound method：100ml blood preserations agent 1 is prepared, is calculated by said components concentration, prepares material：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
The finished product of blood preseration agent 1 is obtained eventually.
Blood preseration agent 1 obtained by the present embodiment is used as collection 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, such as processing 5ml blood samples should add 125 μ
L blood preserations agent 1.
The blood preseration agent 2 that the present embodiment is provided includes following components weight as control, every 100 milliliters of protective agents 2
(g)：
| EDTAP dipotassium ethylene diamine tetraacetate | 8.8g |
| The bromo- 2- nitros -1,3- propane diols of 2- | 3.2g |
| Urea | 8.0g |
| Glucose | 9.6g |
| Mannitol | 5.0g |
| Adenine | 0.3g |
Compound method：100ml blood preserations agent 2 is prepared, is calculated by said components concentration, prepares material：Ethylenediamine tetrem
Sour dipotassium：The bromo- 2- nitros -1,3- propane diols 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
The finished product of blood preseration agent 2 is obtained eventually.
Blood preseration agent 2 obtained by the present embodiment is used as collection 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, such as processing 5ml blood samples should add 125 μ
L blood preserations agent 2.
Blood preseration agent 1 can be as one kind epsilon-polylysine containing natural antiseptic agent of the invention, without chemical preservative, first
The blood preseration agent of aldehyde releasing agent.
Blood preseration agent 2 can as the control of the present embodiment blood preseration agent, containing the poisonous bromo- 2- of chemical preservative 2-
Nitro -1,3- propane diols.
(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
It is reverse to mix 10 times, make blood and preservative agent into homogeneous system.It is again that the blood sample after mixing is quiet at ambient temperature respectively
Put preservation 0 day (be no more than 6 hours), 4 days, 7 days, 14 days, each time point takes the 1 pipe blood sample of each volunteer to be divided
From blood plasma.
Separated plasma method：Blood sample is put into centrifuge, 4 DEG C, 1600g centrifugation 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 centrifugations 10min, 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
(extracts kit can select auspicious and health Biotechnology Ltd. the plasma DNA extracts kit (magnetic bead of Beijing shellfish
Method), or Beijing Genmagbio Biotechnology Co., Ltd. free serum DNA extracts kit etc.), the blood plasma extracted is swum
It is dissolved in from DNA in 40 μ lTris-HCl buffer solutions (pH8.0), obtains plasma DNA sample.
Take the plasma DNA sample of 3 μ l said extracteds as template progress Q-PCR tests, utilize β-actin genes
CT values come detect in blood sample dissociate amount of DNA change.
The Q-PCR reaction systems are：
The Q-PCR reaction conditions：
The primer sequence is：
Primer-F：CTGGGAAGGTTACAGGAAGA；
Primer-R：AATTGGCTCAAACAACGTGAAT.
The plasma DNA sample for standing the 6 volunteer's blood samples extractions of preservation number of days identical is mixed in equal volume, 1 μ is taken
The plasma DNA sample that l is mixed detects the piece of dissociative DNA in blood sample with Agilent 2100DNA high sensitivity chip
Duan great little.
6 volunteers respectively extract 4 pipe 5ml blood samples and are placed in vacuum blood collection tube 2 (containing 125 μ l preservations again respectively to more than
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 be stored at room temperature preservation 0 day (be no more than 6 hours), 4 days, 7 days, the test result of 14 days is as control.
To 6 volunteers above, 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), preservation 0 day (being no more than 6 hours), the test result of 4 days are stored at room temperature using EDTA-2K heparin tube samples and are used as blank
Control.
(2) experimental result：
0 day, 4 days, 7 days, 14 days vacuum blood collection tube 1 (contain 125 μ l preservative agents 1) blood sample is 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 tube 2 (contain 125 μ l preservative agents 2) blood sample is preserved to being stored at room temperature
Dissociative DNA, the CT values detected using Q-PCR, as shown in table 4.
Dissociative DNA to being stored at room temperature the EDTA-2K heparin tube blood samples for preserving 0 day, 4 days, is detected using Q-PCR
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 is preserved 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 stable, do not carry out leukocytes release lots of genes group DNA pollution.As a result referring to table 3 and table
4。
The free DN and vacuum blood collection tube 1 for being stored at room temperature the EDTA-2K heparin tube blood samples for preserving 0 day, 4 days (contain 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
Put preserve 0 day, 4 days blood sample dissociative DNA CT values all relatively, about 30；But EDTA-2K heparin tube room temperatures are quiet
(CT values difference is obviously reduced than preserving (about 30) of 0 day in the CT values (about 26) for putting 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 present
Carry out leukocytes release lots of genes group DNA pollution.
The DNA fragmentation size of each heparin tube sample refer to Fig. 1 to Figure 10, and (biological analyser of Agilent 2100 is big to DNA
The analysis of small and concentration).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 left in 180bp
The right side, meets Circulating DNA fragments size, plasma DNA is completely without degraded；More than 200bp illustrates not deposit without obvious miscellaneous peak
Fragment after genomic DNA degraded, in the absence of 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
DNA peak is about 180bp, and plasma DNA may degrade；There are the multiple very high peaks of more than 200bp, illustrate in the presence of big
Measure the fragment after genomic DNA degraded.The plasma DNA for standing preservation 4 days using the temperature of EDTA-2K heparin tubes room 1 is unstable
And receive the pollution of genomic DNA.
In summary：It was stored at room temperature at 14 days in the holding time, plasma DNA is in the vacuum blood collection tube 1 containing preservative agent 1
Middle preservation effect is preferably, 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.Normal temperature preserve blood sample when using the blood preseration agent 1 containing natural antiseptic agent 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
The normal temperature trafficking experiments of embodiment 2：
Blood preseration agent 1 and the component and its ratio be the same as Example 1 of blood preseration agent 2 that the present embodiment is provided.
(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
It is reverse to mix 10 times, make blood and preservative agent into homogeneous system.The blood sample of each volunteer is respectively in sampling (6 hours 0 day
It is interior) and normal temperature transport after 4 days (96 hours), respectively take 1 pipe to carry out separated plasma, often manage and only take 600 μ l blood plasma as plasma sample,
The method of separated plasma can be the same as Example 1.
The extraction of plasma DNA：Plasma DNA (extracts reagent is extracted to the 600 μ l plasma samples of each volunteer
Box can select auspicious and health Biotechnology Ltd. the plasma DNA extracts kit (paramagnetic particle method) of Beijing shellfish or north
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.
3 μ l plasma DNAs samples are taken as template and carry out Q-PCR tests, are detected using the CT values of β-actin genes
The change of free amount of DNA in blood sample, Q-PCR reaction systems, reaction condition and primer sequence can be the same as Examples 1.
The plasma DNA sample for transporting the 6 volunteer's blood samples extractions of number of days identical is mixed in equal volume, takes 1 μ l to mix
Even plasma DNA sample is big come the fragment for detecting dissociative DNA in blood sample with Agilent 2100DNA high sensitivity chip
It is small.
6 volunteers extract 2 pipe 5ml blood samples and are placed in vacuum blood collection tube 2 (containing 125 μ l preservative agents again respectively to more than
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)
Normal temperature transports the sample of 0 day (being no more than 6 hours) and the sample tests of normal temperature transport 4 days (96 hours) are used as control.
(2) experimental result：
The dissociative DNA of 4 days vacuum blood collection tube 1 (containing 125 μ l preservative agents 1) blood sample is transported to 0 day and normal temperature, is used
The CT values of Q-PCR detections, as shown in table 6.
The dissociative DNA of 4 days vacuum blood collection tube 2 (containing 125 μ l preservative agents 2) blood sample is transported to 0 day and normal temperature, is used
The CT values of Q-PCR detections, as shown in table 7.
After normal temperature is transported 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 is preserved 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
It is stable, do not carry out leukocytes release lots of genes group DNA pollution.
The DNA fragmentation size of each heparin tube sample refer to Figure 11 to Figure 14, and (biological analyser of Agilent 2100 is big to DNA
The analysis of small and concentration).It can be seen that after normal temperature is transported 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 left in 180bp
The right side, meets Circulating DNA fragments size, plasma DNA is completely without degraded；More than 200bp illustrates not deposit without obvious miscellaneous peak
Fragment after genomic DNA degraded, in the absence of the pollution of genomic DNA.
In summary：In 4 days normal temperature haulage times, plasma DNA is preserved in the vacuum blood collection tube containing preservative agent 1
Effect is preferably, consistent with the preservation effect in the vacuum blood collection tube containing preservative agent 2.Used when i.e. normal temperature transports blood sample containing natural
The blood preseration agent 1 of preservative and protecting effect one of the blood preseration agent 2 of the preservative containing toxic chemical to plasma DNA
Cause, but the blood preseration agent 1 of the present invention is safer, and the operating personnel and environment of the contact preservative agent in detection process are more endangered
Evil is smaller.
Table 6
Table 7
Elapsed time proves, formula, proportioning involved by the specific embodiment of the invention, operating method, test method etc.
It is applied to Tea Polyphenols (＞ 95%) and natamycin, and can basically reaches the beneficial effect of epsilon-polylysine, herein no longer
Repeat.
Embodiment described above only expresses the several embodiments of the present invention, and it describes more specific and detailed, but simultaneously
Therefore the limitation to the scope of the claims of the present invention can not be interpreted as.It should be pointed out that for one 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 scope.Therefore, the protection domain of patent of the present invention should be determined by the appended claims.
Claims (9)
1. a kind of blood preseration agent for being used to protect dissociative DNA, it is characterised in that every 100 milliliters of preservative agents include following
Component：
2. blood preseration agent according to claim 1, it is characterised in that the anti-coagulants be EDTAP dipotassium ethylene diamine tetraacetate,
Ethylenediamine tetra-acetic acid tripotassium, disodium ethylene diamine tetraacetate, citric acid receive in one or more.
3. blood preseration agent according to claim 1, it is characterised in that the natural antiseptic agent is epsilon-polylysine, tea
One kind in polyphenol (＞ 95%), natamycin.
4. blood preseration agent according to claim 1, it is characterised in that the dnase inhibitor be urea, ammonium sulfate,
One or more in lauryl sodium sulfate, guanidinium isothiocyanate.
5. blood preseration agent according to claim 1, it is characterised 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
Kind, the pH value range of buffer solution is 6.8~8.0.
6. blood preseration agent according to claim 1, it is characterised in that the antihemolytic be mannitol, sorbierite or
Sucrose.
7. blood preseration agent according to claim 1, it is characterised in that the purine is one in adenine, guanine
Plant or two kinds of combinations.
8. the preservative agent described in claim 1-7 any one, it is characterised in that the preservative agent be used as collection blood sample,
Transport blood sample dissociative DNA preservative agent when blood sample or storage blood sample.
9. blood preseration agent according to claim 8, it is characterised in that the volume of the blood preseration agent and blood sample
Than for：1:30~1:45.
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| CN109609596A (en) * | 2018-12-17 | 2019-04-12 | 依科赛生物科技（太仓）有限公司 | A kind of agent of dissociative DNA blood preseration and its application preventing haemolysis |
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| CN114045285A (en) * | 2021-11-15 | 2022-02-15 | 佛山市顺德区德维医疗科技有限公司 | Preservation method for free DNA |
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| CN112359091A (en) * | 2020-10-12 | 2021-02-12 | 华南理工大学 | Whole blood free DNA preservative, vacuum blood collection tube and preparation method thereof |
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