CN109182332A - A method of the fast and convenient extraction nucleic acid from clinical sample - Google Patents
A method of the fast and convenient extraction nucleic acid from clinical sample Download PDFInfo
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- CN109182332A CN109182332A CN201811153723.XA CN201811153723A CN109182332A CN 109182332 A CN109182332 A CN 109182332A CN 201811153723 A CN201811153723 A CN 201811153723A CN 109182332 A CN109182332 A CN 109182332A
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- 238000000034 method Methods 0.000 title claims abstract 13
- 108020004707 nucleic acids Proteins 0.000 title claims abstract 8
- 150000007523 nucleic acids Chemical class 0.000 title claims abstract 8
- 102000039446 nucleic acids Human genes 0.000 title claims abstract 8
- 238000000605 extraction Methods 0.000 title abstract 2
- 238000010438 heat treatment Methods 0.000 claims abstract 9
- 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 claims abstract 3
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims abstract 3
- 229920002527 Glycogen Polymers 0.000 claims abstract 3
- PZBFGYYEXUXCOF-UHFFFAOYSA-N TCEP Chemical compound OC(=O)CCP(CCC(O)=O)CCC(O)=O PZBFGYYEXUXCOF-UHFFFAOYSA-N 0.000 claims abstract 3
- 229940096919 glycogen Drugs 0.000 claims abstract 3
- SNRUBQQJIBEYMU-UHFFFAOYSA-N dodecane Chemical compound CCCCCCCCCCCC SNRUBQQJIBEYMU-UHFFFAOYSA-N 0.000 claims 2
- 108010077895 Sarcosine Proteins 0.000 claims 1
- 210000004369 blood Anatomy 0.000 claims 1
- 239000008280 blood Substances 0.000 claims 1
- 210000002381 plasma Anatomy 0.000 claims 1
- 210000003296 saliva Anatomy 0.000 claims 1
- 210000002966 serum Anatomy 0.000 claims 1
- 229940048098 sodium sarcosinate Drugs 0.000 claims 1
- ZUFONQSOSYEWCN-UHFFFAOYSA-M sodium;2-(methylamino)acetate Chemical compound [Na+].CNCC([O-])=O ZUFONQSOSYEWCN-UHFFFAOYSA-M 0.000 claims 1
- 206010064571 Gene mutation Diseases 0.000 abstract 1
- 230000003416 augmentation Effects 0.000 abstract 1
- 238000001514 detection method Methods 0.000 abstract 1
- 244000000010 microbial pathogen Species 0.000 abstract 1
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- 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
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
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Abstract
The present invention relates to the methods for extracting nucleic acid fast and convenient from clinical sample.It is proportionally added into clinical sample and sarcosyl, TCEP, Tris-HCl, EDTA and glycogen is added, in the first step heat: 37-50 DEG C heating 10-30 minutes, carry out second step heating again: 60-70 DEG C of heating 2-10 minutes i.e. extractable nucleic acid can be used in PCR and RPA augmentation detection pathogenic microorganism, gene mutation, special target nucleic acids etc..For this method without instrument, step is few, and the time is short, improves nucleic acid extraction efficiency.
Description
Technical field
The invention belongs to nucleic acid extraction fields, and in particular to the method for nucleic acid is extracted in clinical sample.
Background technique
In order to extract nucleic acid from clinical sample, counting method big absolutely is needed by kit and centrifuge, Er Qiecao at present
Make many and diverse, is not suitable for household, field or hospital operation.Have some nucleic acid extraction sides for exploring simplicity in the prior art
Method, for example, Myhrvold et al., (Field-deployable viral diagnostics using CRISPR-
Cas13.Science 360,444-448,2018) employed in method extract wherein add TCEP, EDTA in the sample
RNA by being used for RPA after reverse transcription.And the research of people finds the above method according to the present invention, cannot effectively extract core
Acid is for carrying out PCR (reference examples provided referring to specification embodiment part) after reverse transcription.This method is appropriate only for extracting
RNA cannot still know whether effectively extract DNA.
In addition, in Masaki Ishizawa et al. (Simple procedure of DNA isolation from
Human serum.Nucleic Acids Research, Vol.19, No.20, p5792) provide a kind of rapidly extracting sample
The method of middle DNA, wherein needing to add NaI, EDTA and N- sodium lauroyl sarcosine salt, glycerol and Tris- as carrier
HCl, adjusting pH value is 8, in 60 DEG C of incubation 15min.This method is also appropriate only for extracting DNA, cannot still know whether to extraction RNA
Effectively.Therefore, it is necessary to further research and develop that the method that nucleic acid includes DNA and RNA simple and effective can be extracted, without special
Instrument.
Summary of the invention
It is easy to operate object of the present invention is in view of the deficiencies of the prior art, provide, it is only necessary to reagent be added, do not have to by instrument
The method that device can extract nucleic acid.Inventor passes through various concentration for the method in above-mentioned Myhrvold et al.
The research of TECP, EDTA to RNA for the influence of RT-PCR amplification, determines its suitable concentration range, particularly in view of more
Sarcosyl, Tris-HCl and glycogen is added in the segregating nucleic acid from sample well.The study found that adding in reagent
Add above-mentioned substance, nucleic acid extraction efficiency can be greatly improved.
The present invention provides a kind of method for extracting nucleic acid fast and convenient from clinical sample, it is characterised in that in clinical sample
Middle addition sarcosyl, TCEP, Tris-HCl, EDTA and glycogen heat: 37-50 DEG C of heating 10-30 in the first step
Minute, then carry out second step heating: 60-70 DEG C of heating 2-10 minutes i.e. extractable nucleic acid.
Preferably, the final concentration of 10mM-1M of the TCEP of addition, preferably 10-100mM, most preferably 10mM.
Preferably, the final concentration of 0.1-0.5% (weight) of the sarcosyl of addition, preferably 0.2-
0.4% (weight), most preferably 0.3% (weight).
Wherein, the final concentration of 0.1-2mM of the EDTA of addition, preferably 0.5-1.5mM, most preferably 1mM.
Wherein, the final concentration of 10mM-25mM of the Tris-HCl of addition, preferably 15-20mM, most preferably 15mM.
Wherein, based on 100 μ l of system, the amount of the glycogen of addition is 2-10 μ g, preferably 4-8 μ g, most preferably 5 μ g.
Further, first step heating is preferably 42 DEG C in 40-45 DEG C, heats 15-25min, preferably 20min.
In addition, second step heating be in；62-66 DEG C, preferably 64 DEG C heat 3-8min, preferably 5min.
Wherein, clinical sample is blood, blood plasma, serum, urine or saliva, preferably serum.
The present invention can also can certainly by research and verifying, provided method for extracting nucleic acid effective for RT-PCR
For RPA method, DNA and RNA can be effectively extracted.Thus, it solves the deficiencies in the prior art and provides and only need to
Reagent is added, without the method by instrument with regard to easy rapidly extracting nucleic acid.
Detailed description of the invention
The influence electrophoretogram that Fig. 1 various concentration EDTA and/or TCEP expands RT-PCR.
Fig. 2 extracts RNA and post transcription cloning electrophoretogram from cow's serum.
Fig. 3 is added the substances such as sarcosyl and extracts RNA and post transcription cloning electrophoretogram from cow's serum.
Fig. 4 extracts DNA from cow's serum and expands electrophoretogram.
Fig. 5 extracts RNA and post transcription cloning electrophoretogram from blood and saliva.
Fig. 6 extracts DNA from blood and saliva and expands electrophoretogram.
RNA and post transcription cloning result are extracted in Fig. 7 known references.
Specific embodiment
Below by specific embodiment, the present invention is further illustrated, is not construed as limiting the invention.
The influence that embodiment one: TCEP and EDTA expand RT-PCR
In order to study the influence that TCEP and EDTA expand RT-PCR, following experiment is carried out, with the optimal TCEP of determination
With EDTA concentration.
The first step is separately added into 9 μ l RNA in multiple PCR pipes；
Second step is separately added into final concentration of 10mM EDTA, 1mM EDTA, 0.1mM EDTA；1M TCEP；100mM
TCEP；10mM TCEP；10mM EDTA+100mM TCEP；1mM EDTA+10mM TCEP, 1mM EDTA+100mM TCEP；
Third step, in 42 DEG C of heating 20min；64 DEG C of heating 5min；
4th step, is used for reverse transcription and PCR after processing, the electrophoresis result after PCR is as shown in Figure 1.
From experimental result it is known that TCEP reagent too high levels will affect PCR amplification efficiency.Wherein it can be concluded that being added
TCEP final concentration of 10mM-100mM, and be added the final concentration of 0.1-10mM of EDTA within the scope of be all it is feasible,
Middle TCEP too high levels then will affect subsequent PCR efficiency, but optimal concentration is still 1mM EDTA and 10mM TCEP.
Embodiment two: RNA is extracted from cow's serum with 1mM EDTA and 10mM TCEP
The nucleic acid in cow's serum is extracted as steps described below, and carries out reverse transcription and PCR
100 μ l cow's serums are added in the first step in 2ml centrifuge tube；
Final concentration of 1mM EDTA and 10mM TCEP is added in second step；
Third step, in 42 DEG C of heating 20min；64 DEG C of heating 5min；
4th step is extracted and is completed, in 95 DEG C of processing 10min inactivation of viruses, then after temperature drops to room temperature, for subsequent
RT-PCR.RT-PCR reaction step and reaction system are referring to TaKaRa company PrimeScriptTM One Step RT-PCR Kit
Ver.2 (Dye Plus) product description (article No.: RR057A), upstream primer sequence 5 '
GGGGAGTCGTCAGTGGTTCG3 ', 5 ' GTGCCATGTACAGCAGAGATT3 ' of downstream primer sequence.
Electrophoresis result after its PCR is as shown in Figure 2.
Embodiment three: with after 1mM EDTA, 10mM TCEP, sarcosyl and Tris-HCl be from cow's serum
Extract RNA
Carried out according to the step identical as embodiment two, difference be only that second step be added final concentration of 1mM EDTA and
After 10mM TCEP, sarcosyl is added and Tris-HCl, final concentration are respectively 0.3%, 15mM；Add 5 μ g sugar
Member.
Experiment in the embodiment simultaneously with embodiment two compares experiment.
Electrophoresis result after its PCR as shown in figure 3, can determine whether from band brightness, be added sarcosyl and
Tris-HCl helps to extract RNA from cow's serum.
Example IV: EDTA and TCEP is added and is conducive to extract DNA from cow's serum
It carries out according to the step identical as embodiment three and (directly carries out PCR after extracting nucleic acid), difference is only that second step
0.3% sarcosyl, 15mM Tris-HCl, 5 μ g glycogen and final concentration of 1mM EDTA and 10mM is added
TCEP。
In the embodiment simultaneously be added 0.3% sarcosyl in second step, 15mMTris-HCl, 5 μ g sugar
Member compares experiment.
Electrophoresis result after its PCR as shown in figure 4, can determine whether from band brightness, wherein be added have 1mM EDTA and
10mM TCEP helps to extract DNA from cow's serum.
Embodiment five: RNA is extracted from blood and saliva
The mRNA of blood and saliva β-actin gene is extracted according to the step identical as embodiment three, for subsequent
Reverse transcription and PCR.Reverse transcription system and step are referring to HaiGene company Golden 1st cDNA Synthesis Kit reagent
(product article No.: D0401, the gDNA Remover component of the product is directly degraded removes remaining genomic DNA dirt in RNA to box
Dye).PCR reaction step and reaction system are referring to TaKaRa company's T aqTMVersion 2.0plus dye product description (goods
Number: RR901A), 5 ' TCCATCCTGGCCTCGCTGT 3 ' of upstream primer sequence, downstream primer sequence 5 '
GCTGTCACCTTCACCGTTC 3'.Electrophoresis result after its PCR is as shown in Figure 5.
Embodiment six: DNA is extracted from blood and saliva
The DNA of GAPDH gene in blood and saliva is extracted according to the step identical as example IV, for subsequent
PCR.PCR reaction step and reaction system are referring to TaKaRa company's T aqTMVersion 2.0plus dye product description (goods
Number: RR901A), 5 ' GTTCTTTGAAAACTGAAT 3 ' of upstream primer sequence, downstream primer sequence 5 '
GCATCCACCAAAAACTCT 3'.Electrophoresis result after its PCR is as shown in Figure 6.
Comparative examples
Method according to known references report is to extract nucleic acid (Myhrvold et al., Science 360,444-448
(2018) method employed in):
100 μ l cow's serums are added in the first step in 2ml centrifuge tube；
Second step, is separately added into TCEP, EDTA, and final concentration is respectively 100mM, 1mM；
Third step, 42 DEG C of heating 20min；64 DEG C of heating 5min；
4th step is used for subsequent RT-PCR by the step in embodiment one in 95 DEG C of processing 10min after the completion of extraction.Its
Electrophoresis result fails effectively to extract nucleic acid as shown in fig. 7, it does not amplify bands visible.It can be seen that based on above-mentioned known
The nucleic acid effect of method, extraction is undesirable, cannot detect effective for RT-PCR.
As seen from the above embodiment, it is unsatisfactory that nucleic acid is directly extracted according to known methods, experiments prove that in document
100mM TCEP excessive concentration influence PCR amplification, cannot be effective for PCR amplification.And by means of the present invention, energy
It is enough effectively to extract RNA, after reverse transcription, it is able to carry out effective PCR amplification, band is high-visible, shows this hair
Bright method is practical.
Claims (10)
1. a kind of method for extracting nucleic acid fast and convenient from clinical sample, it is characterised in that dodecane is added in clinical sample
Base sodium sarcosinate, TCEP, Tris-HCl, EDTA and glycogen are heated in the first step: 37-50 DEG C heating 10-30 minutes, then carry out
Second step heating: 60-70 DEG C of heating 2-10 minutes i.e. extractable nucleic acid.
2. the method according to claim 1, wherein the final concentration of 10mM-1M, preferably 10- of the TCEP being added
100mM, most preferably 10mM.
3. the method according to claim 1, wherein the final concentration of 0.1- for the sarcosyl being added
0.5% (weight), preferably 0.2-0.4% (weight), most preferably 0.3% (weight).
4. the method according to claim 1, wherein the EDTA final concentration of 0.1-10mM, preferably 0.5- that are added
1.5mM, most preferably 1mM.
5. according to the method described in claim 5, it is characterized in that, the final concentration of 10mM-25mM for the Tris-HCl being added, excellent
Select 15-20mM, most preferably 15mM.
6. according to the method described in claim 5, it is characterized in that, the final concentration of 2-10 μ g/100ul for the glycogen being added, excellent
Select 4-8 μ g/100ul, most preferably 5 μ g/100ul.
7. being heated the method according to claim 1, wherein first step heating is preferably 42 DEG C in 40-45 DEG C
15-25min, preferably 20min.
8. the method according to claim 1, wherein second step heating be in；62-66 DEG C, preferably 64 DEG C, heating
3-8min, preferably 5min.
9. method according to any one of claims 1 to 8, which is characterized in that clinical sample be blood, blood plasma, serum or
Saliva.
10. according to the method described in claim 9, it is characterized in that, the nucleic acid is RNA or/and DNA.
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| CN201811153723.XA CN109182332A (en) | 2018-09-30 | 2018-09-30 | A method of the fast and convenient extraction nucleic acid from clinical sample |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111254141A (en) * | 2020-04-28 | 2020-06-09 | 博奥生物集团有限公司 | Nucleic acid extraction composition, application thereof, reagent containing nucleic acid extraction composition and kit |
| CN112280904A (en) * | 2020-11-25 | 2021-01-29 | 中国人民解放军军事科学院军事医学研究院 | A method for rapid detection of novel coronavirus nucleic acid |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111254141A (en) * | 2020-04-28 | 2020-06-09 | 博奥生物集团有限公司 | Nucleic acid extraction composition, application thereof, reagent containing nucleic acid extraction composition and kit |
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