CN105734044A - Rinsing liquid for nucleic acid extraction purification - Google Patents
Rinsing liquid for nucleic acid extraction purification Download PDFInfo
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- CN105734044A CN105734044A CN201410740622.8A CN201410740622A CN105734044A CN 105734044 A CN105734044 A CN 105734044A CN 201410740622 A CN201410740622 A CN 201410740622A CN 105734044 A CN105734044 A CN 105734044A
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Abstract
The invention discloses a rinsing liquid for nucleic acid extraction purification. The rinsing liquid comprises a buffer reagent, polyethylene glycol and a salt. The rinsing liquid reduces cross contamination in sample nucleic acid extraction purification, is free of an air drying process in extraction, reduces nucleic acid extraction purification time and improves nucleic acid extraction purification efficiency. A preparation method of the rinsing liquid has simple processes and can be operated easily.
Description
Technical field
The present invention relates to a kind of rinsing liquid, particularly relate to a kind of rinsing liquid for nucleic acid extraction purification.
Background technology
Nucleic acid is one of most basic material of life, comprise DNA (deoxyribonucleic acid) (DNA) and ribonucleic acid (RNA) two class, it is widely present in all life bodies such as animal and plant cells, antibacterial, virus, plays a part to store and convey hereditary information.
Along with nucleic acid for detecting the development of the molecular diagnostic techniques of object, detection of nucleic acids has been widely used for the fields such as Clinical Laboratory, inspection and quarantining for import/export, forensic.One of key link of detection of nucleic acids, namely extracts from various biological samples and the detection of nucleic acids success in downstream is played vital effect by purification of nucleic acid, nucleic acid extraction efficiency and purity.
The extraction purification of nucleic acid is primarily referred to as and is separated with biomacromolecule materials such as protein, polysaccharide, fat by nucleic acid.The method of nucleic acid extraction purification experienced by a period of time development, carry out manual extraction process from organic solvents such as traditional employing phenol/chloroforms gradually and develop into the centrifugal column method adopting pellosil absorption and the method that employing paramagnetic particle method carries out full-automatic nucleic acid extraction purification.
Paramagnetic particle method nucleic acid extraction purification process, generally with nano silicon-based magnetic bead for carrier, adsorbs nucleic acid by magnetic bead in high level salt solution and carries out nucleic acid extraction purification in low salt solutions amplifying nucleic acid from the principle of magnetic bead surfaces desorption.Owing to nanometer magnetic bead has the features such as bigger serface, selective absorption nucleic acid, superparamagnetism, quick magnetic response and physico-chemical property be stable so that it is be highly suitable for automatization.The biotech company that the whole world is famous, as the companies such as Roche, Qiagen, Thermo, Chemagen all have the nucleic acid extracting reagent based on paramagnetic particle method principle and full-automatic extraction purification system on sale.
The method of paramagnetic particle method nucleic acid extraction purification generally comprises lysis release nucleic acid and magnetic bead absorption nucleic acid, rinsing is adsorbed with the magnetic bead of nucleic acid, by nucleic acid from the several key steps eluted magnetic bead.According to existing open source literature data it can be seen that the method for lysis at present is typically based on the difference of sample type, mechanism, the mode such as chemical action and ferment treatment can be adopted.Mechanism, namely includes the physical disruption methods such as hypotonic lysis, ultrasonic degradation, microwave cracking, freezing-thawing and cracking and grain breakage；Chemical action, namely under certain pH environment and Denaturing, cell rupture, protein denaturation precipitation, nucleic acid is released to aqueous phase.Denaturing can obtain by heating, add surfactant (SDS, TritonX-100, Tween20, NP-40, CTAB etc.) or strong ionic agent (guanidinium isothiocyanate, guanidine hydrochloride).PH environment is then provided by the highly basic (NaOH) added or buffer (TE, STE etc.).Under certain pH environment, surfactant or strong ionic agent can make lysis, protein and polysaccharide precipitation, some metal ion chelation agents (EDTA etc.) in buffer can chelating to metal ions M g necessary to nuclease2+、Ca2+, thus suppressing the activity of nuclease, protection nucleic acid is not degraded；Enzyme effect mainly by adding lysozyme or protease so that cell rupture, nucleic acid discharges, as in lysate add protease can protein degradation matter, inactivation nuclease (DNase and RNase), add DNase or RNase be also used for removing unwanted nucleic acid.
Nucleic acid is adsorbed for magnetic bead, according to existing known open source literature it can be seen that in high level salt solution, nucleic acid absorption is to nano silicon-based magnetic bead surfaces, and chaotropic salt sodium iodide or sodium perchlorate can promote the combination of nucleic acid and silica-based magnetic bead.Chinese patent " the paramagnetic particle method method for extracting nucleic acid of a kind of enhancing " (patent No.: 201110124322.3) describes a kind of cracking containing isopropanol with in conjunction with liquid, and wherein isopropanol acts the effect strengthening magnetic bead absorption nucleic acid.
For the rinsing of magnetic bead, its objective is to wash away the impurity such as protein and lipid of non-specific binding on magnetic bead, and reduce the loss of the nucleic acid being adsorbed on magnetic bead surfaces as far as possible, so that the follow-up nucleic acid purity eluted from magnetic bead surfaces is higher.Presently commercially available paramagnetic particle method nucleic acid extracting reagent, the rinsing liquids adopted containing ethanol or isopropanol carry out rinsing 1～2 time more, the formula of different company's reagent is likely to different, but substantially containing the composition such as ethanol and isopropanol, especially a step before carrying out eluting often adopts 60%～75% ethanol or isopropanol to rinse.Can referring to the description of following Chinese patent application and patent: " nucleic acid extraction purification process and test kit based on nanometer magnetic bead " (application number: 201410062784.0), " the paramagnetic particle method method for extracting nucleic acid of a kind of enhancing " (patent No.: 201110124322.3)；" a kind of method of extraction purification DNA " (patent No.: 200910090336.0)；" test kit of paramagnetic particle method extraction bacteria plasmid DNA and extracting method thereof " (patent: 201110204053.1) etc..Owing to follow-up molecular biology is reacted (such as PCR by the residual of known ethanol and isopropanol, enzyme action etc.) significantly inhibit effect, so, after adopting ethanol or rinsing liquid that isopropanol is key component that magnetic bead is rinsed, need that magnetic bead carries out " drying " to process 2～15 minutes, make the ethanol of magnetic bead surfaces or isopropanol fully volatilize.Owing to dry process adds the open-assembly time of magnetic bead, the stability of the nucleic acid of extraction may be affected, additionally, some commercially available automatization's extraction apparatuses are in order to accelerate the volatilization of ethanol or isopropanol, fan with heating function is set, making periphery of magnetic beads air accelerate circulation, the nucleic acid product which increasing extraction forms aerosol and produces the risk of cross-contamination between sample.
For nucleic acid from eluting magnetic bead, the buffer being generally adopted without or containing very low concentrations salinity, for instance TE (pH8.0) or Tris-HCl (pH8.0) etc., also have and directly adopt pure water as the test kit of eluent.In order to improve elution efficiency, often adopting heating type of elution, general heating is preferred to 50～60 DEG C of eluting effects.The nucleic acid extraction purified product of eluting can be directly used for follow-up molecular biology experiment analysis, such as PCR, RT-PCR, enzyme action, order-checking etc..If extracting product not use immediately, should being placed under-20 DEG C of conditions and storing, long-term preservation should be placed under-70 DEG C of conditions.
But, in order to reduce the chance of cross-contamination in sample nucleic acid extraction purification process, " drying " step saved in extraction process, the time reducing nucleic acid extraction purification and improve the efficiency etc. of nucleic acid extraction purification, in addition it is also necessary to the rinsing liquid for nucleic acid extraction purification is carried out necessity of research further.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of rinsing liquid for nucleic acid extraction purification.This rinsing liquid is without ethanol or isopropanol, thus is a kind of rinsing liquid processing step in nucleic acid extraction purification without carrying out " drying ".Thus, the rinsing liquid using the present invention has the advantages such as the efficiency of the chance of cross-contamination in minimizing sample nucleic acid extraction purification process, " drying " step saved in extraction process, the time reducing nucleic acid extraction purification and raising nucleic acid extraction purification, and for this rinsing liquid, its preparation method is simple and easily operated.
For solving above-mentioned technical problem, the rinsing liquid for nucleic acid extraction purification of the present invention, it comprises: buffer reagent, Polyethylene Glycol and salt.
The pH of described rinsing liquid is preferably 6.0～7.0.
Described buffer reagent includes: HEPES buffer or Tris-HCl buffer.Preferably, described HEPES buffer is the HEPES buffer of 10mM～200mM, and its pH is 6.0～7.0；Tris-HCl buffer is the Tris-HCl buffer of 10mM～200mM, and its pH is 6.0～7.0.
Described Polyethylene Glycol is to be formed with water or ethylene glycol progressively addition polymerization by oxirane, and Polyethylene Glycol product is nontoxic, nonirritant, mildly bitter flavor, has good water solublity, and has good intermiscibility with many organic matter components.In the present invention, it is preferred to the Polyethylene Glycol that molecular weight is 6000 or 8000, and the concentration range that Polyethylene Glycol is in rinsing liquid is preferably 5g/100mL～20g/100mL.
Described salt is preferably inorganic salt, including: sodium chloride or potassium chloride.This inorganic salt concentration in rinsing liquid is preferably 0.1M～0.5M.
The rinsing liquid of the present invention, is an important component part in paramagnetic particle method nucleic acid extraction purified reagent, and it can use with other agents coordinate in nucleic acid extraction purified reagent, to play the effect of nucleic acid extraction purification.
Therefore, the present invention also provides for a kind of test kit for nucleic acid extraction purification, comprising:
(1) cracking of nucleic acid with in conjunction with liquid, comprising: chaotropic salt, strong denaturant, detergent and strengthen the reagent etc. of magnetic bead absorption nucleic acid ability；
Wherein, chaotropic salt includes: the sodium chloride of 0.5M～3.0M, sodium perchlorate or sodium iodide etc.；
Strong denaturant includes: guanidinesalt etc.；
Detergent includes: SDS or Triton-X100 etc.；
The reagent strengthening magnetic bead absorption nucleic acid ability includes: ethanol or isopropanol etc..
Cracking herein is with in conjunction with liquid, and as described in background technology, it is for cracking tissue in biological specimen, cell, antibacterial, virus etc., and discharges nucleic acid, promotes that nucleic acid adsorbs with magnetic bead.
(2) pre-rinses, it contains above-mentioned chaotropic salt, strong denaturant, detergent and strengthens the reagent of magnetic bead absorption nucleic acid ability；In the present invention, in order to obtain highly purified nucleic acid, it is generally adopted repeatedly rinsing and is adsorbed with the step of the magnetic bead of nucleic acid, except rinsing liquid provided by the invention, it is also possible to adopt this pre-rinses；
(3) the above-mentioned rinsing liquid of the present invention；
(4) eluent；
For eluent, it is therefore an objective to make the Nucleic Acid Elution being combined in magnetic bead surfaces get off, the buffer being generally without or containing low concentration of salt, as included: TE buffer, Tris-HCl buffer or pure water.Wherein, this TE buffer can be the pH8.0 buffer (i.e. 10mMTris-HCl, 1mMEDTA, pH8.0) containing 10mMTris-HCl, 1mMEDTA, Tris-HCl buffer is the pH8.0 buffer (10mMTris-HCl, pH8.0) containing 10mMTris-HCl.
For mentioned reagent box, the rinsing liquid of the present invention, in scope of the present invention, the concentration of its component may according to the use of cracking be adjusted from conjunction with the different of liquid, pre-rinses and eluent so that the overall performance of extraction purification test kit reaches the best.
Through test, the rinsing liquid of the present invention, the extraction purification for DNA (deoxyribonucleic acid) (DNA) and the big class nucleic acid of ribonucleic acid (RNA) two all has excellent performance.Owing to there is no ethanol or isopropanol, the step that this rinsing liquid processes without " drying " in extraction purification process.Dry step owing to eliminating, decrease the overall time of extraction purification, the chance being greatly reduced between sample cross-contamination, eliminate ethanol that may be present or the isopropanol residual inhibitory action to subsequent reactions, improve extraction efficiency.
Further, since ethanol and isopropanol belong to the hazardous chemical very easily fired, the risk in production, transport and use procedure is very big, and adopts rinsing liquid provided by the invention, and in it, component is general chemicals, without security risk hidden danger.
Furthermore, use the rinsing liquid of the present invention can be substantially reduced production cost.
Accompanying drawing explanation
Below in conjunction with accompanying drawing, the present invention is further detailed explanation with detailed description of the invention:
Fig. 1 is that HBV (hepatitis B virus) international standard substance (1000IU/ml) adopts rinsing liquid of the present invention and without the amplification figure drying step extraction product, wherein, S2～S8 is HBV plasmid standards for quantitation, its concentration respectively S2:3.0+E2IU/ml；S4:3.0+E4IU/ml；S6:3.0+E6IU/ml；S8:3.0+E8IU/ml, the implication of " S2～S8 " in lower Fig. 2～5 is identical with Fig. 1；It addition, the implication of 1E3 in Fig. 1 and HBV theoretical concentration are 1.0E+03IU/mL (1000IU/ml), the method for expressing of other figure is identical with this.
Fig. 2 is that HBV international standard substance (100IU/ml) adopts rinsing liquid of the present invention and without the amplification figure drying step extraction product, and wherein, namely " 1E2 " in Fig. 2 refer to HBV international standard substance (100IU/ml)；
Fig. 3 is that HBV international standard substance (50IU/ml) adopts rinsing liquid of the present invention and without the amplification figure drying step extraction product；
Fig. 4 is that HBV international standard substance (20IU/ml) adopts rinsing liquid of the present invention and without the amplification figure drying step extraction product；
Fig. 5 is that HBV international standard substance (10IU/ml) adopts rinsing liquid of the present invention and without the amplification figure drying step extraction product；
Fig. 6 is that HCV (hepatitis C virus) international standard substance (1000IU/ml) adopts rinsing liquid of the present invention and without the amplification figure drying step extraction product, wherein, S2～S8 is HCV plasmid standards for quantitation, its concentration respectively S2:3.0+E2IU/ml；S4:3.0+E4IU/ml；S6:3.0+E6IU/ml；S8:3.0+E8IU/ml, the implication of " S2～S8 " in lower Fig. 7～10 is identical with Fig. 6；It addition, namely " 1E3 " in Fig. 6 refer to HCV (hepatitis C virus) international standard substance (1000IU/ml)；" NTC " is negative control；
Fig. 7 is that HCV international standard substance (100IU/ml) adopts rinsing liquid of the present invention and without the amplification figure drying step extraction product；
It addition, namely " 1E2 " in Fig. 7 refer to HCV (hepatitis C virus) international standard substance (100IU/ml)；" NTC " in Fig. 7 is negative control；
Fig. 8 is that HCV international standard substance (50IU/ml) adopts rinsing liquid of the present invention and without the amplification figure drying step extraction product；Wherein, " NTC " in Fig. 8 is negative control；
Fig. 9 is that HCV international standard substance (30IU/ml) adopts rinsing liquid of the present invention and without the amplification figure drying step extraction product；Wherein, " NTC " in Fig. 9 is negative control；
Figure 10 is that HCV international standard substance (15IU/ml) adopts rinsing liquid of the present invention and without the amplification figure drying step extraction product.Wherein, " NTC " in Figure 10 is negative control.
Detailed description of the invention
The term used in the present invention, except as otherwise noted, generally has the implication that those of ordinary skill in the art are generally understood that.Also with reference to data, the present invention is described in further detail below in conjunction with specific embodiment.Should be understood that these embodiments present invention solely for the purpose of illustration, but not the scope being intended to limit the present invention in any manner.
The application in hepatitis B virus (HBV) nucleic acid quantification detects of embodiment 1. rinsing liquid of the present invention
The present embodiment adopt rinsing liquid provided by the invention replace the rinsing liquid in commercially available HBV nucleic acid quantification reagent, that saves rinsing liquid in nucleic acid extraction process dries step, HBV international standard substance is carried out detection by quantitative, judging that whether rinsing liquid provided by the invention is feasible from the accurately fixed of testing result and sensitivity two aspect, concrete research material is as follows:
(1) " hbv nucleic acid detection kit (fluorescence quantitative PCR method) " (the medical apparatus and instruments registration certificate number: state's food medicine prison tool (standard) word 2013 the 3402077th) that Shanghai Haoyuan Biotechnology Co., Ltd. produces；This product comprises nucleic acid extracting reagent, HBV nucleic acid amplification agents and plasmid standards for quantitation and reference substance, and wherein nucleic acid extracting reagent comprises suspension containing magnetic beads, extract, washing liquid A (pre-rinses), washing liquid B (rinsing liquid) and eluent；Nucleic acid amplification agents comprises fluorescence HBV reactant liquor A and fluorescence HBV reactant liquor B；Plasmid standards for quantitation and reference substance comprise plasmid standards for quantitation S2～S8 and yin and yang attribute comparison.
(2) rinsing liquid provided by the invention: concrete, its formula is HEPES (20mM, pH6.5), PEG6000 (13%, i.e. 13g/100ml), NaCl (0.3M)；
(3) HBV nucleic acid quantification international standard substance NIBSC10/264 (8.5E+05IU/mL, GenotypeA), from United Kingdom National biological products assay institute (NationalInstituteforBiologicalStandardsandControl, NIBSC)；
(4) HBV feminine gender human normal plasma；
(5) the EZ-Beadsystem-32 instrument for extracting nucleic acid of U.S. TBG company；
(6) the grand stone SLAN-96P quantitative real time PCR Instrument in Shanghai.
Concrete research method:
By HBV nucleic acid quantification international standard substance NIBSC10/264 (8.5E+05IU/mL, GenotypeA) serial dilution is carried out with blood plasma negative for HBV and serum respectively, the sample of 1.0E+03IU/mL, 1.0E+02IU/mL, 5.0E+01IU/mL, 2.0E+01IU/mL, 1.0E+01IU/mL concentration is carried out 8 replications respectively, and accuracy requirement is that the logarithm value of quantitative values differs less than ± 0.45lg scope with the logarithm value of standard substance theoretical value；Sensitivity requirement all should all detect in minimum detectability concentration.According to product description operation, and rinsing liquid provided by the invention being replaced " the washing liquid B " in test kit, delete drying step in extraction procedure, concrete operation step is as follows:
1. the extraction of the HBV plasmid standards for quantitation after adopting the EZ-Beadsystem-32 instrument for extracting nucleic acid of TBG company of the U.S. to be diluted:
Nucleic acid extracting reagent, reference substance, plasmid standards for quantitation are taken out by 1.1, and balance is to room temperature and mixes.
1.2 at sample preparation pre-subpackage reagent of according to the form below 1 layout in 96 hole depth reaction disk of the hole:
Table 1. reaction tray reagent subpackage layout table
Negative control in the 1.3 HBV nucleic acid quantification standard substance taking gradient dilution to be measured and test kit, weak positive control, robust positive control, each 200 μ l of plasmid standards for quantitation S8～S2 join in reaction tray A2～H2 or the A8～H8 hole of pre-subpackage reagent.Every deblocking reaction dish can process 16 samples.Every instrument can be put into 2 reaction tray simultaneously and process 32 samples.
1.4 A6～H6 and the A12～H12 row bottoms that bonding jumper is directed at reaction tray, and reaction tray and bonding jumper are fixed on EZbeadSystem-32 instrument for extracting nucleic acid, according to the form below 2 program performs, and wherein dries Step Time and is disposed as 0, namely deletes and dry step.
Table 2EZbead nucleic acid extraction program
1.5 after EP (end of program), takes off reaction tray, draws eluted product and carries out pcr amplification reaction.
2. adopt the grand stone SLAN-96P quantitative real time PCR Instrument in Shanghai to expand
The 2.1 total systems of amplification are 60 μ l, and wherein fluorescence HBV reactant liquor A and fluorescence HBV reactant liquor B is respectively 10 μ l, nucleic acid extraction product 40 μ l.Pcr amplification reaction pipe use Axygen0.2ml flat cover eight union, CatNo: pipe PCR-0208-C, lid PCR-2CP-RT-C.
2.2 amplification programs are following table, and wherein " ★ " place is fluorescence signal acquisition point, and acquisition channel is set to FAM and ROX.
3. data analysis
In Table 3, HBV international standard substance detection by quantitative data and statistical analysis.Can be seen that from accompanying drawing 1～5 and table 3, the HBV international standard substance concentration of serial dilution recall rate from 20IU/ml to 1000IU/ml all reaches 100% (8/8), amplification curve is in typical " S " type, and the logarithm value of the logarithm value and standard substance theoretical value all meeting quantitative values differs the requirement less than ± 0.45lg scope.Additionally, especially interesting it is, under the lowest detection lower limit 20IU/ml that this HBV nucleic acid quantification test kit is claimed, increase the standard substance of detection 8 example 10IU/ml concentration values, its recall rate also can reach 100% (8/8), and prompting adopts the rinsing liquid of the present invention to significantly improve the detection sensitivity of reagent.In a word, without drying step after above-mentioned experiment prompting employing rinsing liquid of the present invention rinsing, and it is directly entered eluting link, the amplification efficiency of its eluted product is very good, prove to adopt the rinsing liquid without drying process step provided by the invention to be applicable to this HBV nucleic acid quantitative determination reagent kit, and show the analytical performance of excellence.
Table 3HBV international standard substance detection by quantitative data and statistical analysis
Note: the unit of the HBV plasmid standards for quantitation concentration in table 3 is " IU/mL "；Ct is period；The unit of measured concentration is " IU/mL "；NTC (NoneTemplateControl) is no template control, i.e. negative control；Namely Noct without amplification, is not detected by purpose target.
The application in hepatitis C virus (HCV) nucleic acid quantification detects of embodiment 2 rinsing liquid of the present invention
The present embodiment adopts rinsing liquid provided by the invention to replace the rinsing liquid in commercially available HCV nucleic acid quantification reagent, HCV international standard substance is carried out detection by quantitative by the step of drying saving rinsing liquid in nucleic acid extraction process, judging that whether rinsing liquid provided by the invention is feasible from the accurately fixed of testing result and sensitivity two aspect, concrete research material is as follows:
(1) " hepatitis C virus nucleic acid detection kit (fluorescence quantitative PCR method) " (the medical apparatus and instruments registration certificate number: state's food medicine prison tool (standard) word 2011 the 3400554th) that Shanghai Haoyuan Biotechnology Co., Ltd. produces；This product comprises nucleic acid extracting reagent, HCV nucleic acid amplification agents and plasmid standards for quantitation and reference substance, and wherein nucleic acid extracting reagent comprises suspension containing magnetic beads, extract, washing liquid A (pre-rinses), washing liquid B (rinsing liquid) and eluent；Nucleic acid amplification agents comprises fluorescence HCV reactant liquor A, fluorescence HCV reactant liquor B and reaction liquid C；Plasmid standards for quantitation and reference substance comprise plasmid standards for quantitation S2～S8 and yin and yang attribute comparison.
(2) rinsing liquid provided by the invention: concrete, its formula is HEPES (20mM, pH6.5), PEG6000 (13%, i.e. 13g/100ml), NaCl (0.3M)；
(3) HCV nucleic acid quantification international standard substance NIBSC06/102 (2.6E+05IU/mL, Genotype1a), from United Kingdom National biological products assay institute (NationalInstituteforBiologicalStandardsandControl, NIBSC)；
(4) HCV feminine gender human normal plasma；
(5) the EZ-Beadsystem-32 instrument for extracting nucleic acid of U.S. TBG company；
(6) the grand stone SLAN-96P quantitative real time PCR Instrument in Shanghai.
Concrete research method:
By HCV nucleic acid quantification international standard substance NIBSC06/102 (2.6E+05IU/mL, Genotype1a) serial dilution is carried out with blood plasma negative for HCV and serum respectively, the sample of 1.0E+03IU/mL, 1.0E+02IU/mL, 5.0E+01IU/mL, 3.0E+01IU/mL, 1.5E+01IU/mL concentration is carried out several times replication respectively, and accuracy requirement is that the logarithm value of quantitative values differs less than ± 0.45lg scope with the logarithm value of standard substance theoretical value；Sensitivity requirement all should all detect in minimum detectability concentration.According to product description operation, and rinsing liquid provided by the invention being replaced " the washing liquid B " in test kit, delete drying step in extraction procedure, concrete operation step is as follows:
1. nucleic acid extraction link is identical with embodiment 1.
2. adopt the grand stone SLAN-96P quantitative real time PCR Instrument in Shanghai to expand
2.1 to expand total systems be 62 μ l, and wherein fluorescence HCV reactant liquor A is 10 μ l, fluorescence HCV reactant liquor B be 9 μ l, HCV reaction liquid C is 3 μ l, nucleic acid extraction product 40 μ l.Pcr amplification reaction pipe use Axygen0.2ml flat cover eight union, CatNo: pipe PCR-0208-C, lid PCR-2CP-RT-C.
2.2 amplification programs are following table, and wherein " ★ " place is fluorescence signal acquisition point, and acquisition channel is set to FAM and HEX.
3. data analysis
Table 4HCV international standard substance detection by quantitative data and statistical analysis
Note: the NTC (NoneTemplateControl) in table 4 is no template control, i.e. negative control；Namely Noct without amplification, is not detected by purpose target.
Can be seen that from accompanying drawing 6～10 and table 4, the HCV international standard substance concentration of serial dilution recall rate from 30IU/ml to 1000IU/ml all reaches 100%, amplification curve is in typical " S " type, and the logarithm value of the logarithm value and standard substance theoretical value all meeting quantitative values differs the requirement less than ± 0.45lg scope.Additionally, especially interesting, under the lowest detection lower limit 50IU/ml that this HCV nucleic acid quantification test kit is claimed, increase the standard substance of detection 8 example 30IU/ml and 8 example 15IU/ml concentration values, wherein 30IU/ml recall rate also can reach 100% (8/8), and 15IU/ml recall rate also can reach 87.5% (7/8), prompting adopts the rinsing liquid of the present invention to significantly improve the detection sensitivity of reagent.In a word, without drying step after above-mentioned experiment prompting employing rinsing liquid of the present invention rinsing, and it is directly entered eluting link, the amplification efficiency of its eluted product is very good, prove to adopt the rinsing liquid without drying process step provided by the invention to be applicable to this HCV nucleic acid quantitative determination reagent kit, and show the analytical performance of excellence.
In a word, rinsing liquid for the present invention, due to different with the rinsing liquid containing ethanol or isopropanol commonly used at present, it does not contain inhibiting component to subsequent reactions, so without drying step in extraction purification step, such that it is able to shorten extraction time, and decrease and dry the chance of cross-contamination between step sample.
In addition, it is necessary to it is to be noted that: above-described embodiment only for technology design and the feature of the present invention are described, its object is to allow person skilled in the art will appreciate that present disclosure and to implement according to this, can not limit the scope of the invention with this.All equivalences made according to spirit of the invention change or modify, and all should be encompassed within protection scope of the present invention.
Claims (10)
1. the rinsing liquid for nucleic acid extraction purification, it is characterised in that comprise: buffer reagent, Polyethylene Glycol and salt.
2. rinsing liquid as claimed in claim 1, it is characterised in that: the pH of described rinsing liquid is 6.0～7.0.
3. rinsing liquid as claimed in claim 1, it is characterised in that: described buffer reagent includes: HEPES buffer or Tris-HCl buffer.
4. rinsing liquid as claimed in claim 3, it is characterised in that: described HEPES buffer is the HEPES buffer of 10mM～200mM, and its pH is 6.0～7.0；
Tris-HCl buffer is the Tris-HCl buffer of 10mM～200mM, and its pH is 6.0～7.0.
5. rinsing liquid as claimed in claim 1, it is characterised in that: described Polyethylene Glycol is the Polyethylene Glycol of 6000 or 8000.
6. rinsing liquid as claimed in claim 1, it is characterised in that: described Polyethylene Glycol concentration range in rinsing liquid is 5g/100mL～20g/100mL.
7. rinsing liquid as claimed in claim 1, it is characterised in that: described salt is inorganic salt；
This inorganic salt concentration in rinsing liquid is 0.1M～0.5M.
8. rinsing liquid as claimed in claim 7, it is characterised in that: described salt includes: sodium chloride or potassium chloride.
9. the test kit for nucleic acid extraction purification, it is characterised in that including:
(1) cracking of nucleic acid with in conjunction with liquid, comprising: chaotropic salt, strong denaturant, detergent and strengthen the reagent of magnetic bead absorption nucleic acid ability；
Wherein, chaotropic salt includes: the sodium chloride of 0.5M～3.0M, sodium perchlorate or sodium iodide；
Strong denaturant includes: guanidinesalt；
Detergent includes: SDS or Triton-X100；
The reagent strengthening magnetic bead absorption nucleic acid ability includes: ethanol or isopropanol；
(2) pre-rinses, it contains above-mentioned chaotropic salt, strong denaturant, detergent and strengthens the reagent of magnetic bead absorption nucleic acid ability；
(3) rinsing liquid as described in any one of claim 1～8；
(4) eluent, comprising: TE buffer, Tris-HCl buffer or pure water.
10. test kit as claimed in claim 9, it is characterised in that: described TE buffer is the pH8.0 buffer containing 10mMTris-HCl, 1mMEDTA；
Tris-HCl buffer is the pH8.0 buffer containing 10mMTris-HCl.
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| CN109897851A (en) * | 2019-04-12 | 2019-06-18 | 武汉科技大学 | Small molecule DNA purified reagent |
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| CN114507662A (en) * | 2022-03-22 | 2022-05-17 | 合肥欧创基因生物科技有限公司 | Binding solution and supercoiled plasmid large-extraction kit based on same |
| CN114657231A (en) * | 2022-04-27 | 2022-06-24 | 珠海宝锐生物科技有限公司 | Rapid DNA extraction kit with magnetic beads for fluorescent quantitative PCR detection and extraction method thereof |
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