CN107090505A - A kind of buffer system of improvement applied to PCR or RT PCR and application - Google Patents
A kind of buffer system of improvement applied to PCR or RT PCR and application Download PDFInfo
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- CN107090505A CN107090505A CN201710322610.7A CN201710322610A CN107090505A CN 107090505 A CN107090505 A CN 107090505A CN 201710322610 A CN201710322610 A CN 201710322610A CN 107090505 A CN107090505 A CN 107090505A
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- pcr
- buffer system
- improvement
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
Abstract
The invention discloses a kind of buffer system of the improvement applied to PCR or RT PCR, the buffer system of the improvement is mutually mixed and formed for 3 (N morpholines) propane sulfonic acid, Tris and trisodium citrates, and the solution state is alkalescence；Described 3 (N morpholines) propane sulfonic acid：Tris：The mol ratio of trisodium citrate is 20 100：10‑50：15, the pH of the buffer system of the improvement is 8.3 9.The buffering stability of the present invention is stronger, and amplification efficiency is higher, and detection sensitivity and precision are good.
Description
Technical field
The present invention relates to Molecular Detection field there is provided a kind of buffer system of improvement applied to PCR or RT-PCR and
Using.
Background technology
At present, round pcr is widely used in biology, medical treatment, the field such as food.One stable PCR or RT- are provided
PCR buffer systems are the key factors that various enzymes are efficiently carried out during PCR.Tris-HCl buffer systems are most classical PCR
Working reaction system, ensures the function such as pH stability in PCR work.But Tris-HCl buffer solutions are a kind of dual-polarized ions
Buffer solution, pKa is 8.3 (20 DEG C), and △ pKa are 0.021/ DEG C, and relative temperature is more sensitive, is reduced with the rise pH of temperature.
Under typical thermal cycle conditions, when elongating temperature is at 72 DEG C, real pH value between 6.8-7.8, pH be less than 7.5 with
Under, buffer capacity extreme difference.For many thermal starting enzymes, pH changes cause enzymatic activity to decline or even inactivate, and influence PCR or RT-
PCR detection sensitivity and precision.
In order to be improved to prior art, people have carried out long-term exploration, it is proposed that various solutions.
For example, Chinese patent literature discloses a kind of PCR amplification buffer [application numbers of high GC content gene：CN 105861491
A], the invention discloses a kind of PCR amplification buffers of high GC content gene.The PCR amplification bufferings of the high GC content gene
Liquid includes following component：Tris-Hcl 150mmol/L、(NH4)2SO440mmol/L, a water glycine betaine 2.6mol/L, emulsifying agent
Tween 20 0.02%, PCR increased responses agent 20%, with postponing, cross elimination bacterium, -20 DEG C of preservations.Invention additionally discloses one kind
The amplification method of PCR amplification buffers based on high GC content gene and application.Present invention uses (NH4)2SO4It instead of
KCL, to increase the specificity that primer is combined with template, and can compatible broader spectrum of Mg2+ scopes.
Although such scheme solves the deficiencies in the prior art to a certain extent, global design is reasonable not enough.
At present, PCR or RT-PCR buffer systems and the application of a kind of good improvement of detection sensitivity are still lacked.
The content of the invention
Regarding the issue above, the present invention provides a kind of detection sensitivity it is good be applied to PCR or RT-PCR
Improvement buffer system and application.
To reach above-mentioned purpose, present invention employs following technical proposal：One kind of the present invention is applied to PCR or RT-PCR
Improvement buffer system, the buffer system of the improvement mutually mixes for 3- (N- morpholines) propane sulfonic acid, Tris and trisodium citrate
Conjunction is formed；3- (N- morpholines) propane sulfonic acid：Tris：The mol ratio of trisodium citrate is 20-100：10-50：1-5.
Further, the solution state is alkalescence.
Further, the pH of the buffer system of the improvement is 8.3-9.
Further, the pH of the buffer system of the improvement is 8.8.
The buffer system of improvement of the present invention applied to PCR or RT-PCR is in PCR or RT-PCR detection
Using.
Amplification of the buffer system of improvement of the present invention applied to PCR or RT-PCR in the DNA or RNA of pathogen
Application in experiment.
Further, the pathogen is hepatitis b virus hbv, human immunodeficiency virus HIV or tubercle bacillus TB
In any one.
Beneficial effect：The buffering stability of the present invention is stronger, and amplification efficiency is higher, and detection sensitivity and precision are good.
Compared with prior art, the invention has the advantages that：
(1) present invention is using a kind of chemical substance MOPS for being usually used in preparing neutral buffer systems, and Chinese name is also known as 3- (N-
Morpholine) propane sulfonic acid, solution state is prepared into a kind of Tris-MOPS buffer systems into alkalescence with Tris, and the system combines MOPS
Between advantage, a kind of neutral buffered liquid relatively stablized of MOPS, pKa 72 (25 DEG C) buffering range 6.5-7.9, two dimension is widely used in
The electrolyte system composition of isoelectric focusing electrophoresis in gel electrophoresis, it may also be used for Northern hybridize, as RNA separation and turn
Buffer solution during film, reducing buffer system, pH changes are increased to the sensitiveness of temperature.
(2) present invention also with the addition of a kind of weak acid strong alkali salt sodium citrate, sodium citrate in Tris-MOPS buffer systems
With coupling Mg2+Ion energy, it is allowed to which system adds more Mg2+。Mg2+Effect be mainly dNTP-Mg2+With nucleic acid backbone
Interact and can influence Polymerase activity, typically in the case of Mg2+Concentration adjusted between 0.5-5mM, Mg from
Sub- concentration influences very big to PCR amplification efficiencies, and excessive concentration can reduce the specificity of PCR amplifications, and the too low then influence PCR of concentration expands
Units increased in production even makes PCR amplification failures without going out amplified band.Sodium citrate coupling Mg first2+, but as PCR carries out process
In, Mg2+Ion is consumed, the Mg of coupling2+The Mg of slow release supplement consumption2+, ensure that PCR high efficiency is carried out, while again will not be because
For Mg2+Excessive concentration, influences PCR atopics.
(3) the Tris-MOPS- buffered sodium citrates system described in is compared to Tris-HCl buffer systems buffering stability more
By force, amplification efficiency is higher, for reaction system in PCR or RT-PCR courses of reaction, improves PCR or RT-PCR detection sensitivity
And precision, for nucleic acid molecules diagnostic field, it is with a wide range of applications.
Brief description of the drawings
Fig. 1 is that figure buffer system of the present invention contrasts amplification curve diagram with conventional buffer system；
Fig. 2 HBV, HIV, TB amplified production gel electrophoresis figures, 1 represents control group amplified production gel electrophoresis figure, and 2 represent in fact
A group amplified production gel electrophoresis figure is tested, MK is DL500, most upper is 500bp, most bright is 200bp.
Embodiment
The present invention is described further with reference to the drawings and specific embodiments.It should be understood that these embodiments are merely to illustrate
Purpose, rather than the limitation scope of the invention.
Embodiment 1
A kind of buffer system of improvement applied to PCR or RT-PCR of the present invention, the buffer system of the improvement is 3-
(N- morpholines) propane sulfonic acid, Tris and trisodium citrate is mutually mixed and formed；3- (N- morpholines) propane sulfonic acid：Tris：Citric acid
The mol ratio of trisodium is 20：50：2.
The solution state is alkalescence.
The pH of the buffer system of the improvement is 8.3.
The buffer system of improvement of the present invention applied to PCR or RT-PCR is in PCR or RT-PCR detection
Using.
Embodiment 2
The difference of embodiment 2 and embodiment 1 is：A kind of buffering of improvement applied to PCR or RT-PCR of the present invention
System, the buffer system of the improvement is mutually mixed and formed for 3- (N- morpholines) propane sulfonic acid, Tris and trisodium citrate；The 3-
(N- morpholines) propane sulfonic acid：Tris：The mol ratio of trisodium citrate is 60：10：1.
The pH of the buffer system of the improvement is 8.8.
Amplification of the buffer system of improvement of the present invention applied to PCR or RT-PCR in the DNA or RNA of pathogen
Application in experiment.
The pathogen is tubercle bacillus (TB).
Embodiment 3
The difference of embodiment 3 and embodiment 1 is：A kind of buffering of improvement applied to PCR or RT-PCR of the present invention
System, the buffer system of the improvement is mutually mixed and formed for 3- (N- morpholines) propane sulfonic acid, Tris and trisodium citrate；The 3-
(N- morpholines) propane sulfonic acid：Tris：The mol ratio of trisodium citrate is 100：40：5.
The pH of the buffer system of the improvement is 9.
Amplification of the buffer system of improvement of the present invention applied to PCR or RT-PCR in the DNA or RNA of pathogen
Application in experiment.
The pathogen is hepatitis type B virus (HBV).
Embodiment 4
The difference of embodiment 4 and embodiment 1 is：The buffering of improvement of the present invention applied to PCR or RT-PCR
Application of the system in the DNA or RNA of pathogen amplification experiment.
The pathogen is human immunodeficiency virus (HIV).
Experiment 1
As shown in figure 1, the present invention's is nucleic acid-templated：
HIV-1 pseudovirions, RNA is extracted using the Qiaamp Viral RNA kit of Qiagen companies, standby；
Primed probe：
Sense primer 5-ATTAGTCCTATTGAAACTGTGCCAG-3 HPLC are pure
Anti-sense primer 5-ATACTGGAGTATTGTATGGATTTTCAG-3 HPLC are pure
Probe FAM-TGAAGCCAGGAATGGATGGCCCA-BHQ1 HPLC are pure
Fluorescence quantitative RT-RCR is expanded：
Each test reaction system is formulated as follows, 50 μ L systems, includes PCR Mix (setting experimental group and control group) 27.5
μ l, enzyme mixation (setting experimental group and control group) 2.5 μ L brief centrifugations, then add the μ l of template 20 to be detected；Equally press
Positive and negative control is set according to above-mentioned system, positive quality control product is added or the feminine gender μ l of quality-control product 20 is expanded.
The PCR Mix (experimental group) are that the RT-PCR buffer systems used is embodiments 1 shown in table 1：
Table 1
The PCR Mix (control group) are shown in table 2：
Table 2
Each reaction tube is put into the reactive tank of quantitative PCR instruments, sets the title and fluorophor species of each detection (to set
The reporter group for putting target gene is FAM, quenching group selection none), set cycling condition：
Bole's CFX96 fluorescent PCR instrument cycling condition be 45 DEG C 40 minutes, 94 DEG C 2 minutes；94 DEG C 15 seconds, 65 DEG C of 30 seconds, 72
DEG C 30 seconds, 8 circulations；94 DEG C 15 seconds, 60 DEG C 30 seconds, 72 DEG C 30 seconds, 8 circulation；94 DEG C 15 seconds, 53 DEG C 50 seconds, 40 circulation.
Fluorescence is collected at 55 DEG C.
Interpretation of result and judgement are as shown in Figure 1：Fig. 1 data displays, what is kept left is the Tris- of the experimental group correspondence present invention
The RT-PCR amplification curves (being repeated 1 times) that MOPS- buffered sodium citrates system is carried out, that keep right is control group correspondence Tris-HCl
Buffer system carries out RT-PCR amplification curves.The RT-PCR amplifications that experimental group is carried out shift to an earlier date compared to control group amplification Ct values, explanation
Experimental group compares control group, and for RT-PCR amplifications, sensitivity increases.
Experiment 2
Application
(1) selected pathogens HBV, HIV, viral load levels are about 1 × 103copies/mL；Pathogen TB, bacteria concentration
For 1 × 103Individual bacterium/mL, the DNA or RNA of pathogen are extracted using extracts kit；
(2) experimental group reaction system HBV and HIV uses the buffer system of embodiment 3, and TB uses the buffer body of embodiment 2
System；Other components are consistent with experiment 1.Control group reaction system is shown in experiment 1
(3) expanded on regular-PCR instrument.
As shown in Fig. 2 point sample order is respectively MK from left to right, negative control, control group HBV DNA cloning products are real
Test a group HBV DNA cloning products, control group HIV RNA amplification products, experimental group HIV RNA amplification products, control group TB DNA
Amplified production, experimental group TB DNA cloning products.Data illustrate that the system invented is applied to various viral or pathogen
DNA or RNA amplification, HIV RNA or TB DNA control groups system correspondence loading wells are without amplified production band in addition, and experimental group
System has obvious amplified production band, further illustrates that invented Tris-MOPS- buffered sodium citrates system is used for PCR
Or RT-PCR, detection sensitivity is better than control group system.
Specific embodiment described herein is only to marrow of the present invention explanation for example.Technology neck belonging to of the invention
The technical staff in domain can be made various modifications or supplement to described specific embodiment or be replaced using similar mode
Generation, but without departing from marrow of the invention or surmount scope defined in appended claims.
Claims (7)
1. a kind of buffer system of improvement applied to PCR or RT-PCR, it is characterised in that：The buffer system of the improvement is 3-
(N- morpholines) propane sulfonic acid, Tris and trisodium citrate is mutually mixed and formed；3- (N- morpholines) propane sulfonic acid：Tris：Citric acid
The mol ratio of trisodium is 20-100：10-50：1-5.
2. the buffer system of the improvement according to claim 1 applied to PCR or RT-PCR, it is characterised in that：It is described molten
Liquid status are alkalescence.
3. the buffer system of the improvement according to claim 2 applied to PCR or RT-PCR, it is characterised in that：It is described to change
The pH of good buffer system is 8.3-9.
4. the buffer system of the improvement according to claim 3 applied to PCR or RT-PCR, it is characterised in that：It is described to change
The pH of good buffer system is 8.8.
5. the buffer system of the improvement applied to PCR or RT-PCR described in claim any one of 1-4 is PCR's or RT-PCR
Application in detection.
6. the buffer system of the improvement applied to PCR or RT-PCR described in claim any one of 1-4 pathogen DNA or
Application in RNA amplification experiment.
7. application according to claim 6, it is characterized in that the pathogen is hepatitis b virus hbv, human immunity lacks
Fall into any one in virus HIV or tubercle bacillus TB.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710322610.7A CN107090505B (en) | 2017-05-09 | 2017-05-09 | Improved buffer system applied to PCR or RT-PCR and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710322610.7A CN107090505B (en) | 2017-05-09 | 2017-05-09 | Improved buffer system applied to PCR or RT-PCR and application |
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| Publication Number | Publication Date |
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| CN107090505A true CN107090505A (en) | 2017-08-25 |
| CN107090505B CN107090505B (en) | 2020-12-04 |
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| CN201710322610.7A Active CN107090505B (en) | 2017-05-09 | 2017-05-09 | Improved buffer system applied to PCR or RT-PCR and application |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006090987A1 (en) * | 2005-02-28 | 2006-08-31 | Bioquest, Inc. | Methods for performing direct enzymatic reactions involving nucleic acid molecules |
| CN103814140A (en) * | 2011-04-26 | 2014-05-21 | 长角牛疫苗和诊断有限责任公司 | Compositions and methods for detecting and identifying nucleic acid sequences in biological samples |
-
2017
- 2017-05-09 CN CN201710322610.7A patent/CN107090505B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006090987A1 (en) * | 2005-02-28 | 2006-08-31 | Bioquest, Inc. | Methods for performing direct enzymatic reactions involving nucleic acid molecules |
| CN103814140A (en) * | 2011-04-26 | 2014-05-21 | 长角牛疫苗和诊断有限责任公司 | Compositions and methods for detecting and identifying nucleic acid sequences in biological samples |
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| Publication number | Publication date |
|---|---|
| CN107090505B (en) | 2020-12-04 |
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