CN107502600A - A kind of double thermal starting archaeal dna polymerases and PCR amplification detection methods containing nano antibody - Google Patents
A kind of double thermal starting archaeal dna polymerases and PCR amplification detection methods containing nano antibody Download PDFInfo
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Abstract
The invention discloses a kind of PCR amplification detection methods, it comprises the following steps：(1) PCR reaction systems are built, including：Buffer solution；DATP solution；DCTP solution；DGTP solution；DTTP solution；Complementary sense primer, anti-sense primer and probe with detection sample；Double thermal starting archaeal dna polymerases, hot start Taq polymerase is closed according to concentration 1 by chemical modification hot start Taq polymerase and nano antibody:1 ratio mixes；(2) PCR reaction systems and detection sample are mixed into performing PCR amplification；(3) cycle threshold after PCR amplifications is obtained, and the amount of the thing to be detected for detecting sample is confirmed according to cycle threshold.Thermal starting Taq polymerase is prepared from nano antibody and forms double thermal starting archaeal dna polymerases, not only heat start PCR can be applied to replica polymerization enzyme monoclonal antibody, additionally it is possible to functionally substitute the monoclonal antibody of stability difference, and greatly reduce preparation cost.
Description
【Technical field】
The invention belongs to the archaeal dna polymerase technical field of fluorescent quantitative PCR detection, opened more particularly to a kind of double heat
Dynamic archaeal dna polymerase and PCR amplification detection methods.
【Background technology】
When archaeal dna polymerase enters performing PCR reaction, the process of high temperature (94 DEG C~95 DEG C) is risen to by low temperature (20 DEG C~37 DEG C)
In, still there is the enzymatic activity remained, low-level non-specificity is annealed and extended in reaction and starts to occur with during thermal cycle,
This result in the formation of the non-specific amplification product in subsequent PCR cycle again in turn, eventually through amplification needed for reduction
Signal or signal are covered by high background value and make sensitivity and the yield reduction of reaction.Heat start PCR is in the initial denaturation stage
After start PCR enzymatic reactions, by thermal starting reduce primer dimer formation, primer mispairing and non-specific amplification, so as to
Increase PCR specificity and sensitiveness, improve PCR yield.Since thermal starting is prolonged as blocking dna polymerase at a lower temperature
Since a kind of means stretched, for the basis such as Mg of reaction2+, archaeal dna polymerase, primer and dNTP, also develop many sides
Method.1. being wrapped up Taq archaeal dna polymerases by paraffin method and low melting point agarose embedding method, separated with other PCR reactive components,
Until temperature is increased to certain temperature (65 DEG C~70 DEG C), Taq polymerase is released and played a role.Although this method letter
Just, it is but time-consuming, and pollution may be introduced.2. using the monoclonal antibody for Taq polymerase, under normal temperature, monoclonal resists
Body is attached to Taq enzyme avtive spot, and the activity of enzyme is suppressed, and after temperature reaches 70 DEG C, antibody inactivation, Taq enzyme is released.
It is with strong points using enzyme antibody, but antibody cycle length, cost height are prepared, and also at a given temperature, antibody differs surely
All denaturation is fallen.3. the Taq archaeal dna polymerases of chemical modification, the activated centre of polymerase is closed, Taq is blocked to gather at room temperature
Synthase activity, and polymerase activity recovers in first PCR pre-degeneration step.This method does not have animal sources DNA pollution.
KT-C3DNA polymerases are modified using citraconic anhydride, close the enzymatic activity of KT-C3 archaeal dna polymerases, during PCR, only work as temperature
After degree rises to 95 DEG C, acid anhydrides just dissociates from polymerase, enzyme activity recovery, realizes thermal starting.4. Mdification primer.One kind uses two
The method of the individual normal single-stranded primer of double-chain primer substitution, the single stranded oligonucleotide that each double-chain primer includes two equal lengths draw
Thing, one is referred to as " primer strand ", for expanding；Another is referred to as " competition chain ", complementary with " primer strand ", contains one or more
The nucleotides of mispairing, it can not extend in PCR reactions.Under cryogenic, " primer strand " it is double to be attached to formation on " competition chain "
Chain, only under suitable amplification condition, " competition chain " can just be substituted by template, normally be expanded." competition chain " it is competing
The effect of striving adds the specificity that " primer strand " combines DNA profiling.5. dNTP is modified, with 2 ' on ether and ester substitution dNTP-de-
Oxygen ribonucleotide and 3 '-triphosphoric acid group, prevent dNTP from playing a role, after (95 DEG C) preheatings of high temperature, the dNTP of modified
Normal dNTP can be changed into, participates in PCR reactions.6. using golden nanometer particle (AuNP) and quantum dot (QD) at low temperature with
Pfu archaeal dna polymerases interact, and suppress polymerase activity, so as to reduce non-specific amplification, are dissociated at high temperature from enzyme,
PCR reactions are made to be carried out by normal mode.Generally speaking, heat start PCR is to reduce or eliminate non-specific amplification product and primer
The effective ways that dimer is formed, it can be applied to the amplification of genomic DNA single-copy sequence, LA-PCR expands and multiple
In PCR amplifications.
In the prior art, the thermal starting enzyme applied to PCR reactions is often only a kind of, otherwise it is antibody closing, or be
Chemical modification.The thermal starting enzyme of antibody closing is rapid-action, can be fully active in initial reaction stage, plays maximum activity, but rear power
Deficiency；And the thermal starting enzyme of chemical modification then slow heat, the slow release activity during high-temperature denatured, so as to ensure all the time
Special amplification.
Chinese Patent Application No. is No. CN201510707423.1 and discloses one kind containing dyestuff EvaGreen and double heat
Start the quantifying PCR method of enzyme, double thermal starting enzymes include in the patent：HotStarTaq archaeal dna polymerases and Anti Taq DNA
Polymerase, the Anti Taq archaeal dna polymerases are common monoclonal antibody, the thermal starting Taq polymerase of monoclonal antibody closing,
Although high specificity, its preparation process is complicated, time-consuming, and price is costly.
On the whole, the stability of above-mentioned PCR amplification method, sensitivity be not still still high, therefore, it is necessary to provide
A kind of new double thermal starting archaeal dna polymerases solve the above problems.
【The content of the invention】
It is a primary object of the present invention to provide a kind of double thermal starting archaeal dna polymerases and PCR amplification containing nano antibody
Detection method, to improve the stability of PCR amplification detection methods, sensitivity.
To achieve the above object, the present invention adopts the following technical scheme that：A kind of double thermal starting DNA containing nano antibody gather
Synthase, hot start Taq polymerase is closed according to concentration 1 by chemical modification hot start Taq polymerase and nano antibody:1 ratio mixes.
To achieve the above object, the present invention also adopts the following technical scheme that a kind of PCR amplification detection methods, and it includes as follows
Step：
(1) PCR reaction systems are built, including：Buffer solution；DATP solution；DCTP solution；DGTP solution；DTTP is molten
Liquid；Complementary sense primer, anti-sense primer and probe with detection sample；Double thermal starting archaeal dna polymerases, by chemical modification thermal starting
Taq enzyme and nano antibody close hot start Taq polymerase according to concentration 1:1 ratio mixes；
(2) PCR reaction systems and detection sample are mixed into performing PCR amplification；
(3) cycle threshold after PCR amplifications is obtained, and the thing to be detected for detecting sample is confirmed according to cycle threshold
Amount.
The PCR reaction systems also have reverse transcriptase, and the concentration of reverse transcriptase is the 20 of double thermal starting archaeal dna polymerase concentration
Times.
DATP, dCTP, dGTP, dTTP concentration are 0.5mmol/L, and sense primer, the concentration of anti-sense primer are 0.5 μ
Mol/L, the concentration of probe is 0.125 μm of ol/L, chemical modification hot start Taq polymerase and nano antibody closing hot start Taq polymerase
Concentration is 0.5U/ul.
DATP, dCTP, dGTP, dTTP concentration are 0.5mmol/L, and sense primer, the concentration of anti-sense primer are 0.5 μ
Mol/L, the concentration of probe is 0.125 μm of ol/L, chemical modification hot start Taq polymerase and nano antibody closing hot start Taq polymerase
Concentration is 0.5U/ul, reverse transcriptase concentrations 10U/ul.
Sense primer：5′-CGTGATCCTTTGCCAGACACT-3′；
Anti-sense primer：5′-TCGGTCGGGCTACACAGGGTCA-3′；
Probe：5′-FAM-ATCCGACTGTGGCATACGTGCACGTG-BHQ1-3′.
Compared with prior art, a kind of beneficial effect of double thermal starting archaeal dna polymerases containing nano antibody of the present invention is：
Thermal starting Taq polymerase, which is prepared, from nano antibody forms double thermal starting archaeal dna polymerases, not only can be with replica polymerization enzyme monoclonal
Antibody is applied to heat start PCR, additionally it is possible to functionally substitutes the monoclonal antibody of stability difference, and greatly reduces preparation
Cost.
【Brief description of the drawings】
Fig. 1 is the PCR amplification curve comparison diagrams that three kinds of hot start Taq polymerases detect TB in first embodiment of the invention.
【Embodiment】
The present invention is a kind of double thermal starting archaeal dna polymerases containing nano antibody, and this pair of hot start Taq polymerase is chemical modification
Hot start Taq polymerase and nano antibody close hot start Taq polymerase according to concentration 1:1 ratio mixes.
Enter performing PCR amplification to each sample using this pair of hot start Taq polymerase.
Embodiment one：
The TB clinical samples for taking 1mL concentration to be 1e+6copies/mL, are given birth to using Qiagen Bioengineering (Shenzhen) Co., Ltd.
" nucleic acid extraction or the purified reagent progress nucleic acid extraction of production.The TB nucleic acid solutions of acquisition, with water gradient dilution to 1e+
5copies/mL, 1e+4copies/mL, 1e+3copies/mL, obtain the TB nucleic acid solutions of various concentrations.
Build PCR reaction systems
Wherein：Sense primer：5′-CGTGATCCTTTGCCAGACACT-3′
Anti-sense primer：5′-TCGGTCGGGCTACACAGGGTCA-3′
Probe：5′-FAM-ATCCGACTGTGGCATACGTGCACGTG-BHQ1-3′.
Respectively using double hot start Taq polymerases, chemical modification hot start Taq polymerase, nano antibody closing hot start Taq polymerase, press
TBPCR reaction systems are prepared according to above-mentioned formula, wherein, chemical modification hot start Taq polymerase and nano antibody closing thermal starting Taq
Enzyme, both are according to concentration 1:1 ratio is mixed to form double hot start Taq polymerases.
Nano antibody is the Novel engineering antibody only with variable region transformed through genetic engineering, and it lacks light chain, point
Son amount is small, stability is good, easy expression.Thermal starting Taq polymerase, which is prepared, from nano antibody forms double thermal starting archaeal dna polymerases,
Not only heat start PCR can be applied to replica polymerization enzyme monoclonal antibody, can functionally substitute the monoclonal of stability difference
Antibody, and greatly reduce preparation cost.
The TBPCR reaction systems prepared mix with the TB nucleic acid solutions of various concentrations, are examined in fluorescent quantitative PCR
Survey：
| Reagent | Final concentration |
| PCR reaction systems | 20μL |
| TB nucleic acid solutions | 30μL |
| Final volume | 50μL |
Three kinds of hot start Taq polymerase detection TB Ct values (cycle threshold) result
Detailed Ct values result and PCR amplification curves are referring to upper table and Fig. 1.
As a result show, add the experimental group of double hot start Taq polymerases, with chemical modification hot start Taq polymerase or received compared to addition
Meter Kang Ti closing hot start Taq polymerases are the experimental group of the Taq enzyme of one-component, and Ct values are smaller, and fluorescence increasing degree is higher.
Embodiment two：
The HIV clinical samples for taking 1.2mL concentration to be 1e+7IU/mL, are given birth to using Qiagen Bioengineering (Shenzhen) Co., Ltd.
" nucleic acid extraction or the purified reagent " of production, automatic nucleic acid extraction is carried out in QIAsymphony SP/AS self-reacting devices platform.
The HIV nucleic acid solutions of acquisition, with water gradient dilution to 1e+6IU/mL, 1e+4IU/mL, 100IU/mL, 50IU/mL, 20IU/mL,
Obtain the HIV nucleic acid solutions of various concentrations.
Build RT-PCR reaction systems
| Reagent | Final concentration |
| Buffer solution | 2.5× |
| dATP | 0.5mmol/L |
| dCTP | 0.5mmol/L |
| dGTP | 0.5mmol/L |
| dTTP | 0.5mmol/L |
| Sense primer | 0.5μmol/L |
| Anti-sense primer | 0.5μmol/L |
| Probe | 0.125μmol/L |
| Reverse transcriptase | 10U/ul |
| Hot start Taq polymerase | 0.5U/ul |
Wherein：Sense primer：5′-ATCAAGCAGCCATGCAAATGCT-3′
Anti-sense primer：5′-TGAAGGGTACTAGTAGTTCCTGCTATGTC-3′
Probe：5′Fam-ATGAGGAGGCTGCAGAITGGGA–MGB 3′
Respectively using double hot start Taq polymerases, chemical modification hot start Taq polymerase, nano antibody closing hot start Taq polymerase, press
RT-PCR reaction solutions are prepared according to above-mentioned formula, wherein, chemical modification hot start Taq polymerase and nano antibody closing hot start Taq polymerase,
Both are according to concentration 1:1 ratio is mixed to form double hot start Taq polymerases.The concentration of reverse transcriptase is that double thermal starting archaeal dna polymerases are dense
20 times of degree.The reaction system prepared mixes with HIV nucleic acid solutions, is expanded on quantitative real time PCR Instrument in different condition
Increase detection：
| Reagent | Final concentration |
| RT-PCR reaction solutions | 20μL |
| HIV nucleic acid solutions | 30μL |
| Final volume | 50μL |
Three kinds of hot start Taq polymerase detection HIV Ct value results
As a result show, add the experimental group of double hot start Taq polymerases, with chemical modification hot start Taq polymerase or received compared to addition
Meter Kang Ti closing hot start Taq polymerase for one-component Taq enzyme experimental group, Ct values are smaller, and detection 25IU/mL with
As 50IU/mL during low concentration sample, recall rate is higher, shows more preferable sensitivity.
Therefore, by detecting the sample of nucleic acid of TB concentration gradients, relatively pair hot start Taq polymerases and the heat of two kinds of single components
Start Taq enzyme, the testing result Ct values of double hot start Taq polymerases are smaller, and fluorescence increasing degree is higher, shows more excellent Detection results；
By detecting the sample of nucleic acid of HIV concentration gradients, the thermal starting of relatively more double hot start Taq polymerases and two kinds of single components
Taq enzyme, the testing result Ct values of double hot start Taq polymerases are smaller, can detect more low concentration samples, show more preferable spirit
Sensitivity.
Above-described is only some embodiments of the present invention.For the person of ordinary skill of the art, not
On the premise of departing from the invention design, various modifications and improvements can be made, these belong to the protection model of the present invention
Enclose.
Claims (6)
- A kind of 1. double thermal starting archaeal dna polymerases containing nano antibody, it is characterised in that：By chemical modification hot start Taq polymerase and Nano antibody closes hot start Taq polymerase according to concentration 1:1 ratio mixes.
- 2. a kind of PCR amplification detection methods, it is characterised in that comprise the following steps：(1) PCR reaction systems are built, including：Buffer solution；DATP solution；DCTP solution；DGTP solution；DTTP solution；Complementary sense primer, anti-sense primer and probe with detection sample；Double thermal starting archaeal dna polymerases, hot start Taq polymerase is closed according to concentration by chemical modification hot start Taq polymerase and nano antibody 1:1 ratio mixes；(2) PCR reaction systems and detection sample are mixed into performing PCR amplification；(3) cycle threshold after PCR amplifications is obtained, and the amount of the thing to be detected for detecting sample is confirmed according to cycle threshold.
- 3. PCR amplification detection methods as claimed in claim 2, it is characterised in that：The PCR reaction systems also have reverse transcription Enzyme, the concentration of reverse transcriptase are 20 times of double thermal starting archaeal dna polymerase concentration.
- 4. PCR amplification detection methods as claimed in claim 2, it is characterised in that：DATP, dCTP, dGTP, dTTP concentration is 0.5mmol/L, sense primer, the concentration of anti-sense primer are 0.5 μm of ol/L, and the concentration of probe is 0.125 μm of ol/L, chemical modification The concentration of hot start Taq polymerase and nano antibody closing hot start Taq polymerase is 0.5U/ul.
- 5. PCR amplification detection methods as claimed in claim 3, it is characterised in that：DATP, dCTP, dGTP, dTTP concentration is 0.5mmol/L, sense primer, the concentration of anti-sense primer are 0.5 μm of ol/L, and the concentration of probe is 0.125 μm of ol/L, chemical modification The concentration of hot start Taq polymerase and nano antibody closing hot start Taq polymerase is 0.5U/ul, reverse transcriptase concentrations 10U/ul.
- 6. the PCR amplification detection methods as described in claim 4 or 5, it is characterised in that：Sense primer：5′-CGTGATCCTTTGCCAGACACT-3′；Anti-sense primer：5′-TCGGTCGGGCTACACAGGGTCA-3′；Probe：5′-FAM-ATCCGACTGTGGCATACGTGCACGTG-BHQ1-3′.
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Cited By (6)
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| CN109628423A (en) * | 2018-12-06 | 2019-04-16 | 北京春雷杰创生物科技有限公司 | A kind of method of thermal starting Taq archaeal dna polymerase Combinatorial Optimization molecular agents |
| CN110878124A (en) * | 2018-09-05 | 2020-03-13 | 深圳华大生命科学研究院 | Antibody fragment of DNA polymerase, antibody and application thereof |
| CN111349167A (en) * | 2018-12-20 | 2020-06-30 | 东莞市朋志生物科技有限公司 | Antibody for resisting Taq DNA polymerase and application thereof |
| CN111647645A (en) * | 2020-04-13 | 2020-09-11 | 武汉翌圣医疗科技有限公司 | Method for screening Family B DNA Polymerase antibody closed Polymerase activity |
| CN111808197A (en) * | 2020-06-15 | 2020-10-23 | 北京全式金生物技术有限公司 | Taq DNA polymerase monoclonal antibody combination and reaction system and application thereof |
| CN112574971A (en) * | 2020-12-29 | 2021-03-30 | 益善生物技术股份有限公司 | Taq DNA polymerase mutant, PCR reaction reagent and kit |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110878124A (en) * | 2018-09-05 | 2020-03-13 | 深圳华大生命科学研究院 | Antibody fragment of DNA polymerase, antibody and application thereof |
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| CN111647645A (en) * | 2020-04-13 | 2020-09-11 | 武汉翌圣医疗科技有限公司 | Method for screening Family B DNA Polymerase antibody closed Polymerase activity |
| CN111647645B (en) * | 2020-04-13 | 2023-05-05 | 武汉翌圣生物科技有限公司 | Method for screening Family B DNA Polymerase antibody blocking polymerase activity |
| CN111808197A (en) * | 2020-06-15 | 2020-10-23 | 北京全式金生物技术有限公司 | Taq DNA polymerase monoclonal antibody combination and reaction system and application thereof |
| CN111808197B (en) * | 2020-06-15 | 2021-03-30 | 北京全式金生物技术有限公司 | Taq DNA polymerase monoclonal antibody combination and reaction system and application thereof |
| CN112574971A (en) * | 2020-12-29 | 2021-03-30 | 益善生物技术股份有限公司 | Taq DNA polymerase mutant, PCR reaction reagent and kit |
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Application publication date: 20171222 |