CN103966323A - Dry-powder isothermal amplification detection reagent capable of being transported at normal temperature, and preparation method of detection reagent - Google Patents
Dry-powder isothermal amplification detection reagent capable of being transported at normal temperature, and preparation method of detection reagent Download PDFInfo
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- CN103966323A CN103966323A CN201410173060.3A CN201410173060A CN103966323A CN 103966323 A CN103966323 A CN 103966323A CN 201410173060 A CN201410173060 A CN 201410173060A CN 103966323 A CN103966323 A CN 103966323A
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- detection reagent
- amplification detection
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- normal temperature
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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
Abstract
The invention discloses a dry-powder isothermal amplification detection reagent capable of being transported at normal temperature, and a preparation method of the detection reagent. The detection reagent can be prepared by the following steps: adding a freeze-drying protective additive into a liquid reagent, and adopting a vacuum freeze drying method to freeze-dry components of the liquid isothermal amplification detection reagent to be solid dry powder. The solid reagent can be transported at normal temperature; during reaction, all that is needed is to add a liquid reaction substrate to be dissolved; the reactivity is excellent, the operation is convenient, and the dry-powder isothermal amplification detection reagent can be widely used in on-site quick detection of various pathogenes, food microorganisms, transgenic products and the like.
Description
Technical field
The present invention relates to a kind of can normal temperature the dry powder constant-temperature amplification detection reagent and preparation method thereof of transport.
Background technology
Loop-mediated isothermal amplification technique is the constant temperature nucleic acid amplification method of a kind of novelty of being developed by Japanese Eiken Chemical for 2000, be characterized in the 4 kinds of special primers of 6 zone design for target gene, utilize a kind of strand displacement archaeal dna polymerase at isothermal condition (63 DEG C of left and right) insulation 30-60 minute, can complete nucleic acid amplification reaction.Compared with conventional PCR, do not need the processes such as thermally denature, temperature cycle, electrophoresis and the ultraviolet visualization of template.Ring mediated isothermal amplification method is a kind of brand-new nucleic acid amplification method, has feature simple, quick, high specificity.This technology can match in excellence or beauty and even be better than round pcr in the indexs such as sensitivity, specificity and sensing range, does not rely on any special plant and instrument and realizes on-the-spot high-throughput rapid detection, and testing cost is far below quantitative fluorescent PCR.
Loop-mediated isothermal amplification technique need to possess following condition:
(1) DNA profiling that will be replicated;
(2) Auele Specific Primer designing for DNA profiling;
(3)
bstarchaeal dna polymerase;
(4) reaction solution;
（5） dNTP；
（6） Mg2+；
But these materials all need refrigerated storage, especially
bstarchaeal dna polymerase, need to be-20 DEG C of preservations, it can not multigelation, therefore, uses to the transport of reagent and client and has brought great trouble, increase transportation cost, meanwhile, each experiment all will be prepared respective reaction system, uses also very inconvenient, easily cause because operator operates the error causing, and easily cause the dirt intersection between experiment to dye.
Summary of the invention
The object of the invention is to overcome existing deficiency in prior art, provide a kind of can normal temperature the dry powder constant-temperature amplification detection reagent and preparation method thereof of transport.
The technical solution used in the present invention is:
A kind of lyophilized vaccine, it contains raffinose, bovine serum albumin, PEG 20000 and L-threonine.
As preferably, described lyophilized vaccine contains 3～10 parts of raffinoses, 0.1～1 part of bovine serum albumin, 0.5～2 part of PEG 20000 and 0.01～0.1 part of L-threonine, by mass parts.
Can normal temperature the dry powder constant-temperature amplification detection reagent of transport, it comprises constant-temperature amplification detection reagent and above-mentioned lyophilized vaccine.
Described constant-temperature amplification detection reagent comprise constant temperature PCR reaction solution,
bstarchaeal dna polymerase, primer and fluorescence dye.
As preferably, the concentration of described lyophilized vaccine in whole system is 3.61%～13.1%.
Can normal temperature the preparation method of dry powder constant-temperature amplification detection reagent of transport, comprise the steps:
(1) prepare lyophilized powder stoste by the formula of claim 3～5 any one;
(2) by the freeze-drying of lyophilized powder stoste.
The invention has the beneficial effects as follows:
1) dry powder constant-temperature amplification detection reagent of the present invention can increase the stability of constant-temperature amplification detection reagent, the long-term rear freeze-dried detection sensitivity and detection time and the new detection reagent difference configuring of placing is little, and freeze-dried reagent can transport by normal temperature, save energy and transportation cost.
2) dry powder constant-temperature amplification detection reagent of the present invention combines each non-variable reagent, greatly reduces experimental procedure, easy to use, reduces experimental error simultaneously.
Brief description of the drawings
Fig. 1 is the constant-temperature amplification detection reagent after vacuum lyophilization.
Embodiment
Below in conjunction with embodiment, the present invention is further illustrated, but be not limited to this.If no special instructions, " % " below all refers to mass percent.
Dry powder constant-temperature amplification detection reagent of the present invention, component is in table 1.After following system freeze-drying, only need to add 23 μ l ultrapure waters to dissolve, then add 2 μ l nucleic acid-templated, can complete the detection to cause of disease.
Table 1
And reference examples is set compares, the component of each reference examples is in table 2.
Table 2
IMNV primer sequence is in table 3.:
Table 3
Prepare after above-mentioned system, be placed in vacuum freeze drier, freeze-drying program routinely carry out vacuum lyophilization, obtain can normal temperature the dry powder constant-temperature amplification detection reagent of transport.
Each group of dry powder constant-temperature amplification detection reagent normal temperature transport in table 13 days, at 4 DEG C, place again after 50 days, utilize it to detect the plasmid DNA that contains prawn infectivity muscle necrosis virus (IMNV) specific nucleic acid fragment (Accession No:AY570982), and the constant temperature fluorescence detection reagent kit that increases liquid in contrast.
Contrast index comprises: 1, the sensitivity of dry powder reagent; 2, the detection time of dry powder reagent to same concentrations nucleic acid.
Sample to be checked is the plasmid DNA that contains prawn infectivity muscle necrosis virus (IMNV) specific nucleic acid fragment (plasmid DNA of 10pg/ μ l, the plasmid DNA of 1pg/ μ l, the plasmid DNA of 100fg/ μ l, the plasmid DNA of 10fg/ μ l and the plasmid DNA of 1fg/ul) and a negative control of three concentration gradients.
Adopt ABI7500 quantitative real time PCR Instrument to carry out constant-temperature amplification, amplification program is 63 DEG C, 60min.Detected result is as follows:
1, the comparison of sensitivity
Test the minimum detectable concentration that detects each group reagent in table 4 through gradient.
Table 4
2, the comparison of detection time
The fastest detection time of each group reagent is in table 5.
Table 5
Above experimental data shows, dry powder constant-temperature amplification detection reagent of the present invention can increase the stability of constant-temperature amplification detection reagent.The long-term rear freeze-dried detection sensitivity and detection time and the new liquid detecting reagent difference of preparing of placing is little.
Above embodiment is only for introducing preferred case of the present invention, and to those skilled in the art, any apparent changes and improvements of carrying out in the scope that does not deviate from spirit of the present invention, all should be regarded as a part of the present invention.
<110> Guangzhou Deaou Biotechnology Co., Ltd.
<120> can normal temperature the dry powder constant-temperature amplification detection reagent and preparation method thereof of transport
<130>
<160> 6
<170> PatentIn version 3.5
<210> 1
<211> 22
<212> DNA
<213> artificial sequence
<400> 1
ggaatgagat atcagtgaca gg 22
<210> 2
<211> 18
<212> DNA
<213> artificial sequence
<400> 2
ttgtctgacg tgtctcga 18
<210> 3
<211> 42
<212> DNA
<213> artificial sequence
<400> 3
ggactccaag gttgtggatt ccgtggacat gtagaggaat ag 42
<210> 4
<211> 40
<212> DNA
<213> artificial sequence
<400> 4
gtcgaaacaa atgcaagcaa caagtgtaat tgttgaagtt 40
<210> 5
<211> 22
<212> DNA
<213> artificial sequence
<400> 5
atggttttcg ttgacttatg cc 22
<210> 6
<211> 22
<212> DNA
<213> artificial sequence
<400> 6
ttagaaagac gagcaggaat tg 22
Claims (6)
1. a lyophilized vaccine, it contains raffinose, bovine serum albumin, PEG 20000 and L-threonine.
2. lyophilized vaccine according to claim 1, is characterized in that, described lyophilized vaccine contains 3～10 parts of raffinoses, 0.1～1 part of bovine serum albumin, 0.5～2 part of PEG 20000 and 0.01～0.1 part of L-threonine, by mass parts.
3. can normal temperature the dry powder constant-temperature amplification detection reagent of transport, it comprises the lyophilized vaccine described in constant-temperature amplification detection reagent and claim 1 or 2.
According to claim 3 can normal temperature the dry powder constant-temperature amplification detection reagent of transport, it is characterized in that, the concentration of described lyophilized vaccine in whole system is 3.61%～13.1%.
According to claim 3 can normal temperature the dry powder constant-temperature amplification detection reagent of transport, it is characterized in that, described constant-temperature amplification detection reagent comprise constant temperature PCR reaction solution,
bstarchaeal dna polymerase, primer and fluorescence dye.
6. can normal temperature the preparation method of dry powder constant-temperature amplification detection reagent of transport, comprise the steps:
(1) prepare lyophilized powder stoste by the formula of claim 3～5 any one;
(2) by the freeze-drying of lyophilized powder stoste.
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| CN201410173060.3A CN103966323A (en) | 2014-04-25 | 2014-04-25 | Dry-powder isothermal amplification detection reagent capable of being transported at normal temperature, and preparation method of detection reagent |
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| CN201410173060.3A CN103966323A (en) | 2014-04-25 | 2014-04-25 | Dry-powder isothermal amplification detection reagent capable of being transported at normal temperature, and preparation method of detection reagent |
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| CN103966323A true CN103966323A (en) | 2014-08-06 |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105349529A (en) * | 2015-12-09 | 2016-02-24 | 江苏正大天创生物工程有限公司 | Freeze-drying protective agent applied to nucleic acid amplification system |
| CN108754022A (en) * | 2018-06-12 | 2018-11-06 | 广州和实生物技术有限公司 | A kind of kit for detecting HBV-pgRNA |
| CN108753968A (en) * | 2018-06-12 | 2018-11-06 | 广州和实生物技术有限公司 | A kind of kit for detecting cervical carcinoma PAX1 gene methylations |
| CN108796126A (en) * | 2018-06-12 | 2018-11-13 | 广州和实生物技术有限公司 | A kind of kit for detecting HPV |
| CN108796125A (en) * | 2018-06-12 | 2018-11-13 | 广州和实生物技术有限公司 | A kind of kit for detecting HIV-1 |
| CN112481359A (en) * | 2020-11-30 | 2021-03-12 | 河南智泰生物科技有限公司 | Freeze-dried microsphere of LAMP isothermal amplification reagent and preparation method and application thereof |
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| CN102084005A (en) * | 2008-07-02 | 2011-06-01 | 恩尼格马诊断有限公司 | Freeze-dried compositions for biochemical reactions |
| CN103492407A (en) * | 2011-02-09 | 2014-01-01 | 葛兰素史密斯克莱有限责任公司 | Lyophilized formulations |
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2014
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102084005A (en) * | 2008-07-02 | 2011-06-01 | 恩尼格马诊断有限公司 | Freeze-dried compositions for biochemical reactions |
| CN103492407A (en) * | 2011-02-09 | 2014-01-01 | 葛兰素史密斯克莱有限责任公司 | Lyophilized formulations |
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105349529A (en) * | 2015-12-09 | 2016-02-24 | 江苏正大天创生物工程有限公司 | Freeze-drying protective agent applied to nucleic acid amplification system |
| CN108754022A (en) * | 2018-06-12 | 2018-11-06 | 广州和实生物技术有限公司 | A kind of kit for detecting HBV-pgRNA |
| CN108753968A (en) * | 2018-06-12 | 2018-11-06 | 广州和实生物技术有限公司 | A kind of kit for detecting cervical carcinoma PAX1 gene methylations |
| CN108796126A (en) * | 2018-06-12 | 2018-11-13 | 广州和实生物技术有限公司 | A kind of kit for detecting HPV |
| CN108796125A (en) * | 2018-06-12 | 2018-11-13 | 广州和实生物技术有限公司 | A kind of kit for detecting HIV-1 |
| CN108754022B (en) * | 2018-06-12 | 2021-09-10 | 广州和实生物技术有限公司 | Kit for detecting HBV-pgRNA |
| CN108796125B (en) * | 2018-06-12 | 2021-09-10 | 广州和实生物技术有限公司 | Kit for detecting HIV-1 |
| CN108796126B (en) * | 2018-06-12 | 2021-09-10 | 广州和实生物技术有限公司 | Kit for detecting HPV |
| CN108753968B (en) * | 2018-06-12 | 2022-04-12 | 广州和实生物技术有限公司 | Kit for detecting methylation of cervical cancer PAX1 gene |
| CN112481359A (en) * | 2020-11-30 | 2021-03-12 | 河南智泰生物科技有限公司 | Freeze-dried microsphere of LAMP isothermal amplification reagent and preparation method and application thereof |
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Application publication date: 20140806 |