CN103725769A - Method for detecting different mutation types on same mutation site by tubular common primer method - Google Patents
Method for detecting different mutation types on same mutation site by tubular common primer method Download PDFInfo
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Abstract
The invention relates to a method or a theory for detecting different mutation types on the same mutation site by tubular method based on a loop-mediated isothermal amplification (LAMP) technology. According to the method, different mutation basic groups on the same mutation site can be detected at the same time in the same nucleic acid detection reaction tube. 4-6 primers are needed for the LAMP design, through the LAMP kit probe design, only primers covering the mutation site are changed and primers covering other sites are not changed into common primers (the adding amount also remains) for different mutation types of the same mutation site, so that cross reaction between the primer and common primer covering the mutation site is avoided, only verifying whether cross reaction occurs between the primers of different mutation types at the same mutation site, and reaction does not occur if only one to two alkali groups are different. This is the theoretical basis of the detection mode. The method can not only reduce cost and improve efficiency, but also greatly improve the detection sensitivity when multiple mutation types exist at the same time.
Description
Technical field
One tubular type general primer method detects the different mutation types in identical mutation site, and this invention can be applied to ring mediated isothermal amplification detection technique field, the detections such as tubercule bacillus, hepatitis B virus, HIV virus, tumor drug resistance.
Background technology
Ring mediated isothermal amplification method (loop-mediated isothermal amplification, LAMP) be the constant temperature nucleic acid amplification method of a kind of novelty of exploitation in 2000, be characterized in the 4 kinds of special primers of 6 zone design for target gene, utilize a kind of strand displacement DNA polysaccharase at isothermal condition (63 ℃ 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.LAMP is a kind of brand-new nucleic acid amplification method, has feature simple, quick, high specificity.This technology can 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.
In the Drug Resistance Detection of the most widely used model of industry, be drug sensitive experiment and biochip technology at present, drug sensitive experiment is consuming time oversize, although gene chip can be realized high throughput testing preparation cost and testing cost is all more expensive, sensitivity is low, unsuitable spread.Therefore be badly in need of a kind of low, the highly sensitive practicable method of testing cost.
Summary of the invention
The object of the invention is: provide a kind of while one tubular type to detect the loop-mediated isothermal amplification detection method of the different mutation types in identical mutation site.
Technical scheme of the present invention is: the method can detect the different mutating alkali yls in same mutational site in same nuclease assay reaction pipe simultaneously.LAMP design needs 4-6 primer, by in loop-mediated isothermal amplification detection kit probe design, for the different mutation types of identical mutation point, only change the primer that covers catastrophe point place, the primer that covers other site constant (add quantity also constant), guaranteed like this, between the primer of the primer of catastrophe point and other some position, there is not cross reaction, only need checking to cover identical mutation and put between the primer at different mutation types place whether cross reaction occurs, cover so this primer herein and only have one to two base difference, also can not react, the theoretical basis of this detection mode that Here it is.
embodiment 1:
526 point mutation of the resistance to Rifampin of Mycobacterium tuberculosis (RFP) medicine Nucleotide have following several situation:
If adopt existing ring mediated isothermal amplification detection technique, need six test tubes to detect one of these six kinds of sudden change modes, can determine 526 resistances, adopt genechip detection also to need to have six corresponding check points different mutant forms just can be detected, but suppose that they are the same in resistance rank, which does not just need to detect their specifically which kind of mutation type, only needing to detect has above gene to exist, this point is resistance, just based on this, the art of this patent adopts loop-mediated isothermal amplification technique, the art of this patent has designed the 6 kinds of different primers (as follows) that cover corresponding catastrophe point for 6 kinds of 526 positions mutation type, between these 6 primers, only has 1 to 2 base difference, therefore between, can there is not cross reaction, every the primer that covers other primer (general primer) of putting position and covering catastrophe point is all arranged in pairs or groups, they can be separately in a pipe reaction detection determine different catastrophe points, that is to say that cross reaction can not occur for every catastrophe point primer and general primer, between general primer, certainly there is not cross reaction yet, this is all just definite when in design, can not there is cross reaction in all like this reaction links, therefore it is no problem they being added to a tube reaction, as long as there is an above site of sudden change, corresponding primer will combine with Tubercle DNA together with general primer, react, the reaction that other mutant primer is not participated, so just can determine 526 existence sudden changes, it is resistance, can reach like this object that is simultaneously detected various mutations by a reaction tubes.
we are defined as above method: LAMP mono-tubular type general primer method detects the different mutation types of identical mutation point.
If so He one pipe only detects a mutation type and contrasts which type of is best primer add-on? of course not simple only by 6 pipe liquid mixing, the add-on of every general primer is constant (it is the same only examining a mutation type with a pipe), the amount of DNA profiling is also constant, each is also identical with a mutation type of single tube inspection for mutant primer, that is to say no matter DNA exists above which mutation type or which mutation type, and they can go to combine with corresponding mutant primer and general primer.But reaction efficiency and sensitive be different, we suppose the above 6 kinds of sudden changes of existence respectively in 526DNA, and content is identical, but the concentration of every kind of sudden change is all lower than LAMP lowest detection line, if detect so respectively and just can not examine mutator gene, but their tubular type is detected, be equivalent to total reaction volume substantially do not become situation under, effectively mutator gene concentration has improved nearly 6 times, so just completely likely sudden change is arrived in inspection, therefore this technology is not only and is reduced costs, the more important thing is and can improve greatly the simultaneous detection sensitivity of multimutation type.
For 6 kinds of sudden change modes, design primer as follows:
Six corresponding primers in mutational site:
Primer 1, TGACCTACAAGCGCCGACT
Primer 2, TGACCGACAAGCGCCGACT
Primer 3, TGACCCTCAAGCGCCGACT
Primer 4, TGACCCCCAAGCGCCGACT
Primer 5, TGACCGTCAAGCGCCGACT
Primer 6, TGACCAACAAGCGCCGACT
General primer:
526B3?CGACCACCTTGCGGTACG
526FIP?GTAGTGCGACGGGTGTTTTGGTCTGTCACGTGAGCGT
526BIP?ACCCCTGAGGGGCCCAACATTTTTTTCGATGAACCCGAACGG
526LB?TGTACGCGCGGGTCAA
Reaction system (reaction cumulative volume be still 25 μ l)
| Composition | Mother liquid concentration | (μ l) for consumption | Final concentration |
| Nucleic acid-templated | ? | 2.0 | ? |
| Primer 1 | 5μM | 1.0 | ? |
| Primer 2 | 5μM | 1.0 | ? |
| Primer 3 | 5μM | 1.0 | ? |
| Primer 4 | 5μM | 1.0 | ? |
| Primer 5 | 5μM | 1.0 | ? |
| Primer 6 | 5μM | 1.0 | ? |
| B3 | 5μM | 1.0 | 0.8 μM |
| FIP | 40μM | 1.0 | 1.6 μM |
| BIP | 40μM | 1.0 | 1.6 μM |
| LB | 20μM | 1.0 | 0.2 μM |
| dNTP | 150 mM | 2-4 | ? |
| Indicator | 10 mM | 1-2.5 | 0.4-1 mM |
| Bst DNA Polymerase Buffer | 10× | 2.5 | ? |
| Mg 2+ | 50 mM | 1.5-2.5 | ? |
| Bst DNA Polymerase | 8U/μl | 1 | 0.32 U/μl |
| ddH 2O | ? | Benefit to 25 μ l | ? |
Amplified reaction
59-65 ℃ of water-bath 20-60 minute.
Result judgement
After reaction finishes, color change is positive, illustrates that 526 sites exist transgenation, and hue preserving is constant negative.526 site yin and yang attribute contrasts as shown in Figure 1.
embodiment 2:
Two corresponding primers in mutational site:
Primer 1, AAGCGCCGACTGTT
Primer 2, AAGCGCCGACTGTG
531B3?CGTCGACCACCTTGCG
531FIP?GCCGTAGTGCGACGGGTGTTTTGCGGTCTGTCACGTGAGC
531BIP?GATCGAAACCCCTGAGGGGCTTTTGAACCCGAACGGGTTGAC
531LB?GATCGAAACCCCTGAGGGGC
Reaction system (reaction cumulative volume be 25 μ l)
| Composition | Mother liquid concentration | (μ l) for consumption | Final concentration |
| Nucleic acid-templated | ? | 2.0 | ? |
| Primer 1 | 5μM | 1.0 | ? |
| Primer 2 | 5μM | 1.0.0 | ? |
| B3 | 5μM | 1.0 | 0.8 μM |
| FIP | 40μM | 1.0 | 1.6 μM |
| BIP | 40μM | 1.0 | 1.6 μM |
| LB | 20μM | 1.0 | 0.2 μM |
| dNTP | 150 mM | 2-4 | ? |
| Indicator | 10 mM | 1-2.5 | 0.4-1 mM |
| Bst DNA Polymerase Buffer | 10× | 2.5 | ? |
| Mg 2+ | 50 mM | 1.5-2.5 | ? |
| Bst DNA Polymerase | 8U/μl | 1 | 0.32 U/μl |
| ddH 2O | ? | Benefit to 25 μ l | ? |
Amplified reaction
59-65 ℃ of water-bath 20-60 minute.
Result judgement
After reaction finishes, color change is positive, illustrates that 531 sites exist transgenation, and hue preserving is constant negative.531 site yin and yang attribute contrasts as shown in Figure 2.
Useful positively effect of the present invention:
The WHO 2009 new tuberculosis 4,400,000 in the annual report whole world, wherein many resistances tuberculosis is 420,000, extensively resistance tuberculosis accounts for 10% of many resistances tuberculosis; China's investigation shows that just controlling Drug Resistance for Tuberculosis rate is 10% left and right, controls tubercular's resistant rate again up to more than 35%.Resistance tuberculosis has become serious public health and has threatened.53 acquired immune deficiency syndrome (AIDS) in one group, South Africa merge resistance tubercular, and dead 52, wherein 70% is dead in 30 days; The treatment of resistance tuberculosis is very difficult with case control, and medical expense is far away higher than common tuberculosis, and simultaneously due to movement of population, resistance tubercular is difficult to find, manages and has blind area, and social crowd is caused to serious threat.
Not only in pulmonary tuberculosis field, in treating hepatitis B, also there is resistance, hepatitis B (hepatitis B) resistance mainly refers to that in body, hepatitis B (hepatitis B) virus morphs, and causes hepatitis B (hepatitis B) medicine of originally selecting no longer to bring into play any therapeutic efficiency.Also have acquired immune deficiency syndrome (AIDS) field all to occur Similar Problems.
Develop so a kind of simple and quick, highly sensitive detection method extremely urgent, this technology is that LAMP detection resistance has found a kind of practicable method.
Patent content of the present invention adopts loop-mediated isothermal amplification technique to put different mutation types to identical mutation and detects, detect sensitivity higher, detection time is short, easy and simple to handle, can realize again high throughput testing simultaneously, not only be applicable to large hospital use and also can use at middle and small hospital, to Pulmonary Tuberculosis Infection, provide molecular diagnosis fast.Simultaneously, it also can to multiple in the patients such as the type of resistance to multi-medicament tuberculosis, B-type hepatitis, acquired immune deficiency syndrome (AIDS) the most common transgenation detect, the early diagnosis of disease resistance contributes to immediately patient to be implemented to high-intensity treatment plan, thereby contributes to control transmission of disease.
Finally it should be noted that: above embodiment is only illustrative rather than definitive thereof application of the present invention, those skilled in the art is to be understood that: nucleic acid isothermal amplification and product that the present invention also can be used for other detect, and includes but not limited to that rolling circle amplification, the isothermal duplication that relies on helicase, chain substitute amplification, the amplification of nicking restriction endonuclease nucleic acid isothermal; Meanwhile, those skilled in the art is also to be understood that: can modify or be equal to replacement the present invention, in any modification or partial replacement that does not depart from the spirit and scope of the present invention, it all should be encompassed in claim protection domain of the present invention.
Claims (5)
1. the present invention is based on LAMP(loop-mediated isothermal amplification detects) tubular type of technology detects method or the theory of the different mutation types in identical mutation site, it is characterized in that: by loop-mediated isothermal amplification detection kit probe design, for the different mutation types of identical mutation point, only change the primer that covers catastrophe point place, the primer that covers other site constant (add quantity also constant), guaranteed like this, between the primer of the primer of catastrophe point and other some position, there is not cross reaction, only need checking to cover identical mutation and put between the primer at different mutation types place whether cross reaction occurs, cover so this primer herein and only have one to two base difference, also can not react, the theoretical basis of this detection mode that Here it is.
2. what according to right 1, require that we protect is that LAMP technology one tubular type detects the different mutation types in identical mutation site; that we propose the detection to different genes type based on LAMP technology first, as long as a tubular type detects different genes mutation type all at our protection domain.
We to protect be also theory and the method that LAMP technology one tubular type detects the different mutation types in identical mutation site simultaneously.
4. this technology also can be extended to detecting different mutational sites according to claim 1, this technology is to detect the basis in multiple mutational sites simultaneously, other primer that multiple sites design of primers is removed covering mutational site designs general primer as far as possible, avoids cross reaction.
5. this method is mainly used in the detection of various sudden change drug resistant genes (as pulmonary tuberculosis, hepatitis B, acquired immune deficiency syndrome (AIDS) etc.), simultaneously also can be used for same site and whether exist the detection of different effective gene forms.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105255863A (en) * | 2015-10-10 | 2016-01-20 | 北京晋祺生物科技有限公司 | Detection primers and compound of SLCO1B1521T>C |
Citations (1)
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| CN102712944A (en) * | 2009-11-05 | 2012-10-03 | 贝克顿·迪金森公司 | Sequence-specific methods for homogenous, real-time detection of lamp products |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102712944A (en) * | 2009-11-05 | 2012-10-03 | 贝克顿·迪金森公司 | Sequence-specific methods for homogenous, real-time detection of lamp products |
Non-Patent Citations (2)
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| CARL W. DIEFFENBACH ET AL.: "《PCR技术实验指南》", 30 September 2000, article "第三章 引物设计 第三节 多重PCR" * |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105255863A (en) * | 2015-10-10 | 2016-01-20 | 北京晋祺生物科技有限公司 | Detection primers and compound of SLCO1B1521T>C |
| CN105255863B (en) * | 2015-10-10 | 2019-08-23 | 北京晋祺生物科技有限公司 | A kind of detection primer and combinations thereof of SLCO1B1 521T > C |
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Application publication date: 20140416 |