CN108660191A - A kind of digitlization multiple nucleic acid detection method based on coding microball reactor - Google Patents
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Abstract
The digitlization multiple nucleic acid detection method based on coding microball reactor that the invention discloses a kind of, including sample nucleic acid extraction, design of primers, primer mark, hybrid capture, droplet generation, amplified reaction and Scanning Detction and etc.；Involved nucleic acid detection method combines the multiple gene detection advantage of hybridization technique and the high sensitivity quantitation detection advantage of digital pcr in the present invention, is filled with the blank in multiple gene quantitative measurement technology field, has far-reaching influence.
Description
Technical field
The present invention relates to Molecular Detection fields, and in particular to a kind of digitlization multiple nucleic acid based on coding microball reactor
Detection method.
Background technology
The principle of multiplex PCR (Polymerase Chain Reaction, PCR) is same in the reaction system
When multipair specific primer is added, while a plurality of target DNA fragment is amplified, since the size of the amplified fragments of each primer exists
Notable difference is detected while being hybridized the multiple microorganisms of realization according to the clip size of product or using molecule.From head in 1988
Since secondary proposition multiplex PCR concept, by development for many years, multiple PCR technique is gradually ripe, the hand of multiplex PCR detection at present
Section mainly has the modes such as Capillary Electrophoresis (or gel electrophoresis), liquid-phase chip, mass spectrum.Capillary Electrophoresis be according to PCR product not
Same amplified fragments size carries out separation detection to it, which is also one of most common technology of molecular biology.
With the appearance of liquid-phase chip technology, multiplex PCR realizes high-throughput detection.Liquid-phase chip technology is with polystyrene
Microballoon is carrier, and liquid-phase chip is characterized in that system is made of many different microspheres (Beads), solid on microsphere
Surely there is different probe molecules, and form special microballoon code identification system, each bead is all equivalent on solid phase chip
One site.These spherulas are suspended in a multiple PCR products and are hybridized, liquid-phase chip analysis system is then passed through
The fluorescence signal for reading each coding microball, using this system, can to the kind amplified production in the same sample simultaneously into
Row detection.
Round pcr is current most widely used nucleic acid detection technique, and main advantage is simplicity, stablizes, using base
Plinth is good.But the primary limitation of round pcr is that multiple gene detects the target gene limited amount that can cover, and to realize Multiple detection
Multitube is generally required to realize.Although some current methods can realize Multiple detection, but still there are problems that, existing
Multiple detection technology is all to first pass through multiplexed PCR amplification, and this more targets are augmented with problems with：Specificity and sensitivity are not
It is ideal；The a certain limited amplification of specific fragment；Due to existing simultaneously several pairs of even tens pairs of primers in multi-PRC reaction system, make
The probability that primer dimer must be formed increases；If nonspecific products preferential amplification, it will largely consume reaction substrate, reduce
Specific amplification efficiency.In view of the above-mentioned problems, researchers develop the multiple PCR technique of various new, such as double startup widow's core
Thuja acid primer, multiple join dependency formula probe amplification, specific targets extend, target sequence is enriched with multiplex PCR, nest-type PRC etc..This
A little technologies improve in some aspects multiplex amplification there are the problem of, but need when detecting and other technologies means such as liquid phase
Chip hybridization technology combination could realize Multiple detection；In addition, existing Multiple detection can not carry out quantitative analysis.
In recent years fast-developing digital pcr (Digital PCR, dPCR) technology is a kind of new nucleic acid quantification detection side
Method, different from traditional quantitative PCR (qPCR) technologies, digital pcr is by the way of absolute quantitation, independent of standard curve and ginseng
This, directly detects the copy number of target sequence in the same old way.Since this detection method is with outstanding than traditional qPCR technologies
Sensitivity, specificity and accuracy, dPCR are widely used rapidly.The digital pcr detection architecture overwhelming majority is using classical
The bis- probe mentalities of designing of TaqMan, the Multiple detection of dPCR either passes through the extension of fluorescence channel, or uses
The method that RainDance companies change probe all can not reliably realize single tube (or single-chip) genetic test again more than 5.
Hybridization technique and the targeted application target of dPCR technologies are entirely different：Hybridization technique protrudes multiple gene inspection
Analysis ability is surveyed, multiplex amplification is realized by traditional approach, although sensitivity can be improved by PCR amplification, is all
Qualitative or half-quantitative detection；DPCR is short slab in terms of Multiple detection, but its absolute quantitation ability and detection sensitivity make
Hybridization technique is unmatch.For the technological gap present in the quantitative context of detection in multiple gene, in combination with hybridization skill
The advantage of art and dPCR technologies is to fill the research direction of this technological gap.
Invention content
For the shortcomings of the prior art, the purpose of the present invention is to provide one kind to be reacted based on coding microball
The digitlization multiple nucleic acid detection method of device improves the single of digital pcr to improve the problem of traditional multiplex PCR interferes with each other
Tuple is detected, realizes multiple digital nucleic acid quantification detection.
The digitlization multiple nucleic acid detection method based on coding microball reactor that the present invention provides a kind of, including following steps
Suddenly：
1) sample nucleic acid extracts：After being pre-processed to biological sample to be measured, by cracking, combination, washs and strip
Journey extracts its amplifying nucleic acid；
2) design of primers：Respective specific primer is designed according to different specific gene segments to be checked；
3) primer mark：The specific primer of same gene to be checked is marked on coding microball of the same race, obtain respectively by
The coding microball of the primer mark of different genes to be checked；
4) hybrid capture：By the biological sample nucleic acid to be measured extracted in step 1) and the specific primer on coding microball into
Row denaturation hybridization, makes the DNA profiling molecule of biological sample to be measured be specifically incorporated on coding microball, obtains capture dna
The coding microball of template molecule, and set up the correspondence of each biological sample to be measured and coding microball；
5) droplet generates：The coding microball obtained in step 4) and pcr reagent are uniformly mixed to form water
Phase generates droplet of the same size using drop generators；Wherein, it contains up to obtain in a step 4) in each droplet
Coding microball；
6) amplified reaction：Polymerase chain amplified reaction is independently carried out in each droplet, mutually fusion reaction does not produce each droplet
Object；
7) Scanning Detction：Droplet or coding microball therein are scanned one by one with analytical instrument, read each compile
The signal of code microballoon is based on digital multiplex polymerase chain reaction principle using coding microball as counting unit, realizes biological sample to be measured
The quantitative and Multiple detection of this amplifying nucleic acid.
Preferably, biological sample to be measured described in step 1) includes a variety of biological samples, especially a variety of different micro-
Biological sample.
Preferably, coding microball described in step 3) is fluorescence-encoded micro-beads, Raman coding microspheres or fluorescence and grain size
Hybrid coding microballoon, material are selected from polystyrene microsphere, silicon dioxide microsphere, polyacrylic acid microballoon or polymethylacrylic acid and shrink
One kind in glycerine ester microsphere.
Preferably, the biological sample nucleic acid to be measured that denaturation hybridization is carried out described in step 4) is DNA or RNA；Wherein, institute
It states DNA and carries out hybrid capture directly as template molecule；The RNA carries out hybridization as template molecule after carrying out reverse transcription and catches
It obtains.
Preferably, pcr reagent described in step 5) includes archaeal dna polymerase, three phosphorus of dezyribonucleoside
Acid, magnesium ion, buffer solution and non-specific fluorescence labeling dye.
Preferably, the droplet generated in step 5) is placed in water-in-oil system.
Preferably, three kinds of droplets are generated in step 5)：Contained coding microball capture DNA profiling molecule droplet,
The droplet of primer, the droplet without coding microball is only marked in contained coding microball.
The beneficial effects of the invention are as follows：Involved nucleic acid detection method combines the multiple base of hybridization technique in the present invention
Because the high sensitivity quantitation of detection advantage and digital pcr detects advantage, it is filled with the sky in multiple gene quantitative measurement technology field
In vain, there is initiative meaning and far-reaching influence.Using the oil-water interfaces droplet comprising microballoon as PCR reactors in this case,
Include a fluorescence-encoded micro-beads in each droplet, which can efficiently realize because of the parallel processing of million magnitude microballoons
Reactor scale, while including enough archaeal dna polymerases in droplet, PCR raw materials required for the reactions, each droplet is equivalent to independence
PCR reaction systems can effectively reduce the non-specific hybridization reaction of a variety of probes in multi-PRC reaction, it is anti-also to can avoid competition
It should lead to the problem of substrate deficiency.
Fluorescence-encoded micro-beads based on different fluorescent dye types and concentration combination structure are formed with specific primer to be mapped
Relationship, each fluorescence-encoded micro-beads contain only one for specific gene for a kind of cls gene to be checked, therefore in each droplet
To primer.Due to containing only pair of primers in each droplet, during PCR, it not will produce primer non-specific amplification etc. and ask
Topic；Meanwhile having enough substrate and enzyme in each droplet, competitive amplification, which will not occur, leads to the problem of substrate deficiency.It summarizes
For, for each droplet similar to a pipe of substance PCR, the amplified reaction of each droplet is relatively independent.
It after the technical solution, can both improve the problem of traditional multiplex PCR interferes with each other, can also improve digital pcr
Single detect tuple, be a kind of very effective multiple digital nucleic acid detection method.
Description of the drawings
Fig. 1 is the overall technology flow chart of amplifying nucleic acid detection method of the present invention；
Fig. 2 is PCR amplification schematic diagram.
Specific implementation mode
Present invention will be described in further detail below with reference to the accompanying drawings, to enable those skilled in the art with reference to specification text
Word can be implemented according to this.
It should be appreciated that such as " having ", "comprising" and " comprising " term used herein are not discharged one or more
The presence or addition of a other elements or combinations thereof.
It is further illustrated the present invention below by way of specific embodiment.But the detail of embodiment is only used for explaining this hair
It is bright, it should not be construed as limited overall technical solution.
The digitlization multiple nucleic acid detection method based on coding microball reactor that the present embodiment provides a kind of, including following steps
Suddenly：
1) sample nucleic acid extracts：The microorganism in various sources is obtained as sample, is dispersed in water phase, suspension is made
Liquid is treated after micrometer biological sample pre-processed, is extracted by magnetic bead and obtain its amplifying nucleic acid, specific extraction is divided into cracking, knot
Close, wash and elute Four processes；
2) design of primers：Respective specific primer is designed according to different specific gene segments to be checked；
3) primer mark：The specific primer of same gene to be checked is marked on same coding microball, is distinguished
By the coding microball of the primer mark of different genes to be checked, wherein coding microball uses fluorescence polymethyl acid glycidyl
Ester microsphere；
4) hybrid capture：By the biological sample nucleic acid to be measured extracted in step 1) and the specific primer on coding microball into
Row denaturation hybridization, makes the DNA profiling molecule of biological sample to be measured be specifically incorporated on coding microball, obtains capture dna
The coding microball of template molecule chain, and set up the correspondence of biological sample and coding microball to be measured；Wherein, biological sample to be measured
This nucleic acid be DNA when can directly as template molecule carry out hybrid capture, when be RNA need elder generation reverse transcription be DNA after be re-used as
Template molecule carries out hybrid capture；
5) droplet generates：By the coding microball obtained in step 4) and archaeal dna polymerase, deoxyribonucleoside triphosphate, magnesium
The pcr reagents such as ion, buffer solution and non-specific fluorescence labeling dye are uniformly mixed to form water phase, utilize droplet
Generator generates droplet of the same size in water-in-oil system；Wherein, it contains up to obtain in a step 4) in each droplet
The coding microball arrived；
6) amplified reaction：Polymerase chain amplified reaction is independently carried out in each droplet, each droplet does not merge mutually；Wherein,
Contained coding microball captures the droplet of DNA profiling molecule after PCR amplification, and template molecule increases significantly, while non-specific
Property labeling dye be embedded in double-stranded DNA；Contained coding microball is only marked the droplet of specific primer and without the micro- of coding microball
Drop does not change after PCR；
7) Scanning Detction：Droplet is scanned one by one with liquid-phase chip analyzer, reads the signal of each droplet, is realized
The quantitative and Multiple detection of biological sample amplifying nucleic acid to be measured.
Although the embodiments of the present invention have been disclosed as above, but its is not only in the description and the implementation listed
With.It can be applied to various suitable the field of the invention completely.It for those skilled in the art, can be easily
Realize other modification.Therefore without departing from the general concept defined in the claims and the equivalent scope, the present invention is simultaneously unlimited
In specific details and embodiment shown here.
Claims (7)
1. a kind of digitlization multiple nucleic acid detection method based on coding microball reactor, which is characterized in that include the following steps：
1) sample nucleic acid extracts：After being pre-processed to biological sample to be measured, carried by cracking, combination, washing and elution process
Take its amplifying nucleic acid；
2) design of primers：Respective specific primer is designed according to different specific gene segments to be checked；
3) primer mark：The specific primer of same gene to be checked is marked on coding microball of the same race, is obtained different respectively
The coding microball of the primer mark of gene to be checked；
4) hybrid capture：The biological sample nucleic acid to be measured extracted in step 1) is become with the specific primer on coding microball
Property hybridization, so that the DNA profiling molecule of biological sample to be measured is specifically incorporated on coding microball, obtain capture dna template
The coding microball of molecule, and set up the correspondence of each biological sample to be measured and coding microball；
5) droplet generates：The coding microball obtained in step 4) and pcr reagent are uniformly mixed to form water phase, profit
Droplet of the same size is generated with drop generators；Wherein, the coding obtained in a step 4) is contained up in each droplet
Microballoon；
6) amplified reaction：Independently carry out polymerase chain amplified reaction in each droplet, the mutual not fusion reaction product of each droplet；
7) Scanning Detction：Droplet or coding microball therein are scanned one by one with analytical instrument, reading each encodes micro-
The signal of ball is based on digital multiplex polymerase chain reaction principle, realizes in biological sample to be measured using coding microball as counting unit
The quantitative and Multiple detection of nucleic acid.
2. detection method according to claim 1, which is characterized in that biological sample to be measured described in step 1) includes a variety of
Biological sample.
3. detection method according to claim 1, which is characterized in that coding microball described in step 3) is fluorescence-encoded micro-
Ball, Raman coding microspheres or fluorescence and grain size hybrid coding microballoon, material are selected from polystyrene microsphere, silicon dioxide microsphere, gather
One kind in acrylic microspheres or poly (glycidyl methacrylate) microballoon.
4. detection method according to claim 1, which is characterized in that carry out the life to be measured of denaturation hybridization described in step 4)
Object sample nucleic acid is DNA or RNA；Wherein, the DNA carries out hybrid capture directly as template molecule；The RNA is being carried out instead
After transcription hybrid capture is carried out as template molecule.
5. detection method according to claim 1, which is characterized in that pcr reagent described in step 5) includes
Archaeal dna polymerase, deoxyribonucleoside triphosphate, magnesium ion, buffer solution and non-specific fluorescence labeling dye.
6. detection method according to claim 1, which is characterized in that the droplet generated in step 5) is placed in water-in-oil system
In.
7. detection method according to claim 1, which is characterized in that generate three kinds of droplets in step 5)：Contained coding
Microballoon captures the droplet of DNA profiling molecule, the droplet of primer is only marked in contained coding microball, without the micro- of coding microball
Drop.
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Cited By (5)
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| CN109536384A (en) * | 2018-12-24 | 2019-03-29 | 中国科学院上海微系统与信息技术研究所 | A kind of digital pcr system and its application for the quick absolute quantitation of nucleic acid |
| CN111206081A (en) * | 2018-11-21 | 2020-05-29 | 思纳福(北京)医疗科技有限公司 | Nucleic acid detection microsphere, preparation method, kit and high-throughput nucleic acid detection method |
| CN112210589A (en) * | 2019-07-09 | 2021-01-12 | 苏州宇测生物科技有限公司 | Quantitative detection method for nucleic acid molecules |
| CN113881791A (en) * | 2021-11-08 | 2022-01-04 | 国科温州研究院(温州生物材料与工程研究所) | Light-responsive gel microsphere for nucleic acid detection by dPCR method and application of light-responsive gel microsphere in detection of escherichia coli |
| CN115993666A (en) * | 2023-03-23 | 2023-04-21 | 成都理工大学 | Preparation method and application of oil-based silicon-coated DNA tracer |
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| CN113881791B (en) * | 2021-11-08 | 2023-11-24 | 国科温州研究院(温州生物材料与工程研究所) | Light-responsive gel microsphere for dPCR method nucleic acid detection and application thereof in escherichia coli detection |
| CN115993666A (en) * | 2023-03-23 | 2023-04-21 | 成都理工大学 | Preparation method and application of oil-based silicon-coated DNA tracer |
| CN115993666B (en) * | 2023-03-23 | 2023-07-07 | 成都理工大学 | Preparation method and application of oil-based silicon-coated DNA tracer |
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Application publication date: 20181016 |