CN103966333A - Biogen high-throughput qualitative detection method for multiple microdroplet polymerase chain reactions - Google Patents

Biogen high-throughput qualitative detection method for multiple microdroplet polymerase chain reactions Download PDF

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CN103966333A
CN103966333A CN201410215717.8A CN201410215717A CN103966333A CN 103966333 A CN103966333 A CN 103966333A CN 201410215717 A CN201410215717 A CN 201410215717A CN 103966333 A CN103966333 A CN 103966333A
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pcr
droplet
polymerase chain
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chain reaction
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赵凯
施伟
赵笑
唐雪明
任方方
吴洋洋
胡瑞丽
刘华
史斌
宋美萱
赵斌安
潘磊
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Shanghai Academy of Agricultural Sciences
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    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]

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Abstract

The invention discloses a biogen high-throughput qualitative detection method for multiple microdroplet polymerase chain reactions. An improved microdroplet PCR (polymerase chain reaction) high-throughput amplification technology is established, wherein the technology is improved on experimental apparatus, experimental method and reagent, experimental steps are optimized, some experimental links are reduced, apparatuses needed in a test are domesticated, so that the method is easier to perform, and needs on labor power, material resources and financial resources are greatly reduced, and therefore, the microdroplet PCR technology can be grasped by common experiment technical staff, and can be developed in most of domestic biological laboratories. Experimental results are clear and reliable, and therefore, the method disclosed by the invention can realize a high-throughput qualitative detection purpose by carrying out high-throughput amplification onto a large number of target DNA (deoxyribonucleic acid) molecules at the same time.

Description

The biological gene high-throughput qualitative checking method of a kind of multiple droplet polymerase chain reaction
Technical field
The invention belongs to biological gene detection technique field, relate to the biological gene high-throughput qualitative checking method to a kind of multiple droplet polymerase chain reaction.
Background technology
Traditional biological gene is qualitative, quantitative PCR detection technique can only detect single a kind of gene at every turn, and for the indefinite sample of gene element, need to be by multiple detection method, carry out many experiments and could determine, the cycle is long, workload is large, and cost is high.Therefore, in following biological gene detects, that the more and more not competent primary treatment of this detection technique contains is several, the special requirement of the sample detection of tens kinds, tens kinds biological genes, and the biological sample of especially large quantity needs more effective, high-flux detection method fast.Although multiple conventional PCR, quantitative PCR can once be tested and can be detected several genes simultaneously; but it is usually very poor in same reaction tubes, to carry out multiplex PCR effect; produce non-specific amplification, short fragment often can preferentially be increased, and the restructuring in homologous region often can produce artificial fragment.Its key is the design of primer, between primer and primer, primer itself, between product and product, can not there is mispairing, complicated multiple PCR primer design must depend on comparatively large-scale database and computer simulation PCR result, and the checking in later stage, this is not the thing that general small-size laboratory can complete.
At present both at home and abroad some mechanisms and company have carried out the research of high-flux detection method, the biochip technology of having developed some transgenosis detection kit etc. and being widely used in clinical study as Bio-Rad company, SGS feeler mechanism.Yet these detection method costs are very high, technical sophistication, personnel require high and need expensive instrument to complete.Current biological gene product inspection method both domestic and external still be take the detection method of single qualitative, quantitative PCR as main.
Droplet PCR(microdroplet PCR) technology is a kind of new and effective high-throughput nucleic acid DNA molecular amplification technique carrying out in emulsifying agent.Generally, in droplet PCR, single DNA molecules is assigned in the droplet of volume minimum (0.5fl is to the volume of 0.5nl), and this permission forms about 10 in the emulsifying agent of 1 microlitre 7individual reaction is parallel increases, and has eliminated the inherent defect of composite PCR.Improve the flux of target amplification, reduced the consumption of reagent and sample simultaneously.Traditional droplet PCR method more complicated, the reagent that requirement of experiment is too rigorous, use is more, and the instrument that needs is more and be mostly import.
In traditional droplet PCR experiment, what breakdown of emulsion process was used is ether method.By after the centrifugal sucking-off oil phase of PCR product, every pipe adds 1ml water saturation ether, vortex vibration, and the centrifugal 5min of room temperature 13000rpm, goes upper oil phase, repeats this step 2-3 time, with vacuum unit Vacuumcentrifuge (Concentrator5301; Eppendorf) vacuum pumps ether, then detects.Ether extraction is larger to the loss of PCR product, affects the collection of PCR product.And ether has danger, can cause the symptoms such as dizziness, headache after sucking in a large number ether, meet naked light explosive, by traditional vacuum device Vacuum centrifuge vacuum, pump the demand that ether increases laboratory apparatus, improved the cost of experiment.
Summary of the invention
The biological gene high-throughput qualitative checking method that the object of this invention is to provide a kind of multiple droplet polymerase chain reaction, the amplification of droplet composite PCR and denaturing gradient gel electrophoresis (DGGE) applied in any combination are detected in product, can greatly improve the flux of nucleic acid DNA analysis of molecules, a kind of means of analyzing as high-throughput DNA molecular in biological study, can detect a plurality of DNA moleculars simultaneously, improve the flux of analyzing, reduced the consumption of valuable sample and reagent.
The biological gene high-throughput qualitative checking method of a kind of multiple droplet of the present invention polymerase chain reaction, is that the water of oil phase and PCR reaction system is mixed, and utilizes the emulsifying technology of " water-in-oil " that PCR reaction system is separated into 10 9individual droplets, each droplet contains one or two templates and primer, each droplet represents a reaction, these reactions can be increased abreast under identical condition, carry out multiplex PCR simultaneously; Because genetically modified PCR product fragment sequence or the length of each different lines are variant, by droplet, PCR increases, different PCR product fragment sequences or size are variant, and then can to biological gene, carry out qualitative detection by denaturing gradient gel electrophoresis (DGGE); Because the product of the multiple different biological gene amplifications in same PCR pipe is different, therefore can detect multiple biological gene simultaneously, and then can carry out high throughput testing.
Specifically, the biological gene high-throughput qualitative checking method of a kind of multiple droplet of the present invention polymerase chain reaction, comprises the steps:
The first step, DNA sequence dna for biological sample to be measured designs respectively PCR Auele Specific Primer (comprising upstream primer or downstream primer), require that each is close to primer Tm value, the length of each target fragment is close as far as possible respectively, thereby be conducive to increase under the PCR parameter being equal to, primer length is generally between 20-25bp, and the object clip size of amplification is between 200-250bp.The specific primer that the present invention is directed to following four kinds of transgenic corns (Bt11, MON863, TC1507, NK603) design is as shown in table 1.
Table 1
Second step: droplet pcr amplification: in droplet PCR, use the upstream and downstream primer of the first step, in water emulsifier, their corresponding genomic dna templates are carried out to droplet pcr amplification.
Being formulated as follows of described water emulsifier:
At 25 ℃, get 200 μ l droplet PCR waters (table 2) in 1.5 minutes, the speed of dripping with 6 seconds/dropwise joins in 400 μ l droplet PCR oil phases (table 3), meanwhile utilize speed governing type magnetic stirring apparatus to mix, after adding, continue to mix 5 minutes, form uniform water emulsifier, it comprises the droplet differing in size, and in this water emulsifier, the diameter of droplet is 1～8 μ m, and the water emulsifier of every microlitre comprises 3 * 10 6to 1 * 10 7individual droplet.
Table 2 droplet PCR water component
Described droplet PCR oil phase is oil-surfactant mixture, at 25 ℃, in mixing each reagent as the ratio in table 3, obtains.
Table 3 droplet PCR oil phase component
Reagent Volume percent
Sorbester p17 4.5%
Tween 80 0.4%
Triton x-100 0.05%
Paraffin oil 95.05%
The 3rd step, PCR product separation and purifying: after droplet PCR completes, collect the water emulsifier of same reaction, and centrifugal 5 minutes of 13000rmp at room temperature, draw the water that lower floor comprises droplet pcr amplification product, and purify this water with PCR product purification test kit, remove primer, enzyme, inorganic ion, dNTPs etc., without with ether extraction with further with vacuum unit, ether is removed.
The 4th step, denaturing gradient gel electrophoresis detects PCR product, DGGE technology is on general polyacrylamide gel basis, the denaturing agent that has added gradient, thereby can be same length but the different DNA fragmentation of sequence make a distinction, thereby the different PCR product fragments in droplet PCR can be distinguished and carry out qualitative, high throughput testing.
Beneficial effect of the present invention:
The present invention has designed target Auele Specific Primer, adopt droplet pcr amplification method and in conjunction with denaturing gradient gel electrophoresis (denatured gradient gel electrophoresis, DGGE) detect the analytical procedure of product, make different DNA profiling molecules parallel amplification in droplet independently, greatly improved analysis efficiency and flux.It is target that the detection sequence of applying transgene crop is take in the present invention, further optimizes amplification system and the condition of the method, and handiness and the usability of more outstanding the inventive method, make it have high-throughput, high specific and highly sensitive.Can overcome the insurmountable non-specific amplification phenomenon of conventional multiplex PCR.
Detection method of the present invention has been optimized experimental procedure, and emulsification is the committed step of droplet PCR, and emulsion process requires to complete in 1.5min, as calculated, within every 6 seconds, drip one, in 1.5 minutes, add 200 μ l, strictly controlled the time, the error that reduces to bring in experimentation;
Experimental technique is simplified, and has reduced some experiment links.After the PCR product obtaining at collection droplet pcr amplification, through centrifugal, take off a layer water, clean PCR product or directly carry out corresponding product analysis, substituted ether method used in traditional droplet PCR experiment, this experimental technique is simple to operate, without with ether extraction with further with traditional vacuum device, ether is removed; Experimental period is short, and the product efficiency after cleaning is higher, the danger of also having avoided ether to bring; The washing lotion that stirrer after emulsification is used by cell cultures is carried out the cracking of nucleic acid, has eliminated the risk of the crossed contamination of DNA profiling.
The instrument using in droplet process of the present invention all domesticizes, and compares external import instrument, has greatly reduced the cost of experiment; Experiment is more easily carried out, greatly reduced the demand of human and material resources and financial resources, make droplet round pcr can be general experimental technique personnel and grasp, most of biology laboratories are carried out at home.
The inventive method not only can be applicable to the analysis of transgene component, and the selection of a new qualitative and high throughput analysis is also provided for DNA molecular analysis in other field.Therefore, this technology is except being applied to the qualitative and high throughput testing of multiple transgenic product, also can be applicable to food borne pathogenic microorganism, animal and veterinary pathogenic microorganism, human disease microorganism, aquatic products pathogenic microorganism, environmental microorganism, plant pathogenic microorganism is qualitative and high throughput testing, also can also for the qualitative and high throughput testing of multiple study of human disease-related gene position and according to the mutant gene detecting for genetic counseling.In a word, the inventive method can be for the qualitative and high throughput testing of all biological genes.
Accompanying drawing explanation
Fig. 1 is droplet PCR schematic flow sheet of the present invention.
Fig. 2 is constant speed, the constant temperature magnetic force heating stirrer 43-2S of Shanghai Gong Hui Trade Co., Ltd..
Fig. 3 is domestic 2ml cillin bottle.
Fig. 4 is domestic 3 * 8mm stirrer.
Fig. 5 is domestic 8 PCR tubules.
Fig. 6 is DDGE electrophoresis chamber.
Fig. 7 is DDGE gel making device.
Fig. 8 is conventional multiplex PCR and PCR result comparison diagram of the present invention; The left side is conventional multiplex PCR result, and the right is droplet multiplex PCR result of the present invention; 1. transgenic corns TC1507; 2. transgenic corns BT176; 3. genetically modified corn MON 863; 4. transgenic corns NK603; 5. transgenic corn BT 11; 6. corn Inner source gene IVR.
Embodiment
Below in conjunction with specific embodiments and the drawings, technical scheme of the present invention is described in further detail, but described embodiment does not limit the scope of the invention.
The test method of unreceipted actual conditions in embodiment below, conventionally according to normal condition, for example Sambrook equimolecular is cloned: laboratory manual (New York:Cold Spring HarborLaboratory Press, 1989) condition described in, or the condition of advising according to manufactory.
In experiment, instrument has been carried out to production domesticization and has replaced, during preparation emulsion, utilized the constant speed of domestic Shanghai Gong Hui Trade Co., Ltd., constant temperature magnetic force heating stirrer 43-2S(to see Fig. 2) replaced constant speed, the constant temperature magnetic force heating stirrer of import; The container that uses domestic cillin bottle (see figure 3) to replace the Cryo Tube bottle of import to prepare as emulsion droplets; Use domestic 3 * 8mm stirrer (see figure 4) to replace the stirrer 3 * 8mm of import; Use domestic PCR eight connecting leg (see figure 5)s to replace PCR eight connecting legs of import.
Embodiment, referring to Fig. 1, detects the specificity of 5 transgenic strains and reference gene, i.e. qualitative detection, and step is as follows:
The first step: vegetable material and DNA extraction thereof
Genetically modified crops: Bt11 corn, MON863 corn, TC1507 corn, NK603 corn, Bt176 corn.
DNA of plants is extracted: the DNA of plants extraction agent box of application Academy of Agricultural Sciences, Shanghai City and Shanghai Entry-Exit Inspection and Quarantine bureau's joint research and development extracts and purifying plant genome DNA, and the DNA that gets the 260ng of each sample carries out droplet PCR as the template of further experiment in this example.
Second step: target Auele Specific Primer design
Design 5 pairs of target Auele Specific Primers (referring to table 1), specific amplification clip size is in 250bp left and right.
The 3rd step: droplet preparation
1) preparation of oil-surfactant mixture
At 25 ℃, in the ratio in table 3, mix each reagent, preparing oil-surfactant mixture is droplet PCR oil phase.
2) preparation of water emulsifier
At 25 ℃, get 200 μ l droplet PCR waters (table 2), in 1.5 minutes, dropwise join in 400 μ l droplet PCR oil phases (table 3), within average every 6 seconds, use 200 μ l liquid-transfering guns to drip one, meanwhile utilize speed governing type magnetic stirring apparatus to mix, the strict period, the error that reduces to bring in experimentation, after being added dropwise to complete, continue to stir 5 minutes, substituted the emulsion drop generator of import, after adding, continued to mix 5 minutes, form uniform water emulsifier, water emulsifier comprises the droplet differing in size.
The 4th step: droplet pcr amplification
The water emulsifier evenly distribute of getting 600 μ l is divided and is installed in 12 PCR pipes, gets in addition 50 μ l droplet PCR waters (table 2) and directly adds in PCR pipe for non-emulsification contrast, by carrying out droplet pcr amplification as the condition of following table 4.
Table 4
Cycle number Temperature (℃) Time
1 94 5min
94 30s
30 56 40s
72 30s
1 72 7min
The 5th step: PCR product separation and purifying
The reaction solution that droplet pcr amplification is completed is collected in the eppendof pipe of a 1.5ml, under 13000rpm condition centrifugal 5 minutes, oil phase wherein and aqueous phase separation are opened, take out the water react liquid 100ul of lower floor, with Anygen AxyPrep PCR cleaning agents box, product is cleaned, effectively remove the remaining primer of reaction and some inorganic ions, enzyme etc., use 60ul ddH 2o wash-out, without with ether extraction with further with vacuum unit, ether is removed.
The 6th step: denaturing gradient gel electrophoresis (denatured gradient gel electrophoresis, DGGE) detects droplet PCR product
Main experimental procedure is as follows:
1) sponge pad is fixed on a gum-making rack, the glue plate system of similar " sandwich " structure is vertically placed on to sponge top, with the eccentric wheel that is distributed in gum-making rack both sides, fix glue plate system (see figure 6);
2) one have three polyethylene suction pipes, wherein two longer, one is shorter, that short root is connected with Y tube, two long being connected with small casing, and being connected on syringe;
3) mark " high density " and " lower concentration " on two syringes, and install relevant accessory, adjust the scale of gradient transfer system to suitable position, be rotated counterclockwise cam to zero position;
4) prepare in acrylamide soln to two centrifuge tube of two kinds of sex change concentration 30% and 70%, every pipe adds 18 μ l TEMED, and 80 μ l10%APS cover rapidly and screw after cap, turns upside down and mixes for several times.With the syringe that is connected with polyethylene tube and indicates " high density ", draw the glue of all high densitys, the same for the glue operation of lower concentration;
5) carefully drive bubble away and rock syringe, promoting solution to the end of polypropylene tube;
6) a correct side that respectively high density and lower concentration syringe is placed on to gradient transfer system fixes, and the polypropylene tube of syringe is connected with Y tube;
7) soft and stably rotating cam transmit solution, keep constant actuating cam lentamente rapidly so that solution constant speed be circulated in gel slab;
8) carefully insert comb, allow gel polymerisation 1 hour, electrophoresis control device is opened, and preheating electrophoretic buffer to 60 ℃ cleans rapidly the equipment being finished;
9) after 1 hour, pull out away comb, glue is placed on to (see figure 7) in electrophoresis chamber, clean point sample hole, covers temperature-control device and makes temperature rise to 60 ℃;
10) point sample, electrophoresis (200V, 5h);
11) after electrophoresis, first push a sheet glass aside, then glue is put into dish, with deionized water rinsing, glue and sheet glass are departed from, outwell deionized water, add in 250ml stationary liquid (10% ethanol, 0.5% Glacial acetic acid), place 15 minutes;
12) outwell stationary liquid, with deionized water rinsing twice, after outwelling, add 250ml silver dye liquor (0.2%AgNO3, with before add 200 μ l formaldehyde), be placed on shaking table and dye 15 minutes;
13) outwell silver-colored dye liquor, with deionized water rinsing twice, after outwelling, add 250ml nitrite ion (1.5%NaOH, 0.5% formaldehyde) colour developing;
14) after occurring, band takes pictures.
The 7th step: qualitative detection and interpretation of result
As shown in Figure 8, can find out that the experimental result effect that the instrument with the improved droplet PCR method of the present invention and production domesticization obtains is very good, picture is very clear, and band is obvious, and background is clear.The conventional multiplex PCR result in the left side than the right droplet multiplex PCR result many a band, be the non-specific amplification of conventional multiplex PCR.Although we have used multiple biosoftware to design when the conventional multiple PCR primer of design, and compare in whole biological gene storehouse, then after synthetic primer, every pair of primers is verified, prove that specificity meets the requirements.6 pairs of transgenic corns primers and template are put together while carrying out conventional multiplex PCR, still produce a non-specific band.Visible droplet multiplex PCR can overcome the insurmountable non-specific amplification phenomenon of conventional multiplex PCR.
Finally should be noted that, above embodiment is only unrestricted in order to technical scheme of the present invention to be described, although the present invention is had been described in detail with reference to preferred embodiment, those of ordinary skill in the art is to be understood that, can modify or be equal to replacement the technical scheme of invention, and not departing from the spirit and scope of technical solution of the present invention, it all should be encompassed in claim scope of the present invention.

Claims (5)

1. a biological gene high-throughput qualitative checking method for multiple droplet polymerase chain reaction, is characterized in that, comprises the following steps:
The first step, designs respectively PCR Auele Specific Primer pair for biological sample to be measured;
Second step, is used above-mentioned Auele Specific Primer to corresponding DNA profiling being carried out to droplet pcr amplification in water emulsifier;
The concrete compound method of described water emulsifier is: at 25 ℃, get the droplet PCR water of 1 parts by volume, the speed of dripping with 6 seconds/, at the uniform velocity splashes in the droplet PCR oil phase of 2 parts by volume, the uniform water emulsifier of formation after mixing, and it comprises the droplet differing in size; In described water emulsifier, the diameter of droplet is 1～8 μ m, and the water emulsifier of every microlitre comprises 3 * 10 6to 1 * 10 7individual droplet;
The 3rd step, PCR product separation and purifying: after the PCR product that collection droplet pcr amplification obtains, through centrifugal, take off a layer water, clean PCR product or directly carry out corresponding product analysis;
The 4th step, detects PCR product, uses denaturing gradient gel electrophoresis to detect.
2. the biological gene high-throughput qualitative checking method of multiple droplet according to claim 1 polymerase chain reaction, is characterized in that, described biological sample to be measured and corresponding Auele Specific Primer are to as follows:
Corn Inner source gene IVRL primer is:
IVRL-fp1：5’-GTATCACAAGGGCTGGTACC-3’
IVRL-fp2：5’-CCGTCTAGAGCATGACGATC-3’。
3. the biological gene high-throughput qualitative checking method of multiple droplet according to claim 1 polymerase chain reaction, is characterized in that, described droplet PCR water consists of:
4. the biological gene high-throughput qualitative checking method of multiple droplet according to claim 1 polymerase chain reaction, it is characterized in that, the volume percent of described each component of droplet PCR oil phase is: sorbester p17 4.5%, tween 80 0.4%, triton x-100 0.05%, paraffin oil 95.05%.
5. the biological gene high-throughput qualitative checking method of multiple droplet according to claim 1 polymerase chain reaction, is characterized in that, in the 3rd step, uses Anygen AxyPrep PCR cleaning agents box to clean PCR product.
CN201410215717.8A 2014-05-20 2014-05-20 Biogen high-throughput qualitative detection method for multiple microdroplet polymerase chain reactions Pending CN103966333A (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106755548A (en) * 2017-03-14 2017-05-31 中国农业科学院生物技术研究所 A kind of transgenic corns high-flux detection method based on oil droplet generation technique
CN107723346A (en) * 2017-11-28 2018-02-23 上海市农业科学院 A kind of droplet PCR for transgenic corns Multiple detection is coupled DGGE analysis methods
CN114752657A (en) * 2022-05-05 2022-07-15 中山大学 Polydisperse liquid drop digital nucleic acid detection method and application thereof

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101948926A (en) * 2010-10-02 2011-01-19 上海交通大学 Method for analyzing droplet polymerase chain reaction based on capillary gel electrophoresis detection
CN103757115A (en) * 2014-01-17 2014-04-30 上海市农业科学院 Biological gene qualitative, quantitative and high-flux detection method based on multiple microdroplet PCR (Polymerase Chain Reaction) coupling fluorescence spectrophotometry

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101948926A (en) * 2010-10-02 2011-01-19 上海交通大学 Method for analyzing droplet polymerase chain reaction based on capillary gel electrophoresis detection
CN103757115A (en) * 2014-01-17 2014-04-30 上海市农业科学院 Biological gene qualitative, quantitative and high-flux detection method based on multiple microdroplet PCR (Polymerase Chain Reaction) coupling fluorescence spectrophotometry

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106755548A (en) * 2017-03-14 2017-05-31 中国农业科学院生物技术研究所 A kind of transgenic corns high-flux detection method based on oil droplet generation technique
CN107723346A (en) * 2017-11-28 2018-02-23 上海市农业科学院 A kind of droplet PCR for transgenic corns Multiple detection is coupled DGGE analysis methods
CN114752657A (en) * 2022-05-05 2022-07-15 中山大学 Polydisperse liquid drop digital nucleic acid detection method and application thereof

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Application publication date: 20140806