CN107164366A - A kind of preparation method and its detection method for possessing double single stranded end PCR primers - Google Patents
A kind of preparation method and its detection method for possessing double single stranded end PCR primers Download PDFInfo
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- CN107164366A CN107164366A CN201710492708.7A CN201710492708A CN107164366A CN 107164366 A CN107164366 A CN 107164366A CN 201710492708 A CN201710492708 A CN 201710492708A CN 107164366 A CN107164366 A CN 107164366A
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- 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
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
Abstract
The present invention relates to a kind of preparation method for possessing double single stranded end PCR primers, comprise the following steps：(1) the forward primer F and reverse primer R sequences designed for amplification purpose nucleic acid, and Tag1 and Tag2 sequences are added respectively at the 5' ends of forward primer F and reverse primer R sequences, separated respectively with some thymidines between Tag1 sequences and forward primer F sequences, between Tag2 sequences and reverse primer R sequences, synthetic primer Tag1 F and Tag2 R；(2) at least 30min is irradiated to primer Tag1 F and Tag2 the R aqueous solution with ultraviolet；(3) mixing PCR reaction systems, which are placed in PCR instrument, is reacted, and obtains the PCR primer for possessing double single stranded ends.The detection of PCR primer of the present invention, it is not necessary to extra modification is carried out to primer, while also eliminating the cumbersome operating procedure such as electrophoresis, making the detection of PCR primer becomes convenient and swift, detection efficiency can be improved, cost is reduced, be particularly suitable for use in detection or remote districts in real time are used.
Description
Technical field
The invention belongs to nucleic acid field, and in particular to a kind of preparation method for possessing double single stranded end PCR primers and
Its detection method.
Background technology
PCR (PCR) is a kind of Protocols in Molecular Biology for being used to expand specific DNA fragmentation, and it can see
Work is the special DNA replication dna of in vitro.Round pcr by by template nucleic acid in high temperature (95 DEG C) time variation into single-stranded, in drop
(being down to 50 DEG C -60 DEG C) after temperature, primer is specifically bound with template according to base pair complementarity；In most suitable extension temperature (72
DEG C or so), archaeal dna polymerase is with the reaction of 5'-3' direction composition complementary strand.Whole reaction by high-temperature denatured, process annealing and
The circular response of thermophilic extension composition is carried out.One circulation is needed 2~4 minutes, target DNA is expanded rapidly within 2~3 hours,
Will millions of times of target gene amplification amplification be expanded.Because round pcr high specificity, sensitivity it is high, easy, it is quick the advantages of and by
Extensive use.Not only it is commonly used for the basic research of nucleic acid, round pcr is also the goldstandard of current detection of nucleic acids, in medical science, life
The fields such as thing engineering are widely used.PCR primer is the double-stranded DNA with flat end, generally comprise by agarose gel electrophoresis,
Polyacrylamide gel electrophoresis etc. is separated, and then needs to be analyzed by dyeing and ultraviolet transmission imaging.Aforesaid operations
Complex steps, time-consuming, and relies on the professional of instrument and special training, is not suitable for high flux, the inspection of quick PCR primer
Survey.
The technology detected using nucleic acid test strip is the new technology of a detection PCR primer, and the technology is generally required
Double modifications or other special processing are carried out to PCR primer sequence：First, double Mdification primers：In order to be pressed from both sides in test strips
The detection of heart method, which is required for carrying out the small molecule that double modifications are usually used in mark, a Biotin, Digoxin, FITC, Cy3 etc.,.These are small
Molecule is modified in the end of primer, and when primer and template annealing extension, small molecule is also incorporated into product end, from
And reach the purpose of mark PCR primer.Such as Wansika when detecting blackspot syndrome and white spot syndrome virus nucleic acid,
Mark Biotin and FITC respectively on two primers, thus Streptavidin (SA) coated collaurum can by SA with
Biotin high-affinity is acted on and capture zone Biotin-Tag amplified production.Because the T lines region in test strips is drawn in advance
There is FITC antibody, the product with FITC-Tag is fixed on by T lines area by the specific effect of FITC antibody and antigen
Domain, realizes the quick detection to viral nucleic acid.Although this modification can realize quick detection, need to carry out primer
Double labelling, adds the cost of detection.2nd, double modifications increase temperature denaturation treatment：Pcr amplification product is typically all that flat end is double
Chain, if not carrying out molecular modification in end, then prominent cohesive end is not used to hybridize；It is high-temperature denatured can will be double
Chain separation and obtain single-stranded, matched and fixed to nanoparticle sublist by the complementary nucleic acid with being marked on nanoparticle surface
Face.By the use of the upper Poly T of colorful polystyrene nanoparticle mark as Tag such as Despina, for capture zone Poly A
Purpose nucleic acid sequence, for detecting corn IVR genes (Fig. 1) rapidly and sensitively.IVR PCR double-stranded segments it is high-temperature denatured into
After single-stranded, hybridize with the short-chain nucleic acids with Poly A, form the double-stranded segment with Poly A tails, be eventually adding Poly T and gather
Styrene nanoparticle is captured；Due to having modified Biotin (biotin) on PCR primer in advance, pass through Biotin
With the SA high-affinities formation nanoparticle-PCR primer-SA compounds in T lines region, visualization band is formed in T lines region.This
Sample could carry out sandwich method detection along with high-temperature denatured by dual chemical modification with test strips, and cost is high and needs high temperature to become
Property it is single-stranded to produce, efficiency is low, influence detection sensitivity.
Existing nucleic acid detection test strip, which need to be modified prime end, can just complete detection, such as patent CN
105301237 A disclose a kind of collaurum mark detection kit for detecting nucleic acid, and just detection line, nature controlling line and probe need to be made
With antigen or haptens, such as biotin, digoxin or fluorescent dye are marked.New collaurum nucleic acid test strip be with
Collaurum is as tracer label thing, and the detection material based on base pair complementarity principle can be used for the detection of nucleic acid.By
In in T lines, C lines area is fixed is nucleic acid, it is possible to use base pair complementarity realizes detection to target sequence.One section of design is with treating
The sequence of survey product (e.g., single stranded sequence) side single-stranded complementary, which is used to draw on standard gold, T lines, to be had and the product opposite side single-stranded complementary
Sequence, by base pair complementarity effect by standard gold sequence capturing so when reaction product reaches gold standard pad, formed product-
The compound of collaurum.This compound is moved forward along test strips in the presence of chromatography power, because T line sequences row can be with product
The single-stranded complete complementary of opposite side and compound is fixed, naked eyes are formed using aggregation of the collaurum at T lines visible
Red stripes.Being drawn on C lines has sequence with standard gold sequence complete complementary, it is captured when unnecessary collaurum reaches C lines and
Aobvious red, therefore the quality control standard that can be normally detected as test strips.Because Standard PCR product only has flat end structure,
The condition of single-stranded hybridization can not be met, this method there is no the detection of method progress PCR primer.
Ultraviolet refers to the general name that electromagnetic spectrum medium wavelength is radiated from 100nm~400nm, can be divided into UVA (wavelength 320
~400nm), UVB (290~320nm of wavelength), UVC (100~290nm of wavelength).There are some researches show ultraviolet radioactive can be to life
Object brings adverse effect, including Mutagenesis, carcinogenesis and cell death etc., produces the possible cause of these adverse effects
It is that photochemical reaction product is generated in DNA molecular.In the presence of UV, the adjacent pyrimidine bases of nucleotide sequence pass through [2+2]
Cycloaddition reaction formation cyclobutane dimer, wherein thymine dimer is main photoproduct..
The content of the invention
The problem of nucleic acid detection method very complicated that is caused for flat end PCR primer, cost are high, sensitivity is low, this
Invention proposes a kind of new solution, without special modification and other operating procedures, it is possible to directly PCR primer is examined
Survey, reach accurate, quick, sensitive, inexpensive, the easy detection demand of operation.
It is an object of the present invention to provide a kind of preparation method for possessing double single stranded end PCR primers, including following step
Suddenly：
(1) the forward primer F and reverse primer R sequences designed for amplification purpose nucleic acid, and in forward primer F and reversely
The 5' ends of primer R sequences respectively add Tag1 and Tag2 sequences, between Tag1 sequences and forward primer F sequences, Tag2 sequences and
Separated respectively with some thymidines between reverse primer R sequences, synthetic primer Tag1-F and Tag2-R；
(2) at least 30min is irradiated to primer Tag1-F and the Tag2-R aqueous solution with ultraviolet；
(3) mixing PCR reaction systems, which are placed in PCR instrument, is reacted, and obtains the PCR primer for possessing double single stranded ends.
Further, the two ends of some thymidines respectively add an adenine or cytimidine respectively in step (1), can enter one
Step improves the formation rate of dimer.Become because TT-CPD (thymine dimer) absorbs photon after excitation state, it is more likely that
Be quenched by neighbouring base, the overlapping geometric center of this migration energy or electric charge that possibly rely on them, such as base and base
It is overlapping to determine electronics coupled, therefore neighbouring base is by influenceing the reply of dimer and influenceing it to ultimately generate rate.Particularly
When neighbouring base is guanine (G), response rate highest, it is minimum to result in rate, and when using adenine or cytimidine,
Formation rate is then higher.
Further, the quantity of thymidine is 6-10 in step (1).
Further, forward primer and reverse primer sequences length are respectively 18-25nt, Tag1 and Tag2 in step (1)
Sequence length is all 12nt.Forward primer and reverse primer sequences, Tag1 and Tag2 sequences can be adjusted according to different genes to be checked
Row can be with general.Tag1 and Tag2 sequences can carry out Random Design according to design of primers principle, as long as without continuous T.
Further, Tag1 and Tag2 sequences are：
Tag1：5'-CTGTACGAGACT-3'
Tag2：5'-TCTAATGCTGAT-3',
Above-mentioned Tag1 and Tag2 sequences have proven to can be general in detection of nucleic acids.
Further, step (2) 100~290nm of middle-ultraviolet lamp optimal wavelength.
It is a further object to provide the above-mentioned detection method for possessing double single stranded end PCR primers, may be selected with
Any of lower two methods：
(1) direct method：Separately design and synthesize the sequence C-Tag1 and C-Tag2 complementary with Tag1 and Tag2, and respectively
Modified at C-Tag1 and C-Tag2 3' ends with sulfydryl (- SH)；C-Tag1 and C-Tag2 after modified is passed through into golden sulfide linkage
(Au-S) it is attached on colloidal gold particle, obtains two kinds of different colloid gold label products：AuNP DNA1 and AuNP DNA2；Will
AuNP DNA1 and AuNP DNA2 press C-Tag1 and C-Tag2 mol ratios 0.8-1.2:0.8-1.2 is mixed and is prepared into red, transparent
Mark colloidal gold solution；PCR primer is added in mark colloidal gold solution and mixed, is visually observed and is detected and judged, is marked
It is feminine gender to remember the unchanged then testing result of colloidal gold solution, and it is the positive that mark colloidal gold solution, which becomes cloudy then testing result,；
(2) test strips method：Separately design and synthesize the sequence C-Tag1 and C-Tag2, C- complementary with Tag1 and Tag2
Tag1 3' ends are modified with sulfydryl (- SH), synthesize Tag1 sequences；C-Tag1 after modification is attached to by golden sulfide linkage (Au-S)
On colloidal gold particle, AuNP DNA1 are obtained, and AuNP DNA1 are sprayed in the gold standard pad of colloidal gold strip；By C-Tag2 sequences
Row are fixed on the detection zone (T) of test strips；Tag1 sequences are fixed on to the quality control region (C) of test strips；PCR primer is added drop-wise to examination
The sample pad of paper slip, is added dropwise sample-loading buffer, waits result to be seen.
Method (one) is using the plasma resonance effect of collaurum, the glue that scattered colloid gold particle takes on a red color and assembled
Body gold grain is in purple, blueness, and is precipitated.Therefore, surface can be had to the collaurum of DNA marker by DNA hybridization
Link together, the distance between " furthering " colloid gold particle, and be assembled into macromolecular particle to produce coagulation, it is now molten
There is Red Shift Phenomena, color change in liquid, and macroscopic precipitation particle occurs, therefore can realize the quick inspection to PCR primer
Survey.The principle of this method passes through on the surface of collaurum as shown in fig. 6, forming both sides has after the PCR primer of single stranded end
Au-S coordinate bonds have modified two kinds of different single stranded DNAs, and they can hybridize with the Tag1 and Tag2 of PCR primer end respectively, and
The above-mentioned change of generation.
Method (two) is complementary with AuNP DNA1 in test strips gold standard pad using PCR primer single stranded end Tag1, Tag2 and examination
C-Tag2 is complementary on paper slip detection zone, by this two Tag sandwich effect, and PCR primer can be captured and be fixed on detection
Red line is produced at area, lacks a Tag and is impossible to red T lines occur；Quality control region fix nucleic acid Tag1 can by with
AuNP DNA1 are complementary and capture unnecessary collaurum and be presented red.Therefore positive is two red lines, and feminine gender is a red line (matter
Control area).
Further, in method (two) using draw film gold spraying instrument by AuNP DNA1, C-Tag2 sequence, the spray of Tag1 sequences or
Draw on colloidal gold strip.
Further, sample-loading buffer is in method (two)：4 × SSC, pH 7.0, dripping quantity is 10-50 μ L.
Further, the applied sample amount of PCR primer is 5-10 μ L in method (two).
It is a further object to provide a kind of nucleic acid detection test strip, it includes sample pad, gold standard pad, nitre successively
Acid cellulose film and adsorptive pads module, every two adjacent module sections are overlapping, and detection zone is disposed with nitrocellulose filter
And quality control region；Its PCR forward primer and the 5' ends of reverse primer sequences respectively add Tag1 and Tag2 sequences, Tag1 sequences and
Separated respectively with some thymidines between forward primer sequence, between Tag2 sequences and reverse primer sequences；By the mutual of Tag1
Complementary series C-Tag1 is prepared into nucleic acid-gold label AuNP DNA1 with collaurum, and AuNP DNA1 are sprayed in gold standard pad；Will
Complementary sequence C-Tag2 is fixed on detection zone with Tag2；Tag1 sequences are fixed on quality control region.
Further, sample pad, gold standard pad, nitrocellulose filter and adsorptive pads are all fixed in PVC board.
It is a further object to provide the preparation method of above-mentioned nucleic acid detection test strip, comprise the following steps：(1)
The nucleic acid prepared-gold label AuNP DNA1 are sprayed on glass fibre membrane with a stroke film gold spraying instrument gold standard pad is made；(2) will
C-Tag2 sequences and Tag1 sequences with film gold spraying instrument stroke is drawn on nitrocellulose filter, respectively constitute detection zone and Quality Control respectively
Area；(3) sample pad, gold standard pad, nitrocellulose filter and adsorptive pads module are respectively assembled in PVC board, per two adjacent moulds
The overlapping 1-3mm of block；(4) that the test strips assembled are cut into 2-5mm is wide.
It is a further object to provide a kind of kit for detecting nucleic acid, including test strips system and/or mark glue
Body gold solution；
The test strips system includes nucleic acid detection test strip and sample-loading buffer；
The nucleic acid detection test strip includes sample pad, gold standard pad, nitrocellulose filter and adsorptive pads module successively, per phase
Two adjacent module sections are overlapping, and detection zone and quality control region are disposed with nitrocellulose filter；Its PCR forward primer F and
The 5' ends of reverse primer R sequences add Tag1 and Tag2 sequences respectively, between Tag1 sequences and forward primer F sequences, Tag2 sequences
Separated respectively with some thymidines between row and reverse primer R sequences；By Tag1 complementary series C-Tag1 and collaurum system
It is standby into nucleic acid-gold label AuNP DNA1, and AuNP DNA1 are sprayed in gold standard pad；By the sequence C-Tag2 complementary with Tag2
It is fixed on detection zone；Tag1 sequences are fixed on quality control region；The sample pad, gold standard pad, nitrocellulose filter and adsorptive pads are all
It is fixed in PVC board；
The sample-loading buffer is：4 × SSC, pH 7.0；
The mark colloidal gold solution presses C-Tag1 and C-Tag2 mol ratios 0.8- by AuNP DNA1 and AuNP DNA2
1.2:0.8-1.2 is mixed and formed；The AuNP DNA1 and AuNP DNA2 are respectively surface modification C-Tag1 and C-Tag2 glue
Body gold particle.
The applicant forms thymine dimer using ultraviolet irradiation in primer, and discovery can obstruct PCR polymerases
Progradation, using this primer pair purpose fragment with thymine dimer enter performing PCR expand, PCR can be produced
Thing end carries single stranded sequence, therefore collaurum nucleic acid test strip can be used to detect it.The present invention is contained by combining
There are the round pcr and nucleic acid test strip technology of thymine dimer primer, establish a kind of simple and efficient detection method, from
And the steps such as electrophoresis are saved, reach efficient, accurate, quick, sensitive, inexpensive, the easy detection demand of operation.This method has
Following advantage：
(1) compared with common nucleic acid detection test strip, the present invention is used as signal with the goldstandard of this detection of nucleic acids of PCR
Amplification means, substantially increase detection sensitivity and versatility.
(2) different from existing barrier PCR reactions, the present invention need not carry out special modification to primer, it is only necessary to letter
Single ultraviolet irradiation processing, it is possible to reach the purpose to form single stranded end PCR primer, processing procedure is simple, without cumbersome
It is modification step, with low cost.
(3) compared with the method for traditional Modify to primer, present invention eliminates the step of modification, after the completion of ultraviolet irradiation just
Performing PCR reaction can be entered, do not limited by conditions such as personnel, areas, instrument is detected or lacked at the scene of being particularly suitable for use in real time
Remote districts are used.If pcr amplification product can directly with mark colloidal gold solution or ELISA test strip, without use antibody or
The albumen such as person's part, have saved testing cost.
(4) Tag1 the and Tag2 sequences in the present invention can with general, as long as changing the primer sequence complementary with target to be measured,
Detection mark colloidal gold solution used or test strips need not adjust, and can be used directly, are a kind of wide spectrum detection platforms, are worth
Promote the use of.
(5) it is easy to use quick, it is easy to detect in real time and remote districts is used, reaction can be completed in 10 minutes；Into
This is low, it is not necessary to special instrument and equipment；Have a wide range of application, be suitable for a variety of detection objects；Collaurum sheet is not required to as red
Chromogenic agents are added, are easy to observation.
Brief description of the drawings
Fig. 1 in the prior art Properties of Polystyrene Nano Particles detect IVR PCR primer schematic diagram；
Fig. 2 embodiments 1 are denatured gel electrophoresis result；
Primer schematic diagram of Fig. 3 embodiments 2 containing thymidine；
Fig. 4 thymine dimer generting machanisms；
PCR courses of reaction in Fig. 5 embodiments 2；
The schematic diagram for marking colloidal gold solution to detect PCR primer is utilized in Fig. 6 embodiments 3；
Colloidal gold solution result of variations in Fig. 7 embodiments 3；
The test strips structure schematic diagram of Fig. 8 embodiments 4；
The amplifying nucleic acid ELISA test strip PCR primer result of Fig. 9 embodiments 4 and principle；
PCR primer electrophoresis result in Figure 10 embodiments 4.
Embodiment
The present invention is described in further details with reference to specific embodiment and accompanying drawing.
The barrier PCR polymerase extensions of the thymine dimer of embodiment 1
Using the nucleotide sequence containing thymidine as template, 2T, 4T, 6T are represented in template containing 2 respectively, 4,6 it is adjacent
Thymidine (as shown in table 1), with AZP1 as primer, carry out primer extension assay, i.e., only a progress single loop (35 DEG C are moved back
Fiery 5min, 72 DEG C of extension 5min).Experimental result is as shown in Figure 2：Product analysis is carried out with denaturation PAGE glue, is not passed through in template
In the case of crossing ultraviolet irradiation, AZP1 can be fully extended (swimming lane 5,7,9) by template of 2T, 4T, 6T；When with 100~290nm
After length ultraviolet line irradiation template 40min, using 4T and 6T as template in the case of (swimming lane 6,8), AZP1 extends some bases
Just do not re-extend afterwards, there is obvious barrier product to be formed, using 6T as template in the case of it is the most obvious.AZP1 is extended about
The position that about 5 bases are parked in 24nt is moved after 5-6 base, pillar location.
Sequence information involved by the embodiment 1 of table 1
Due to containing thymine dimer in template, the base extension under thymidine effect of PCR polymerases is blocked
And the double-strand of complete complementary can not be formed, therefore this primer containing thymine dimer is reacted for PCR, it can produce
There is single-stranded PCR primer both sides.The thymine dimer that can prove ultraviolet irradiation formation from experiment above is obstructed
The extension of PCR polymerases.
The PCR amplifications of the Ebola virus partial sequence of embodiment 2
(1) DNA fragmentation that PCR is expanded, sequence are treated according to announced Ebola virus genome sequence in GenBank is selected
Column information is shown in Table 2, overall length 103nt.Forward primer F and reverse primer R are separately designed, and Tag1 is added respectively at F, R 5' ends
With Tag2 sequences, separated respectively with 6 thymidines between F and Tag1 sequences, between R and Tag2 sequences, respectively obtain Tag1-
F and Tag2-R.Primer and template relation are shown in Fig. 3.
Sequence information needed for the amplification experiment of the Ebola Target single stranded ends PCR primer of table 2
(2) Tag1-F and Tag2-R of the experimental group aqueous solution are irradiated with UVC (100~290nm of wavelength)
30min, makes adjacent thymidine formation dimer, and molecular mechanism is shown in Fig. 4；The primer of non-irradiation control group irradiates without UVC.
(3) mixing PCR reaction systems, which are placed in PCR instrument, is reacted：The blank for being not added with template Ebola Target is set
Control group, experimental group and non-irradiation control group except primer be respectively irradiation and be not irradiated in addition to, remaining condition all same.
PCR reaction systems and PCR cycle parameter are shown in Table 3, table 4 respectively；PCR courses of reaction are as shown in Figure 5.
The PCR experiment reaction system of table 3：
The PCR experiment reaction condition of table 4：
Step 2-4 are circulated 30 times.
The direct method detection of embodiment 3PCR amplified productions
(1) colloid gold particle is prepared using reduction of sodium citrate method：96mL ultra-pure waters are added in 250mL conical flasks, are used
Pipettor pipettes 1mL 1wt% HAuCl4Solution is added thereto, while adding stirring magneton, while stirring, is heated to boiling
Rise.Then, 3mL 1wt% sodium citrate solution is rapidly added, continues to stir and keeps micro-boiling.Solution colour blackening immediately,
Continue heating stirring, solution colour can be observed from depth to shallow, is finally changed into claret from brick-red.It is stable not Deng solution colour
After changing again, continue heating stirring 10min.Continue to stir, treat that solution is down to room temperature, prepare 15 ± 2nm of particle diameter colloid
Gold grain.
(2) it (is standard gold sequence to be coordinated the different single stranded DNAs of two kinds of key label by Au-S on colloid gold particle surface：Point
Not Wei C-Tag1, C-Tag2 (table 2), its 3' end carry out sulfydryl modification).50 μ L standard gold sequences are added in 2mL colloidal gold solutions
(10 μm of ol/L, C-Tag1/C-Tag2), 225 μ L water, 350 μ L 100mmol/L PBS (pH 8.0), 350 μ L 0.1wt%
SDS, 525 μ L 2mol/L NaCl (carry out substep dropwise addition depending on colloidal gold solution stability difference, gradually improved in standard gold solution
NaCl concentration), left at room temperature over night.12000r/min centrifuges 10min, abandons supernatant and washs precipitation with 1mL 0.01wt%SDS,
Repeat twice, precipitation is dissolved in 1mL and preserved in liquid (0.3mol/L NaCl, 10mmol/L PBS, 0.01%SDS) again.Extremely
This, preparing two kinds of surface modifications has the colloidal gold solution of single stranded DNA.
(3) detect that principle is as shown in Figure 6 to PCR primer using surface modification DNA collaurum.20 μ L are taken to implement
The pcr amplification product of example 2, the colloidal gold solution of addition 40 μ L C-Tag1 modifications and the collaurum of 40 μ L C-Tag2 modifications are molten
Liquid, adds and standing is mixed after the ethanol of 1/4 volume fraction, is that visual solution change can be observed to occur through 10-20min.Such as Fig. 7
Shown result, the colloidal gold solution color that PCR primer, blank control group and non-irradiation control group are not added does not change,
And the colloidal gold solution color added after PCR primer changes, illustrate that PCR primer can be served as in colloidal gold solution
Linker effect, can further the distance between collaurum, and assembling forms macromolecular particle, finally sends out colloid gold particle
Raw coagulation, by naked eyes it is observed that the color change and precipitation of solution occur.This method was demonstrated by drawing that UV irradiates
Thing can make PCR primer end formation two single-stranded, and can be detected with this method directly against PCR primer.
The test strips method detection of embodiment 4PCR amplified productions
(1) separately design and synthesize and used with the sequence C-Tag1 of Tag1 and Tag2 complementations and C-Tag2, C-Tag1 5' ends
Sulfydryl (- SH) is modified, and synthesizes Tag1 sequences.
(2) C-Tag1 after modification is attached on colloidal gold particle by golden sulfide linkage (Au-S), obtains nucleic acid-gold mark
Thing AuNP DNA1, and AuNP DNA1 are sprayed in the gold standard pad of colloidal gold strip (glass fibre membrane)；By C-Tag2 sequences
The detection zone (T) of nitrocellulose filter is fixed on stroke film gold spraying instrument；Tag1 sequences are fixed on nitric acid fibre with film gold spraying instrument is drawn
The quality control region (C) of the plain film of dimension.
As shown in figure 8, sample pad, gold standard pad, nitrocellulose filter and adsorptive pads module are respectively assembled in PVC board,
Per the adjacent overlapping 2mm of two modules, it is ensured that chromatography process is smoothed out；By the automatic cutting machine of the test strips assembled, cutting
It is wide into 3mm.
(3) PCR primer of 5 μ L embodiments 2 is added drop-wise to the sample pads of test strips (experimental group and control group is added drop-wise to respectively
Different test strips), 15 μ 4 × SSC of L are added dropwise dropwise, allow sample slowly to be moved ahead in test strips, result is observed after 5 minutes, test
Respectively there are a red stripes in detection zone (T) and quality control region (C) in group, illustrates that PCR reactions are normally carried out and have obtained target production
Thing, testing result and principle are shown in Fig. 9.Non- irradiation control group and blank control group do not occur in detection zone (T) and quality control region (C)
Band.
In addition, the reliability to verify test strips, has carried out traditional electrophoresis detection simultaneously to above-mentioned PCR primer, has such as schemed
Visible in figure shown in 10, non-irradiation control group has specific amplification band in 150bp or so, and experimental group has mesh in 200bp or so
Product is marked, blank control group is without specific band.Fully confirm that above-mentioned direct method and test strips method detect the degree of accuracy of PCR primer
It is high.
Compared to the method for traditional electrophoresis detection PCR primer, method of the invention need not carry out Modify to primer, PCR productions
Thing can be detected directly, and save electrophoresis, the process of dyeing, detect simpler, time saving, the degree of accuracy is high, sensitivity is high,
PCR primer detection consumption can be micro- to 5 μ L, it is only necessary to which 5-10min is that can obtain testing result.
SEQUENCE LISTING
<110>Chinese Marine University
<120>A kind of preparation method and its detection method for possessing double single stranded end PCR primers
<130> 2017
<160> 13
<170> PatentIn version 3.5
<210> 1
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> 2T
<400> 1
tctaatgctg atgttacatc gtcgtcgtcc aaattg 36
<210> 2
<211> 38
<212> DNA
<213> Artificial Sequence
<220>
<223> 4T
<400> 2
tctaatgctg atgttttaca tcgtcgtcgt ccaaattg 38
<210> 3
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> 6T
<400> 3
tctaatgctg atgtttttta catcgtcgtc gtccaaattg 40
<210> 4
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> AZP1
<400> 4
caatttggac gacgacgat 19
<210> 5
<211> 103
<212> DNA
<213> Artificial Sequence
<220>
<223> Ebola Target
<400> 5
gttgacccgt atgatgatga gagtaataat tatcctgact acgaggattc ggctgaaggc 60
accacaggag atcttgatct cttcaatttg gacgacgacg atg 103
<210> 6
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> F
<400> 6
gttgacccgt atgatgatga gagt 24
<210> 7
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> R
<400> 7
catcgtcgtc gtccaaattg 20
<210> 8
<211> 12
<212> DNA
<213> Artificial Sequence
<220>
<223> Tag1
<400> 8
ctgtacgaga ct 12
<210> 9
<211> 12
<212> DNA
<213> Artificial Sequence
<220>
<223> Tag2
<400> 9
tctaatgctg at 12
<210> 10
<211> 44
<212> DNA
<213> Artificial Sequence
<220>
<223> Tag1-F
<400> 10
ctgtacgaga ctatttttta gttgacccgt atgatgatga gagt 44
<210> 11
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Tag2-R
<400> 11
tctaatgctg atatttttta catcgtcgtc gtccaaattg 40
<210> 12
<211> 12
<212> DNA
<213> Artificial Sequence
<220>
<223> C-Tag1
<400> 12
agtctcgtac ag 12
<210> 13
<211> 12
<212> DNA
<213> Artificial Sequence
<220>
<223> C-Tag2
<400> 13
atcagcatta ga 12
Claims (10)
1. a kind of preparation method for possessing double single stranded end PCR primers, it is characterised in that comprise the following steps：
(1) the forward primer F and reverse primer R sequences designed for amplification purpose nucleic acid, and in forward primer F and reverse primer
The 5' ends of R sequences add Tag1 and Tag2 sequences respectively, between Tag1 sequences and forward primer F, Tag2 sequences and reverse primer R
Between separated respectively with some thymidines, synthetic primer Tag1-F and Tag2-R；
(2) at least 30min is irradiated to primer solution Tag1-F and Tag2-R with ultraviolet；
(3) mixing PCR reaction systems, which are placed in PCR instrument, is reacted, and obtains the PCR primer for possessing double single stranded ends.
2. the preparation method according to claim 1 for possessing double single stranded end PCR primers, it is characterised in that in step (1)
The two ends of some thymidines respectively add an adenine or cytimidine respectively.
3. the preparation method according to claim 1 for possessing double single stranded end PCR primers, it is characterised in that in step (1)
The quantity of thymidine is 6-10.
4. the preparation method for possessing double single stranded end PCR primers according to claim 1 or 2 or 3, it is characterised in that
Tag1 and Tag2 sequences are respectively：
Tag1：5’-CTGTACGAGACT-3’
Tag2：5’-TCTAATGCTGAT-3’.
5. the preparation method for possessing double single stranded end PCR primers according to claim 1 or 2 or 3, it is characterised in that step
Suddenly 100~290nm of (2) middle-ultraviolet lamp optimal wavelength.
6. any detection methods for possessing double single stranded end PCR primers of claim 1-5, it is characterised in that it is optional with
Any of lower two methods：
(1) direct method：Separately design and synthesize the sequence C-Tag1 and C-Tag2 complementary with Tag1 and Tag2, and respectively in C-
Tag1 and C-Tag2 3' ends sulfydryl modification；C-Tag1 and C-Tag2 after modified is attached to glue by golden sulfide linkage respectively
In body gold particle, AuNP DNA1 and AuNP DNA2 are obtained；AuNP DNA1 and AuNP DNA2 are rubbed by C-Tag1 and C-Tag2
You compare 0.8-1.2:0.8-1.2 mixes the mark colloidal gold solution for being prepared into red, transparent；PCR primer is added to mark colloid
Mixed in gold solution, visually observe and detected and judged, the unchanged then testing result of mark colloidal gold solution is feminine gender, mark
It is the positive that colloidal gold solution, which becomes cloudy then testing result,；
(2) test strips method：Separately design and synthesize the sequence C-Tag1 complementary with Tag1 and Tag2 and C-Tag2, C-Tag1
3' ends sulfydryl modification, synthesizes Tag1 sequences；C-Tag1 after modification is attached on colloidal gold particle by golden sulfide linkage, obtained
AuNP DNA1, and AuNP DNA1 are sprayed in the gold standard pad of colloidal gold strip；C-Tag2 sequences are fixed on test strips
Detection zone；Tag1 sequences are fixed on to the quality control region of test strips；PCR primer is added drop-wise to the sample pad of test strips, loading is added dropwise
Buffer solution, waits result to be seen.
7. possess the detection method of double single stranded end PCR primers according to claim 6, it is characterised in that in method (two)
Sample-loading buffer is：4 × SSC, pH 7.0.
8. a kind of nucleic acid detection test strip, it is characterised in that it includes sample pad, gold standard pad, nitrocellulose filter and suction successively
Water cushion module, every two adjacent module sections are overlapping, and detection zone and quality control region are disposed with nitrocellulose filter；Its PCR
Forward primer F and reverse primer R sequences 5' ends respectively add Tag1 and Tag2 sequences, Tag1 sequences and forward primer F sequences
Separated respectively with some thymidines between row, between Tag2 sequences and reverse primer R sequences；By Tag1 complementary series C-
Tag1 is prepared into nucleic acid-gold label AuNP DNA1 with collaurum, and AuNP DNA1 are sprayed in gold standard pad；Will be mutual with Tag2
Sequence C-the Tag2 of benefit is fixed on detection zone；Tag1 sequences are fixed on quality control region.
9. the preparation method of nucleic acid detection test strip described in claim 8, it is characterised in that comprise the following steps：(1) it will prepare
Good nucleic acid-gold label AuNP DNA1 are sprayed on glass fibre membrane with a stroke film gold spraying instrument and gold standard pad are made；(2) by C-Tag2
Sequence and Tag1 sequences with film gold spraying instrument stroke is drawn on nitrocellulose filter, respectively constitute detection zone and quality control region respectively；(3) will
Sample pad, gold standard pad, nitrocellulose filter and adsorptive pads module are respectively assembled in PVC board, per the adjacent overlapping 1- of two modules
3mm；(4) that the test strips assembled are cut into 2-5mm is wide.
10. a kind of kit for detecting nucleic acid, it is characterised in that including test strips system and/or mark colloidal gold solution；
The test strips system includes nucleic acid detection test strip and sample-loading buffer；
The nucleic acid detection test strip includes sample pad, gold standard pad, nitrocellulose filter and adsorptive pads module successively, per adjacent
Two module sections are overlapping, and detection zone and quality control region are disposed with nitrocellulose filter；Its PCR forward primer F and reversely
The 5' ends of primer R sequences respectively add Tag1 and Tag2 sequences, between Tag1 sequences and forward primer F sequences, Tag2 sequences and
Separated respectively with some thymidines between reverse primer R sequences；Tag1 complementary series C-Tag1 is prepared into collaurum
Nucleic acid-gold label AuNP DNA1, and AuNP DNA1 are sprayed in gold standard pad；Sequence C-the Tag2 complementary with Tag2 is fixed
In detection zone；Tag1 sequences are fixed on quality control region；The sample pad, gold standard pad, nitrocellulose filter and adsorptive pads are all fixed
In PVC board；
The sample-loading buffer is：4 × SSC, pH 7.0；
The mark colloidal gold solution presses C-Tag1 and C-Tag2 mol ratios 0.8-1.2 by AuNP DNA1 and AuNP DNA2:
0.8-1.2 is mixed and formed；The AuNP DNA1 and AuNP DNA2 are respectively surface modification C-Tag1 and C-Tag2 collaurum
Particle.
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| CN114807331A (en) * | 2022-05-12 | 2022-07-29 | 中国海洋大学 | Nanopore sequencing method of short-chain DNA |
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| CN107164366B (en) | 2020-09-04 |
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