CN106282193A - HPP's aptamer and screening technique thereof and application - Google Patents
HPP's aptamer and screening technique thereof and application Download PDFInfo
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- CN106282193A CN106282193A CN201610646875.8A CN201610646875A CN106282193A CN 106282193 A CN106282193 A CN 106282193A CN 201610646875 A CN201610646875 A CN 201610646875A CN 106282193 A CN106282193 A CN 106282193A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1034—Isolating an individual clone by screening libraries
- C12N15/1093—General methods of preparing gene libraries, not provided for in other subgroups
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
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- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/16—Aptamers
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
- G01N2333/72—Assays involving receptors, cell surface antigens or cell surface determinants for hormones
Abstract
The invention discloses a kind of HPP's aptamer and screening technique thereof and application, HPP's aptamer is with any one in Q10,5 ' Q11 FAM 3 ', 5 ' Q12 FAM 3 ', 5 ' BHQ1 Q13 3 ', 5 ' Q14 BHQ1 3 ', 5 ' SH Q16 3 ', 5 ' SH Q17 3 '.Its screening technique includes synthesizing random library and primer, GO SELEX screening, PCR amplification library, preparation DNA strand library, repeating the step such as screening, negative screening, multi-turns screen.HPP's aptamer of the present invention can be applicable to the test kit of preparation detection HPP, have affinity more higher than protein antibodies and specificity, non-immunogenicity, can chemosynthesis, molecular weight is little, stable, be prone to the advantages such as preservation and labelling.
Description
Technical field
The present invention relates to biological technical field, relate to a kind of aptamer and screening thereof and application, particularly relate to one
Can be used for detecting the aptamer of HPP and screening technique thereof and application.
Background technology
HPP is by a kind of polypeptide hormone being made up of 36 aminoacid of pancreatic secretion.HPP has as follows
Physiological action: (1) suppression cholecystokinin and the discharge of pancreatin, makes smooth muscle of bile vesica relax, it is possible to decrease the pressure in gallbladder, gallbladder
House steward's sphincter anxiety is strengthened, and suppression bile is to duodenal discharge；(2) suppress pancreatic juice and bile secretion after the meal, suppress pancreas
The facilitation to pancreatic secretion such as secretin and cholecystokinin；(3) gastric acid that suppression pentagastrin causes in the gastrointestinal tract
Secretion；(4) suppress the secretion of Plasma Motilin Levels and Colonic Motility, increase the pressure of inferior esophageal sphincter, suppress body of stomach myoelectrical activity.Nucleic acid is fitted
Part (i.e. aptamer), is aglucon phyletic evolution (the systematic evolution of ligands by index concentration
By exponential enrichment) one section of single stranded DNA (DNA (deoxyribonucleic acid)) filtering out or RNA (ribonucleic acid) sequence
Row.Aptamer can self formed specific tertiary structure, with target high specific, combine high-affinity.Additionally, nucleic acid
Aptamers also has the feature that molecular weight is little, nontoxic, is easily-synthesized and modifies.Owing to aptamer can simplicity synthesize rapidly, become
This is relatively low, and size is less, and of many uses, and they have become as the powerful distinguishing intraor extracellular target molecule.Nucleic acid is fitted
The wide range of the discernible object of part, it has developed into a kind of novel molecular probe in recent years, in medical application
Also there is certain progress in field.Therefore, the aptamer of screening HPP is for developing new detection method and studying its physiology
Effect has certain value.
Traditional aptamer screening technique generally requires fixing library or target, and this includes that covalency is fixed and non-covalent
Fix two kinds of approach.Whether which kind of fixing means, the native conformation through fixing library or target the most all can
It is affected.Additionally, also can produce sterically hindered with the combination of target to library for fixing carrier, these are all unfavorable for sieve
Choosing obtains the sequence that binding ability under free state is strong, therefore develops revocable aptamer screening technique and seems the heaviest
Want.Utilize graphene oxide to can be good at the feature adsorbed single stranded DNA and can remove by centrifugation herein, graphene oxide is drawn
Enter during screening, reduce non-specific adsorption, effectively remove unconjugated single stranded DNA, and do not affect aptamer
Combination with target.The method greatly increases the efficiency of screening, has saved labour force and has reduced cost.
Summary of the invention
The technical problem to be solved in the present invention is to overcome the deficiencies in the prior art, it is provided that one has higher than protein antibodies
Affinity and specificity, non-immunogenicity, can chemosynthesis, molecular weight is little, stable, be prone to preserve and can be used for of labelling
The aptamer of detection HPP；Additionally provide the screening technique of above-mentioned aptamer and preparation detection HPP
Test kit in application.
For solving above-mentioned technical problem, it is provided that a kind of HPP's aptamer, described HPP's nucleic acid is adaptive
Body is any one in following sequence 1～7:
Sequence 1 is Q10, and the sequence of described Q10 is the sequence shown in SEQ ID NO.1；
Sequence 2 is 5 '-Q11-FAM-3 ', and the sequence of described Q11 is the sequence shown in SEQ ID NO.2；
Sequence 3 is 5 '-Q12-FAM-3 ', and the sequence of described Q12 is the sequence shown in SEQ ID NO.3；
Sequence 4 is 5 '-BHQ1-Q13-3 ', and the sequence of described Q13 is the sequence shown in SEQ ID NO.4；
Sequence 5 is 5 '-Q14-BHQ1-3 '；The sequence of described Q14 is the sequence shown in SEQ ID NO.5；
Sequence 6 is 5 '-SH-Q16-3 ', and the sequence of described Q15 is the sequence shown in SEQ ID NO.6；
Sequence 7 is 5 '-SH-Q17-3 ', and the sequence of described Q15 is the sequence shown in SEQ ID NO.7.
Particularly as follows:
Sequence 1:5 '-GGCACCGCTGTTTTAGCCTCGGCTGAGACAAGGGC-3 '.
Sequence 2:5 '-GGCACCGCTGTTTTAGCCTCGGCTGAGACAAGGGCT-FAM-3 '.
Sequence 3:5 '-CGTGCAATGTCGAATGCATGAGCAAACATGGCGAT-FAM-3 '.
Sequence 4:5 '-BHQ1-TGGCACCGCTGTTTTAGCCTCGGCTGAGACAAGGGC-3 '.
Sequence 5:5 '-GGCACCGCTGTTTTAGCCTCGGCTGAGACAAGGGCT-BHQ1-3 '.
Sequence 6:5 '-SH-TTTTTTTTTTGGCACCGCTGTTTTAGCCTCGGCTGAGACAAGGGC-3 '.
Sequence 7:5 '-SH-TTTTTTTTTTCGTGCAATGTCGAATGCATGAGCAAACATGGCGAT-3 '.
Above-mentioned HPP's aptamer, it is preferred that a certain position on the nucleotide sequence of described sequence 1～7
Be phosphorylated, methylate, amination, sulfhydrylation or isotopologue.Above-mentioned nucleotide sequence is phosphorylated, methylates, amino
After change, sulfhydrylation or isotopologue, performance will not change, it is also possible to identifies HPP.
Above-mentioned HPP's aptamer, it is preferred that be combined with biology on the nucleotide sequence of described sequence 1～7
Element, digoxin, fluorescent material, nano material or enzyme labelling.Above-mentioned biotin, digoxin, fluorescent material, enzyme labelling can be in conjunction with
Optional position at nucleotide sequence.
Above-mentioned HPP's aptamer, it is preferred that described fluorescent material includes fluorescein, rhodamine, anthocyanidin.
Above-mentioned HPP's aptamer, it is preferred that described nano material is gold nano grain.
Above-mentioned HPP's aptamer, it is preferred that described enzyme labelling is horseradish peroxidase.
Above-mentioned HPP's aptamer, it is preferred that described HPP's aptamer also includes following three kinds
Any one in sequence:
(1) with the homology of the nucleotide sequence of described HPP's aptamer more than 80%；
(2) nucleotide sequence with described HPP's aptamer carries out the sequence hybridized；
(3) RNA sequence that the nucleotide sequence of described HPP's aptamer is transcribed.
Above-mentioned HPP's aptamer, it is preferred that described HPP's aptamer also includes described people's pancreas
The phosphorothioate backbone that the skeleton of the nucleotide sequence of polypeptide-nucleic acid aptamers derives, or described HPP's nucleic acid is suitable
The corresponding peptides nucleic acid that part is transformed into.
As total technology design, present invention also offers the screening side of a kind of above-mentioned HPP's aptamer
Method, comprises the following steps:
S1, synthesis random library and primer:
Random library employing RS35 random library: 5 '-AGCGTCGGATACCACTACTA
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN-ATCATGGAGTTCGTGGTCAG-3 ',
5 ' primers: 5 '-FAM-AGCGTCGGATACCACTACTA-3 ',
3 ' primers: 5 '-Biotin-CTGACCACGAACTCCATGAT-3 '；
S2, GO-SELEX screen: be dissolved in buffer solution by HPP's dry powder, hatch with random sequence；So
The buffer that rear addition contains graphene oxide is hatched, and collects supernatant by centrifugation, is HPP's specific nucleic acid adaptive
Body library；
S3, PCR expand library: described HPP's specific nucleic acid aptamers library is carried out PCR amplification, obtain amplification and produce
Thing；
S4, prepare DNA strand library: the agarose microbeads modified by Streptavidin is centrifugal removes supernatant, then by described expansion
Volume increase thing is hatched at normal temperatures with agarose microbeads；It is subsequently adding alkali liquor in agarose microbeads, reacts under room temperature, be centrifuged, receive
Collection supernatant；Collect the solution dripped after described supernatant is crossed desalination post, be DNA strand library；
S5, repeat screening: the random library substituting in described step S2 by described DNA strand library, and repeat above-mentioned step
The process of rapid S2～S4 is at least one times；
S6, negative screening: bovine serum albumin, human serum albumin, hemoglobin dry powder are dissolved in buffer solution,
Then hatch with described DNA strand library；It is subsequently adding the buffer containing graphene oxide to hatch, discards by centrifugation
Clearly, add buffer solution and graphene oxide is uniformly dispersed again, collect graphene oxide solution now；
S7, multi-turns screen: the graphene oxide solution collected by S6 is substituted the DNA strand library of S5, continue to repeat on
State the operating process of step S5～S6；Until filtering out the identification ability to HPP to meet the aptamer of requirement.
Above-mentioned screening technique, it is preferred that particularly as follows: HPP's dry powder is dissolved in buffer solution in described step S2
In, at 37 DEG C, hatch 2h with random sequence；It is subsequently adding the buffer containing graphene oxide and hatches 25min at 25 DEG C,
Collect supernatant by centrifugation, be HPP's specific nucleic acid aptamers library；Described random sequence and described graphene oxide
Ratio is 1.67nmol/mg.
Above-mentioned screening technique, it is preferred that in described S3 step, the process conditions of described PCR amplification are: 94 DEG C of denaturations
10min, degeneration 30sec at 94 DEG C, 56.9 DEG C of annealing 30sec, 72 DEG C extend 30sec, and circulation 10～20 is taken turns, finally in 72 DEG C
The most finally extend 7min；In described step S4, the time that described amplified production and agarose microbeads are hatched at normal temperatures is 0.5h,
Add alkali liquor in agarose microbeads, under room temperature, react 15min.
Same technology as the present invention is conceived, and present invention also offers a kind of described HPP's aptamer in system
Application in the test kit of standby detection HPP.
Compared with prior art, it is an advantage of the current invention that:
(1) the invention provides a kind of HPP's aptamer, compared to protein antibodies, there is higher affinity
With specificity；Energy specificity and the identification HPP of high-affinity, dissociation constant is within nanomolar range.
(2) the invention provides a kind of HPP's aptamer, non-immunogenicity；Can synthesize by iii vitro chemical, point
Son amount is little, can modify different parts and replace, and sequence is stable, it is easy to preserve；It is easy to labelling and (need not labelling
Two resist).
(3) the invention provides the screening technique of a kind of HPP's aptamer, use graphene oxide auxiliary
HPP's aptamer screens, and can greatly improve the efficiency of screening, thus reduce cost, saves labour force.
(4) the invention provides the application in detection HPP of a kind of HPP's aptamer, operation is more
Simply, rapidly, and the cycle is short, favorable reproducibility.Owing to the synthesis cost of aptamer is low compared with antibody preparation cost, so detection
Cost is the lowest.Its target molecule combined is studied by the aptamer utilizing the present invention, it is also possible to be best understood from
Its physiological function, has good application prospect in terms of the diagnosis of pancreatic polypeptide-producing tumor.
Accompanying drawing explanation
For making the purpose of the embodiment of the present invention, technical scheme and advantage clearer, below in conjunction with the embodiment of the present invention
In accompanying drawing, the technical scheme in the embodiment of the present invention is carried out clear, complete description.
Fig. 1 is principle and the key step schematic diagram of graphene oxide assisting sifting in the embodiment of the present invention, and pattern one is
The process of positive-selecting, pattern two is negative and positive-selecting combines the process carried out.
Fig. 2 is the secondary structure schematic diagram of the Q10 aptamer utilizing IDTDNA software to simulate in the embodiment of the present invention.
Fig. 3 is that the secondary structure of the FAM-Q11 aptamer utilizing IDTDNA software to simulate in the embodiment of the present invention is shown
It is intended to.
Fig. 4 is that the secondary structure of the FAM-Q12 aptamer utilizing IDTDNA software to simulate in the embodiment of the present invention is shown
It is intended to.
Fig. 5 is that the secondary structure of the BHQ1-Q13 aptamer utilizing IDTDNA software to simulate in the embodiment of the present invention is shown
It is intended to.
Fig. 6 is that the secondary structure of the BHQ1-Q14 aptamer utilizing IDTDNA software to simulate in the embodiment of the present invention is shown
It is intended to.
Fig. 7 is the secondary structure signal of the SH-Q16 aptamer utilizing IDTDNA software to simulate in the embodiment of the present invention
Figure.
Fig. 8 is the secondary structure signal of the SH-Q17 aptamer utilizing IDTDNA software to simulate in the embodiment of the present invention
Figure.
Fig. 9 is that in the embodiment of the present invention, native polyacrylamide gel electrophoresis investigates sequence Q10 to HPP's combination
The testing result of situation.
Figure 10 is the testing result that in the embodiment of the present invention, multi-functional microplate reader investigation Q11 is combined situation with HPP.
Figure 11 is the testing result that in the embodiment of the present invention, multi-functional microplate reader investigates Q11 and HPP's binding ability.
Figure 12 is that in the embodiment of the present invention, fluorescence spectrophotometer investigates incubation time to the impact that Q11 is combined with HPP
Testing result.
Figure 13 is that in the embodiment of the present invention, fluorescence spectrophotometer investigates the temperature detection on the impact that Q11 is combined with HPP
Result.
Figure 14 is the difference of multi-functional microplate reader detection Q11 and HPP's antibody combining site.
Figure 15 is the selectivity that multi-functional microplate reader detection Q11 is combined with HPP.
Figure 16 is the pattern that fluorescence spectrophotometer detection Q12 and Q13 is combined with HPP.
Figure 17 is the binding site of fluorescence spectrophotometer detection Q14 and Q15 and HPP.
Figure 18 dynamic light scattering method detection Q16 and Q17 Detection results to HPP.
The selectivity of Figure 19 dynamic light scattering method detection Q16 and Q17 detection HPP.
Detailed description of the invention
Below in conjunction with Figure of description and concrete preferred embodiment, the invention will be further described, but the most therefore and
Limit the scope of the invention.
Embodiment
Material and instrument employed in following example are commercially available.
Embodiment 1:
A kind of aptamer that can be used for detecting HPP, including:
Sequence 1 (Q10):
5’-GGCACCGCTGTTTTAGCCTCGGCTGAGACAAGGGC-3’(SEQ ID NO.1)。
Sequence 2 (FAM-Q11):
5’-GGCACCGCTGTTTTAGCCTCGGCTGAGACAAGGGCT-FAM-3’(SEQ ID NO.2)。
Sequence 3 (FAM-Q12):
5’-CGTGCAATGTCGAATGCATGAGCAAACATGGCGAT-FAM-3’(SEQ ID NO.3)。
Sequence 4 (BHQ1-Q13):
5’-BHQ1-TGGCACCGCTGTTTTAGCCTCGGCTGAGACAAGGGC-3’(SEQ ID NO.4)。
Sequence 5 (BHQ1-Q14):
5’-GGCACCGCTGTTTTAGCCTCGGCTGAGACAAGGGCT-BHQ1-3’(SEQ ID NO.5)。
Sequence 6 (SH-Q16):
5’-SH-TTTTTTTTTTGGCACCGCTGTTTTAGCCTCGGCTGAGACAAGGGC-3’(SEQ ID NO.6)。
Sequence 7 (SH-Q17):
5’-SH-TTTTTTTTTTCGTGCAATGTCGAATGCATGAGCAAACATGGCGAT-3’(SEQ ID NO.7)。
Negative control (FAM-Random):
5’-NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN-FAM-3’。
Closing probe (FAM-Q15):
5’-FAM-AGCCTTTGTTTCATCCGAGG-3’。
Assist probes (SH-Q18):
5’-SH-TTTTTTTTTT-3’。
Note: N represents arbitrary base in A, T, G, C.In the present embodiment above-mentioned bar sequence, seven can be used for detect people's pancreas many
Peptide, respectively Q10, FAM-Q11, FAM-Q12, BHQ1-Q13, BHQ1-Q14, SH-Q16, SH-Q17；FAM-Random is as the moon
Property comparison；FAM-Q15 is as closing probe；SH-Q18 is as assist probes.
Q10, FAM-Q11, FAM-Q12, BHQ1-Q13, BHQ1-Q14, SH-Q16, SH-Q17 mainly use the following side of screening
Method screening obtains, as it is shown in figure 1, specifically include following steps:
The screening technique of HPP's aptamer of a kind of the present embodiment, comprises the following steps:
1, the random single-stranded DNA banks shown in following sequence and primer are synthesized:
Random library RS35:
5 '-AGCGTCGGATACCACTACTA-NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN (N represents A, T,
One in tetra-bases of G, C)-ATCATGGAGTTCGTGGTCAG-3 '；
5 ' primers: 5 '-FAM-AGCGTCGGATACCACTACTA-3 '；
3 ' primers: 5 '-Biotin-CTGACCACGAACTCCATGAT-3 '.
2, the preparation of buffer solution:
The preparation of 2.1 1 × Binding Buffer (pH=7.35): weigh 2.383g HEPES, 3.510g respectively
NaCl, 0.186g KCl, and 0.203g MgCl2·6H2O is dissolved in 200mL aquesterilisa, and regulation pH value, to 7.35, adds water
Be settled to 500mL and obtain 1 × Binding Buffer, wherein the concentration of HEPES be the concentration of 20mM, NaCl be 120mM, KCl
The concentration that concentration is 5mM, MgCl2 be 2mM, then 1 × Binding Buffer is preserved in 4 DEG C of refrigerators.
2.2 concentration are the preparation of the PBS solution (pH=7.5) of 20mM: weigh 0.2940g NaH2PO4·2H2O,
2.8960g Na2HPO4·12H2O, 4.390g NaCl adds aquesterilisa to 200mL, adjusts pH to 7.5, constant volume in 500mL volumetric flask,
4 DEG C of preservations.
2.3 concentration are the preparation of the NaOH solution of 200mM: weigh 0.800g NaOH, add a small amount of aquesterilisa and dissolve, constant volume
In 100mL volumetric flask, 4 DEG C of preservations.
The preparation of 2.4 antibody incubation liquid (20mM PBS, 0.05%Tween-20, pH=7.5): weigh 0.2940g
NaH2PO4·2H2O, 2.8960g Na2HPO4·12H2O, 4.390g NaCl adds aquesterilisa to 200mL, adds 50 μ L Tween-
20, adjust pH to 7.5, constant volume is in 500mL volumetric flask, 4 DEG C of preservations.
2.5 concentration are the preparation of the PBS solution (pH=7.4) of 30mM: weigh 0.0889g NaH2PO4·2H2O,
0.8699g Na2HPO4·12H2O, 1.7532g NaCl, adds aquesterilisa to 60mL, and after fully dissolving, constant volume is in 100mL capacity
Bottle, 4 DEG C of preservations.
3, GO-SELEX screening obtain the fit library of HPP's specific nucleic acid, principle as shown in Figure 1:
3.1 random library pretreatment: the centrifuge tube that will be equipped with 5nmol random library RS35 is placed in a centrifuge, at 9000g
Under be centrifuged 10min, carefully take out centrifuge tube, it is to avoid rock, be rapidly added 1 × Binding Buffer of 400 μ L, by centrifuge tube
After being placed at 95 DEG C heating 5min, take out immediately and ice bath 5min, further take out to put and maintain after 5min obtains pretreatment at room temperature
Random library.
3.2 hatch: dissolving 200 μ g HPP's dry powder with 1 × Binding Buffer of 50 μ L, to obtain HPP molten
Liquid, joins HPP's solution in step 3.1 in pretreated random library, hatches 2h at 37 DEG C.
3.3 dissociate: add graphene oxide dispersion (final concentration of 30 μ of graphene oxide dispersion after having hatched
G/mL), adsorbing 25min at 25 DEG C, then centrifugal 10min under 20800g, collects supernatant, is screening income earner's pancreatic polypeptide
Specific nucleic acid aptamers library.
4, carry out PCR and expand library: the HPP's specific nucleic acid aptamers library taking 100 μ L screening gained carries out routine
PCR expands, and amplification condition is: denaturation 10min at 94 DEG C, degeneration 30sec at 94 DEG C, and anneal at 56.9 DEG C 30sec, at 72 DEG C
Extending 30sec, circulation 10 is taken turns, and finally finally extends 7min at 72 DEG C.Need after first round screening by all HPP
Library expands in advance 10 circulations that specific nucleic acid is fit, then carry out the amplification of this step, obtain amplified production.
5, DNA strand library is prepared: agarose microbeads 2200g modified by 200 μ L Streptavidins is centrifuged removes supernatant, then
With 500 μ L PBS washings, it is centrifuged and removes supernatant；Repeated washing twice.PCR in step 3 is expanded amplified production and the agar of gained
Sugar microsphere is hatched half an hour at normal temperatures, by the parent of the biotin on amplified production with the Streptavidin in agarose microbeads
With acting on, double-stranded DNA is fully captured agarose microbeads surface.Then 2200g is centrifugal removes supernatant, with PBS centrifuge washing three
Secondary；Add 200mM NaOH solution 500 μ L to degenerative treatments for double-stranded DNA in agarose microbeads, normal-temperature reaction
15min, 2200g are centrifuged 5min, collect supernatant；After desalination post washs with 10mL sterilized water, after adding alkaline denaturation, collection obtains
Supernatant, drips off naturally.Adding 1mL sterilized water, collect the solution dripped, this is DNA strand library.
6, screening is repeated: the random library in the DNA strand library alternative steps 3 step 5 obtained, repeat the above steps
Positive-selecting shown in 3～5, PCR expand and produce single-stranded DNA banks process, are repeated 4 times.
7, negative screening: the 5th take turns screening and after, with human serum albumin, bovine serum albumin, hemoglobin be
Comparison, carries out negative screening by screening the DNA strand library obtained after step 5, and the concrete operations of negative screening are: by human serum
Albumin, bovine serum albumin, hemoglobin dry powder are dissolved in buffer solution, then with after step 5, to screen the DNA obtained mono-
Chain library is hatched；It is subsequently adding the buffer containing graphene oxide to hatch, supernatant discarded by centrifugation, adds buffering molten
Graphene oxide is uniformly dispersed by liquid again, collects graphene oxide solution now.
8, multi-turns screen: by the DNA strand library in the graphene oxide solution alternative steps 6 collected by step 7, continues
Repeat the operating process of above-mentioned steps 6～7, after the first round screens, be about for hatching the library amount of screening
500pmol, from the beginning of third round is screened, target begins to decline with the ratio of random library, and ratio drops to 1 from 10；Repeat sieve
By the fluorescence spectrophotometer monitoring gained DNA strand library enhancing situation to HPP's identification ability during choosing, until 18 take turns
After screening, DNA strand library meets requirement to the identification ability of HPP, is analyzed through high flux sequence by products therefrom.To order-checking
After result finishing analysis, seven sequences that final available enrichment is high: Q10, Q11, Q12, Q13, Q14, Q16, Q17, these are seven years old
Bar sequence is the aptamer that can be used for detecting HPP of above-mentioned the present embodiment.
Investigate 1: with IDTDNA software, seven sequences of this present embodiment are carried out Sketch of secondary structure.With IDTDNA software pair
Seven sequences of the present embodiment carry out the structural representation after Sketch of secondary structure as shown in Fig. 2 to Fig. 8, and analog result shows seven
The secondary structure of bar sequence all contains stem loop section, shows that it can be curled into tertiary structure further and be combined with HPP.
Investigate 2: non denatured polyacrylamide gel electrophoresis detects the combination effect of sequence 1 (Q10) and HPP.
Concrete investigation method comprises the following steps: Q10 carries out degenerative treatments: after heating 5min at 95 DEG C, take out immediately
And ice bath 5min, more at room temperature maintain 5min.Experimental group: take 5 μ L concentration and be 5 μMs of degeneration good DNA sequence with 5 μ L concentration is
The HPP of 100 μMs mixes in one 200 μ L PCR pipe；Matched group: taking 5 μ L concentration is the DNA sequence and 5 μ that 5 μMs of degeneration are good
1 × Binding Buffer of L mixes in another 200 μ L PCR pipe, and two PCR pipe hatch 2h the most respectively at 25 DEG C.Incubate
After educating end, (sample-loading buffer contains 0.25% all to add 3 μ L loading buffer in 10 μ L sample of experimental group and matched group
Bromophenol blue, 0.25% dimethylbenzene green grass or young crops FF, 30% glycerol), 2 μ LSYBR GOLD dyestuffs obtain mixed liquor, mixed liquor is mixed
After, take in the loading wells that 10 μ L click and enter 18% non-denaturing polyacrylamide gel, with constant current mode 10mA in 1 × tbe buffer liquid
In carry out electrophoresis, electrophoresis tank answers lucifuge.When purple bromophenol blue band runs to about 1cm bottom gel, stop electrophoresis, take out and clap
According to.
Non-denaturing polyacrylamide gel compound method (10mL system): 30% acrylamide/methylene diacrylamide
6mL, 5 × tbe buffer liquid 2mL, ultra-pure water 1.93mL, 10% Ammonium persulfate. 70 μ L, TEMED 7 μ L.
The compound method of electrophoretic buffer (TBE, 5 ×/500mL): Tris-base 27g, Na2EDTA·2H2O 1.86g,
Boric acid 13.75g, adds 400mL ultra-pure water, is adjusted to pH=8.3, and by solution constant volume in 500mL volumetric flask.
Testing result is as it is shown in figure 9, swimming lane 1 is matched group, and swimming lane 2 is experimental group, and swimming lane 2 exists one relative to swimming lane 1
Location shifting is to affect the secondary structure of Q10 with Q10 after HPP is combined thus affects its electrophoretic velocity, and the present embodiment is described
Q10 aptamer HPP is had identification ability.
Investigate 3: by the combination effect of multi-functional microplate reader, graphene oxide detection sequence 2 (FAM-Q11) and HPP
Really.
According to the method investigating 2, FAM-Q11 is carried out degenerative treatments.By good for degeneration FAM-Q11 with final concentration of 1 μM
HPP mixes in EP pipe, the final concentration of FAM-Q11 is respectively 10,25,50,75,100,150,200,300nM.Mixing
It is placed in well in 25 DEG C of calorstats and hatches 2h, rear add graphene oxide and to control its ratio with FAM-Q11 be 2nmol/mg, then
Hatch 25min.Collect supernatant after being centrifuged 15min with 20800g, utilize the fluorescence of multi-functional microplate reader detection supernatant.With glimmering
Light changing value is vertical coordinate, and the concentration of DNA is abscissa, by Y=Bmax × X/ (Kd+ X) equation simulation curve, obtain this enforcement
The dissociation constant of example FAM-Q11 draws curve such as Figure 10, thus can get dissociation constant KdAs shown in table 1 below.
Table 1: dissociation constant result
| Sequence names | Dissociation constant (nanomole) |
| Q11 | 24.72±13.40 |
As shown in Figure 10, FAM-Q11 meets Michaelis-Menten equation with the combination of HPP, is that typical albumen is combined with part
Pattern；Dissociation constant KdIt is in nanomole rank, shows that FAM-Q11 has stronger affinity with the combination of HPP.
Investigate the combination effect of 4: multi-functional microplate reader, graphene oxide detection aptamer FAM-Q11 and HPP
Fruit and specificity.
Respectively FAM-Q11 and negative control (FAM-Random) are carried out degenerative treatments according to the method investigating 2.By degeneration
Good FAM-Q11, FAM-Random mix FAM-Q11, FAM-with the HPP of variable concentrations in black ELISA Plate
Random final concentration is 50nM, the final concentration of HPP is respectively 0,0,100,250,500,1000,1250,1500nM.
ELISA Plate being placed in 25 DEG C of calorstats after mixing and hatch 2h, rear addition graphene oxide also makes its final concentration of 18 μ g/mL,
(first group of 0nM HPP, as fluorescence initial value, is not added with graphite oxide after hatching to hatch 25min in 25 DEG C of calorstats
Alkene).The fluorescence of multi-functional microplate reader detection FAM is utilized after hatching end.
Result is as shown in figure 11: relative to random sequence FAM-Random, the signal that FAM-Q11 is combined with HPP is more
By force, illustrate that the FAM-Q11 of the present embodiment has strong specific recognition capability to HPP.
Investigate 5: fluorescence spectrophotometer, graphene oxide detection incubation time is to aptamer FAM-Q11 and HPP
The impact of combination effect.
According to the method investigating 2, FAM-Q11 is carried out degenerative treatments.By FAM-Q11 good for degeneration with final concentration of
HPP's mixing of 1500nM, adds the denseest at 25 DEG C after hatching 15min, 30min, 45min, 60min, 90min respectively
Degree is the graphene oxide of 18 μ g/mL, then hatches 25min, and now the final concentration of FAM-Q11 is 50nM.Finally use fluorescence light
The fluorescence of spectrometer detection FAM, and calculate the fluorescence recovery rate under different incubation time.
As shown in figure 12, after 45min, fluorescence recovery rate basically reaches platform to result, and the FAM-of the present embodiment is described
Q11 is very fast with the combination of HPP.
Investigate 6: fluorescence spectrophotometer, graphene oxide detect the temperature knot to aptamer FAM-Q11 Yu HPP
Close the impact of effect.
According to the method investigating 2, FAM-Q11 is carried out degenerative treatments.The FAM-Q11 that degeneration is good is respectively with final concentration
HPP's mixing of 0nM and 1500nM, 4 DEG C, 15 DEG C, 25 DEG C, hatch under 37 DEG C of four different temperatures, the end of DNA sequence
Concentration is 50nM.After hatching 2h, add graphene oxide and make its final concentration of 18 μ g/mL, then hatching 25min.Finally use
The fluorescence of fluorescence spectrophotometer detection FAM, and calculate the fluorescence recovery rate under different temperatures.
As shown in figure 13, the fluorescence recovery rate difference at four temperature is little, illustrates that temperature is to the present embodiment for result
FAM-Q11 is inconspicuous with the combination of HPP impact.
Investigate the difference of 7: multi-functional microplate reader detection aptamer FAM-Q11 and HPP's antibody combining site.
HPP's antibody fixing on magnetic bead: take the 50 μ coated magnetic beads of L protein A, through magnetic field separation supernatant discarded
Liquid.Add 200 μ L antibody incubation liquid 2.4 parts of embodiment 1 (antibody incubation liquid collocation method see) to wash, divide through magnetic field
From abandoning supernatant, cyclic washing magnetic bead three times；Add 5 μ g HPP's antibody after washing, at 37 DEG C, hatch 30min；Incubate
Educate after end through magnetic field separation abandoning supernatant, and with 200 μ L antibody incubation liquid washing magnetic bead three times, magnetic bead is dispersed in the most at last
In 500 μ L antibody incubation liquid.
According to the method investigating 2, FAM-Q11 is carried out degenerative treatments.The good FAM-Q11 of degeneration and final concentration be respectively 0,
100, HPP's mixing of 200nM, the final concentration of FAM-Q11 is 20nM, hatches 1.5h at 25 DEG C.It is subsequently added 20 μ L solid
Reserve the magnetic bead of HPP's antibody, then hatch 30min.Take supernatant through magnetic field separation after hatching end, use multifunctional enzyme mark
The fluorescence of FAM in instrument detection supernatant.
Result is as shown in figure 14: FAM-Q11 and variable concentrations HPP add after hatching and be fixed with HPP's antibody
Magnetic bead, hatch in rear supernatant fluorescence and decline, illustrate that HPP can also be shown by antibody capture after being combined with FAM-Q11
FAM-Q11 is different from the site that antibody and HPP combine with the site that HPP combines.
Investigate 8: multi-functional microplate reader detects the selectivity that aptamer FAM-Q11 is combined with HPP.
According to the method investigating 7, HPP's antibody is fixed on magnetic bead.
According to the method investigating 2, FAM-Q11 is carried out degenerative treatments.The good FAM-Q11 of degeneration respectively with HPP, pancreas
Island element, glucagon, protein kinase A peptide substrate, glutathion mix, and the final concentration of FAM-Q11 is 20nM, HPP
Final concentration be respectively 0,200nM, the final concentration of insulin are respectively 0,200nM, the final concentration of glucagon are respectively 0,
200nM, the final concentration of protein kinase A peptide substrate are respectively 0,200nM, the final concentration of glutathion are respectively 0,200nM, 25 DEG C
Under hatch 1.5h.It is subsequently added 20 μ L and fixes the magnetic bead of HPP's antibody, then hatch 30min.Hatch after end through magnetic field
Separation takes supernatant, detects the fluorescence of FAM in supernatant by multi-functional microplate reader, and calculates change in fluorescence rate.
As shown in figure 15, FAM-Q11 and different material add, after hatching, the magnetic bead being fixed with HPP's antibody to result, only
Have the supernatant fluorescence that HPP organizes to decline, illustrate only HPP after being combined with FAM-Q11 can also antibody capture, have
There is specificity.
Investigate 9: fluorescence spectrophotometer detects two aptamer FAM-Q12 of the present embodiment (sequence 3), BHQ1-Q13 (sequence
Row 4) and the binding pattern of HPP.
According to the method investigating 2, after being mixed by FAM-Q12 and BHQ1-Q13, carry out degenerative treatments.The FAM-good by degeneration
Q12 and BHQ1-Q13 respectively with final concentration of 0,10,20,50, the HPP of 100nM mix, at 25 DEG C, hatch 4h, FAM-
The final concentration of Q12 and BHQ1-Q13 is 100nM.The fluorescence of FAM in fluorescence spectrophotometer detection solution is utilized after hatching end.
Result is as shown in figure 16: along with the increase of HPP's concentration, the fluorescence intensity of solution declines, fluorophor is described
Being furthered with the distance of quenching group, fluorescence is quenched, and this shows that FAM-Q12 and BHQ1-Q13 can tie with HPP simultaneously
Close, form sandwich structure.
Further, in the present embodiment, SH-Q16 and SH-Q17 can also be combined with pancreatic polypeptide simultaneously, forms sandwich knot
Structure.
Investigate 10: fluorescence spectrophotometer detects aptamer BHQ1-Q14 (sequence 5), FAM-Q15 (closing probe) and people
The binding site of pancreatic polypeptide.
BHQ1-Q14 and FAM-Q15 makes annealing treatment: mixes BHQ1-Q14 and FAM-Q15 to be placed in 95 DEG C and adds
Hot 5min, is placed on room temperature and makes its natural cooling 2h.BHQ1-Q14 and FAM-Q15 annealed is respectively 0nM with final concentration
Mixing with the HPP of 50nM, hatch 2h at 25 DEG C, wherein the end of the final concentration of 20nM, FAM-Q15 of BHQ1-Q14 is dense
Degree is 25nM.Separately set the matched group BHQ1-Q14 as final concentration 20nM, also at 25 DEG C, hatch 2h.Fluorescence is used after hatching end
The fluorescence of spectrogrph detection FAM.
Result is as shown in figure 17: the Q14 that marked quenching group is recognition sequence, and the Q15 that marked fluorophor is envelope
Closing sequence, the fluorescence of the fluorescence Q14 more single than matched group after Q14 and Q15 annealing reduces many, annealed process is described
Together with Q11 with Q15 can well hybridize, furthered the distance between fluorophor and quenching group, and fluorescence is quenched.And warp
Q14 and Q15 of annealing, after adding HPP, compares Q14 and Q15 complex, and fluorescence rises, and HPP's energy is described
Be combined with Q14, destroy the hybrid structure of Q14 and Q15 so that the distance of quenching group and fluorophor becomes remote.And due to Q15
Close is the 3 ' ends of Q14, illustrates that HPP may combine with the 3 ' ends of Q14.
Investigate 11: it is (auxiliary that dynamic light scattering method detects the present embodiment SH-Q16 (sequence 6), SH-Q17 (sequence 7), SH-Q18
Help probe) Detection results to HPP.
The synthesis of gold nano grain: after being boiled by the gold chloride of 100mL 0.01%, adds 3mL 1% trisodium citrate molten
Liquid, continues heating and maintains fluidized state, and solution becomes claret, then boils 15 to 20min, can stop heating.Treat that solution is cold
It is settled to 100mL the most afterwards.
SH-Q16, SH-Q17 and SH-Q18 are in the modification on gold nano grain surface: by 10 μ L concentration be 100 μMs of SH-Q16,
10 μ L concentration be 100 μMs of SH-Q18 and 10 μ L concentration be 100 μMs of SH-Q17,10 μ L concentration be that 100 μMs of SH-Q18 are separately added into
It is all that 1mL gold nano grain (does two pipe Duplicate Samples) to volume, at 4 DEG C, hatches 16h.It is subsequently adding 500 μ L 30mM
PBS, then hatch 40h at 4 DEG C.After hatching end, the rotating speed centrifugal 30min at 4 DEG C with 20800g removes supernatant, will precipitation
1 × Binding Buffer of 1mL is uniformly dispersed, then removes supernatant with rotating speed centrifugal 30min at 4 DEG C of 20800g,
Repeat dispersion, be centrifuged and go supernatant step twice.Obtain the co-modified gold grain of SH-Q16, SH-Q18 and SH-Q17, SH-Q18 are common
(SH-16 and SH-17, as detection probe, has recognition reaction, SH-Q18 not to have recognition reaction, is only intended to the gold grain modified
The regulation detection probe modification density on gold grain surface, SH-Q18 and SH-Q16 modifies jointly at Yijin pellet surface, SH-
Q18 and SH-Q17 modifies the most jointly on another gold grain surface).
Take the gold grain of the SH-Q16 that 20 μ L modify and SH-Q17 gold grain mixing that 20 μ L modify, add people's pancreas many
Peptide, the final concentration of HPP is respectively 0,1,2,5,10,15nM, at 25 DEG C, hatch 2h after mixing.Utilize after hatching end
The particle diameter of the gold grain of dynamic light scattering method detection sample.
Result is as shown in figure 18: along with the rising of HPP's concentration, the particle diameter of gold grain increases, HPP's energy is described
SH-Q16 and SH-Q17 on gold grain is combined simultaneously, draws in the distance between gold grain, makes particle diameter become big.And the grain of gold grain
Footpath change is linear with the concentration of HPP, and linear equation is y=0.138+1.229x, and correlation coefficient is 0.9936,
Detection is limited to 37pM.Illustrate to utilize SH-Q16, SH-Q17 and dynamic light scattering method detection HPP to have preferable effect, inspection
Survey limit relatively low.
Investigate 12: dynamic light scattering method detects the selectivity of two aptamer detection HPPs of the present embodiment.
The synthesis of gold nano grain and SH-Q16, SH-Q17 and SH-Q18 modification on the gold particle is with reference to investigating 11.
Take 20 μ L and modified the gold grain of SH-Q16 and 20 μ L have modified the mixing of SH-Q17 gold grain, be separately added into people's pancreas many
Peptide, bovine serum albumin, human serum albumin, immunoglobulin G, insulin, glucagon, the final concentration of six kinds of materials is all
For 15nM, after mixing, at 25 DEG C, hatch 2h.The particle diameter of the gold grain of dynamic light scattering method detection sample is utilized after hatching end.
Result is as shown in figure 19: has modified the gold grain of SH-Q16 and has modified SH-Q17 gold grain only and HPP
Hatch rear change of size more apparent, hatch rear change of size not quite with other material, illustrate that SH-Q16 and SH-Q17 is many with people's pancreas
The combination of peptide has certain specificity.
The above, be only presently preferred embodiments of the present invention, and the present invention not makees any pro forma restriction.Though
So the present invention discloses as above with preferred embodiment, but is not limited to the present invention.Any it is familiar with those skilled in the art
Member, in the case of without departing from the spirit of the present invention and technical scheme, may utilize in method and the technology of the disclosure above
Hold and technical solution of the present invention is made many possible variations and modification, or be revised as the Equivalent embodiments of equivalent variations.Therefore,
Every content without departing from technical solution of the present invention, the technical spirit of the foundation present invention is to any letter made for any of the above embodiments
Single amendment, equivalent, equivalence change and modification, all still fall within the range of technical solution of the present invention protection.
Claims (10)
1. HPP's aptamer, it is characterised in that described HPP's aptamer is following sequence 1～7
In any one:
Sequence 1 is Q10, and the sequence of described Q10 is the sequence shown in SEQ ID NO.1；
Sequence 2 is 5 '-Q11-FAM-3 ', and the sequence of described Q11 is the sequence shown in SEQ ID NO.2；
Sequence 3 is 5 '-Q12-FAM-3 ', and the sequence of described Q12 is the sequence shown in SEQ ID NO.3；
Sequence 4 is 5 '-BHQ1-Q13-3 ', and the sequence of described Q13 is the sequence shown in SEQ ID NO.4；
Sequence 5 is 5 '-Q14-BHQ1-3 '；The sequence of described Q14 is the sequence shown in SEQ ID NO.5；
Sequence 6 is 5 '-SH-Q16-3 ', and the sequence of described Q15 is the sequence shown in SEQ ID NO.6；
Sequence 7 is 5 '-SH-Q17-3 ', and the sequence of described Q15 is the sequence shown in SEQ ID NO.7.
HPP's aptamer the most according to claim 1, it is characterised in that the nucleotides sequence of described sequence 1～7
A certain position on row is phosphorylated, methylates, amination, sulfhydrylation or isotopologue.
HPP's aptamer the most according to claim 1, it is characterised in that the nucleotides sequence of described sequence 1～7
Biotin, digoxin, fluorescent material, nano luminescent material or enzyme labelling it is combined with on row.
HPP's aptamer the most according to claim 3, it is characterised in that described fluorescent material includes fluorescence
Element, rhodamine, anthocyanidin；Described nano material is gold nano grain；Described enzyme labelling is horseradish peroxidase.
HPP's aptamer the most according to any one of claim 1 to 4, it is characterised in that described people's pancreas is many
Peptide nucleic acid(PNA) aptamers also includes any one in following three kinds of sequences:
(1) with the homology of the nucleotide sequence of described HPP's aptamer more than 80%；
(2) nucleotide sequence with described HPP's aptamer carries out the sequence hybridized；
(3) RNA sequence that the nucleotide sequence of described HPP's aptamer is transcribed.
HPP's aptamer the most according to any one of claim 1 to 4, it is characterised in that described people's pancreas is many
Peptide nucleic acid(PNA) aptamers also includes the thiophosphate that the skeleton of the nucleotide sequence of described HPP's aptamer derives
Skeleton, or the corresponding peptides nucleic acid that described HPP's aptamer is transformed into.
7. the screening technique of HPP's aptamer according to any one of a claim 1 to 6, it is characterised in that bag
Include following steps:
S1, synthesis random library and primer:
Random library employing RS35 random library: 5 '-AGCGTCGGATACCACTACTA-
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN-ATCATGGAGTTCGTGGTCAG-3 ',
5 ' primers: 5 '-FAM-AGCGTCGGATACCACTACTA-3 ',
3 ' primers: 5 '-Biotin-CTGACCACGAACTCCATGAT-3 '；
S2, GO-SELEX screen: be dissolved in buffer solution by HPP's dry powder, hatch with random sequence；Then add
Enter the buffer containing graphene oxide to hatch, collect supernatant by centrifugation, be HPP's specific nucleic acid aptamers literary composition
Storehouse；
S3, PCR expand library: described HPP's specific nucleic acid aptamers library is carried out PCR amplification, obtains amplified production；
S4, prepare DNA strand library: the centrifugal supernatant that goes of agarose microbeads modified by Streptavidin, more described amplification is produced
Thing is hatched at normal temperatures with agarose microbeads；It is subsequently adding alkali liquor in agarose microbeads, reacts under room temperature, be centrifuged, in collection
Clear liquid；Collect the solution dripped after described supernatant is crossed desalination post, be DNA strand library；
S5, repeat screening: the random library substituting in described step S2 by described DNA strand library, and repeat the above steps S2
～the process of S4 is at least one times；
S6, negative screening: bovine serum albumin, human serum albumin, hemoglobin dry powder are dissolved in buffer solution, then
Hatch with described DNA strand library；It is subsequently adding the buffer containing graphene oxide to hatch, supernatant discarded by centrifugation,
Add buffer solution to be again uniformly dispersed by graphene oxide, collect graphene oxide solution now；
S7, multi-turns screen: the graphene oxide solution collected by S6 is substituted the DNA strand library of S5, continues to repeat above-mentioned step
The operating process of rapid S5～S6；Until filtering out the identification ability to HPP to meet the aptamer of requirement.
Screening technique the most according to claim 7, it is characterised in that particularly as follows: HPP is done in described step S2
Powder is dissolved in buffer solution, hatches 2h with random sequence at 37 DEG C；It is subsequently adding the buffer containing graphene oxide to exist
Hatch 25min at 25 DEG C, collect supernatant by centrifugation, be HPP's specific nucleic acid aptamers library；Described random sequence with
The ratio of described graphene oxide is 1.67nmol/mg.
Screening technique the most according to claim 7, it is characterised in that in described S3 step, the technique bar of described PCR amplification
Part is: 94 DEG C of denaturations 10min, degeneration 30sec at 94 DEG C, and 56.9 DEG C of annealing 30sec, 72 DEG C extend 30sec, circulate 10～20
Wheel, finally finally extends 7min at 72 DEG C；In described step S4, described amplified production is hatched at normal temperatures with agarose microbeads
Time be 0.5h, add alkali liquor in agarose microbeads, under room temperature, react 15min.
10. HPP's aptamer according to any one of a claim 1 to 6 is at the reagent of preparation detection HPP
Application in box.
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