CN105861669A - Amplificatory detection method for quickly capturing branched-chain DNA (Deoxyribonucleic Acid) signal of hybrid target substance - Google Patents
Amplificatory detection method for quickly capturing branched-chain DNA (Deoxyribonucleic Acid) signal of hybrid target substance Download PDFInfo
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Abstract
The invention relates to the field of biotechnology, and particularly relates to an amplificatory detection method for quickly capturing a branched-chain DNA (Deoxyribonucleic Acid) signal of a hybrid target substance. By using the amplificatory detection method for quickly capturing the branched-chain DNA signal of the hybrid target substance, the detection sensitivity of a method for amplifying the branched-chain DNA signal is increased; the lowest detection sensitivity can be up to 200 to 300 transcripts per time; the sensitivity is increased by 4 times; detection time is extremely shortened, and is shortened to be within 5 to 7 hours from previous OVEN (overnight reaction); compared with a bDNA (branched-DNA) technique of the Siemens, a QuantiMAT (Quantitative Melocular Amplification Technique) is lower in cost and better in convenience. Through surveying the concentration of a 250fg/ml HIV (Human Immunodeficiency Virus) quality control product, a difference among the detectable rates of different magnetic beads is surveyed by setting different hybridization time; the technical route of the QuantiMAT can be used for detecting the target substance quite well; meanwhile, the specificity is guaranteed.
Description
Technical field
The present invention relates to biological technical field, put particularly to a kind of fast Acquisition hybridizing targets thing branch chain DNA signal
Big detection method.
Background technology
For a long time, nucleic acid detection technique is monopolized by PCR, the nucleic acid detection technique grown up so far PCR nothing more than
Covert improvement, the technology such as including LAMP, TMA, but the technology development stagnation, the example relatively that expand based on non-target substance
Such as hybrid capture II technology based on antibody capture (HC2 of Kai Jie company of Germany), NASBA technology based on RNA reverse transcription
(Hao Luojie company of the U.S.), but these technology all extremely rely on the extracting and purifying of nucleic acid, add sample contamination comparatively speaking
Risk.
Summary of the invention
In view of this, the present invention provides a kind of fast Acquisition hybridizing targets thing branch detection method that chain DNA signal amplifies.
The present invention establishes a kind of method of brand-new nucleic acid substances detection, expands detection of nucleic acids product by the foundation of this simple and easy method
In medical treatment and the range of scientific research field.
In order to realize foregoing invention purpose, the present invention provides techniques below scheme:
The invention provides the detection method that a kind of fast Acquisition hybridizing targets thing branch chain DNA signal amplifies, including such as
Lower step:
Step 1: obtain nucleic acid hybridization primer；Nucleic acid purpose fragment is divided into capturing function primer according to functional attributes
District, mark function guiding region, covering function guiding region, respectively according to described capturing function guiding region, described mark function primer
District, described covering function guiding region obtain complementary primer；
The primer in described capturing function district is capturing function district primer；
Step 2: take amido modified universal primer and be combined with the specific groups of magnetic bead surfaces (formation sulfonic group), forms bag
There is the magnetic bead of nucleic acid；
Described amido modified universal primer is combined with described capturing function district primer hybridization；
The described magnetic bead being coated with nucleic acid can specificity capture target substance；
Step 3: obtain nucleic acid branched chain signal amplifying system；System includes primary signal amplifier chain and secondary signal is put
Big chain；
The length of described primary signal amplifier chain is 500～560bp, including the primary preposition homing sequence of amplifier chain and 20 times
The preposition boot section of secondary signal amplifier chain repeated；
The length of described secondary signal amplifier chain is 420～480bp, including the secondary preposition homing sequence of amplifier chain and 20 times
The reporter probe duplicate block repeated；
The described primary preposition homing sequence of amplifier chain combines with the hybridization of mark function guiding region described in step 1；
The secondary signal repeated for 20 times of the described secondary preposition homing sequence of amplifier chain and described primary signal amplifier chain is put
The hybridization of big chain preposition boot section combines；
Step 4: obtain the reporter probe of AP coupling；Amido modified reporter probe primer is combined shape with alkali phosphatase
Becoming nucleic acid/protein complexes, described nucleic acid/protein complexes is miscellaneous with the reporter probe duplicate block of described in step 3 20 repetitions
Knot is closed, it is thus achieved that AP labelling reporter probe, expands the signal value of captured target substance, detection.
In some specific embodiments of the present invention, the nucleotide sequence of the universal primer of described capturing function guiding region
(5 '～3 ') are as shown in SEQ ID No.1.It is specially (5 '～3 '):
NNNNNNNNNNNNNNNNNNNNNNNNNTTTTTAGCTTGGGCTTAGTCGTT。
In some specific embodiments of the present invention, the nucleotide sequence (5 of the universal primer of described mark function guiding region
'-3 ') as shown in SEQ ID No.2.It is specially (5 '-3 '):
GAATCATTCGAATTTTACCGTTTTTNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN
NTTTTTACGTATTCGGATTCGGCTT。
In some specific embodiments of the present invention, the described primary preposition homing sequence of amplifier chain such as SEQ ID No.3
Shown in.It is specially AAGCCGAATCCGAATACGTCGGTAAAATTCGAATGATTC.
In some specific embodiments of the present invention, the preposition boot section of secondary signal amplifier chain that described 20 times are repeated
Nucleotide sequence is as shown in SEQ ID No.4.It is specially
AGGATCGTAAGTAACTAAGGACTACCAGGATCGTAAGTAACTAAGGACTACCAGGATCGTAAGTAACTAAGGACTAC
CAGGATCGTAAGTAACTAAGGACTACCAGGATCGTAAGTAACTAAGGACTACCAGGATCGTAAGTAACTAAGGACTA
CCAGGATCGTAAGTAACTAAGGACTACCAGGATCGTAAGTAACTAAGGACTACCAGGATCGTAAGTAACTAAGGACT
ACCAGGATCGTAAGTAACTAAGGACTACCAGGATCGTAAGTAACTAAGGACTACCAGGATCGTAAGTAACTAAGGAC
TACCAGGATCGTAAGTAACTAAGGACTACCAGGATCGTAAGTAACTAAGGACTACCAGGATCGTAAGTAACTAAGGA
CTACCAGGATCGTAAGTAACTAAGGACTACCAGGATCGTAAGTAACTAAGGACTACCAGGATCGTAAGTAACTAAGG
ACTACCAGGATCGTAAGTAACTAAGGACTACCAGGATCGTAAGTAACTAAGGACTACC。
In some specific embodiments of the present invention, the described secondary preposition homing sequence of amplifier chain such as SEQ ID No.5
Shown in.It is specially GGTAGTCCTTAGTTACTTACGATCCT.
In some specific embodiments of the present invention, the nucleotide sequence of the reporter probe duplicate block that described 20 times are repeated is such as
Shown in SEQ ID No.6.It is specially GAATCCATTGAATCCTGTGATTGAATCCATTGAATCCTGTGATTGAATCCATTGA
ATCCTGTGATTGAATCCATTGAATCCTGTGATTGAATCCATTGAATCCTGTGATTGAATCCATTGAATCCTGTGATT
GAATCCATTGAATCCTGTGATTGAATCCATTGAATCCTGTGATTGAATCCATTGAATCCTGTGATTGAATCCATTGA
ATCCTGTGATTGAATCCATTGAATCCTGTGATTGAATCCATTGAATCCTGTGATTGAATCCATTGAATCCTGTGATT
GAATCCATTGAATCCTGTGATTGAATCCATTGAATCCTGTGATTGAATCCATTGAATCCTGTGATTGAATCCATTGA
ATCCTGTGATTGAATCCATTGAATCCTGTGATTGAATCCATTGAATCCTGTGATTGAATCCATTGAATCCTGTGATT。
In some specific embodiments of the present invention, the sequence such as SEQ ID No.7 institute of described AP labelling reporter probe
Sequence-the AP shown, specially 5 '-AATCACAGGATTCAATGGATTC-AP-3 '.
In some specific embodiments of the present invention, the sequence of universal primer amido modified described in step 2 is
NH2-sequence as shown in SEQ ID No.8, specially 5 '-NH2-AACGACTAAGCCCAAGCTAGTG-3 '.
The invention provides the detection method that a kind of fast Acquisition hybridizing targets thing branch chain DNA signal amplifies, including such as
Lower step: step 1: obtain nucleic acid hybridization primer；By nucleic acid purpose fragment according to functional attributes be divided into capturing function guiding region,
Mark function guiding region, hide function guiding region, respectively according to described capturing function guiding region, described mark function guiding region,
Described covering function guiding region obtains complementary primer；
The primer in described capturing function district is capturing function district primer；
Step 2: take amido modified universal primer and be combined with the specific groups of magnetic bead surfaces (formation sulfonic group), forms bag
There is the magnetic bead of nucleic acid；
Described amido modified universal primer is combined with described capturing function district primer hybridization；
The described magnetic bead being coated with nucleic acid can specificity capture target substance；
Step 3: obtain nucleic acid branched chain signal amplifying system；System includes primary signal amplifier chain and secondary signal is put
Big chain；
The length of described primary signal amplifier chain is 500～560bp, including the primary preposition homing sequence of amplifier chain and 20 times
The preposition boot section of secondary signal amplifier chain repeated；
The length of described secondary signal amplifier chain is 420～480bp, including the secondary preposition homing sequence of amplifier chain and 20 times
The reporter probe duplicate block repeated；
The described primary preposition homing sequence of amplifier chain combines with the hybridization of mark function guiding region described in step 1；
The secondary signal repeated for 20 times of the described secondary preposition homing sequence of amplifier chain and described primary signal amplifier chain is put
The hybridization of big chain preposition boot section combines；
Step 4: obtain the reporter probe of AP coupling；Amido modified reporter probe primer is combined shape with alkali phosphatase
Becoming nucleic acid/protein complexes, described nucleic acid/protein complexes is miscellaneous with the reporter probe duplicate block of described in step 3 20 repetitions
Knot is closed, it is thus achieved that AP labelling reporter probe, expands the signal value of captured target substance, detection.
The present invention improves branch's chain DNA method for amplifying signal detection sensitivity, and minimum detection sensitivity can reach
200～300 transcript/time sensitivity improve 4 times；Shorten the detection time greatly, (the most anti-by OVEN before
Should) foreshorten to 5～7 hours in complete the detection time；QuantiMAT technical costs is lower compared with Siemens bDNA technology
Honest and clean, convenience is more preferable.By investigating the HIV quality-control product concentration of 250fg/ml, different hybridization time is set and investigates different magnetic
The recall rate difference of pearl, the technology path of QuantiMAT can well detect target substance, guarantee specificity simultaneously.
The problem that the present invention (QuantiMAT) solves includes, the target not relying on nucleic acid exponential amplification amplifies, and is not required to
Will be stripped sample, reverse transcription, purification decrease sample contamination risk, the instrument that need not specialty easy and simple to handle, only need
Wanting water-bath or the equipment of other offer constant temperature, the basic unit that is prone to low cost promotes.
Accompanying drawing explanation
In order to be illustrated more clearly that the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing
In having technology to describe, the required accompanying drawing used is briefly described.
Fig. 1 shows the quantitative interval graph of embodiment 3；
Fig. 2 shows the claim 1 technical scheme first step；
Fig. 3 shows claim 1 technical scheme second step；
Fig. 4 shows claim 1 technical scheme the 3rd step；
Fig. 5 shows branched chain signal amplifying system design primary signal amplifier chain design；
Fig. 6 shows branched chain signal amplifying system design secondary signal amplifier chain design；
Fig. 7 shows claim 1 technical scheme the 4th step；
Fig. 8 shows overall evaluation of a technical project schematic diagram.
Detailed description of the invention
The invention discloses the detection method that a kind of fast Acquisition hybridizing targets thing branch chain DNA signal amplifies, this area
Technical staff can use for reference present disclosure, is suitably modified technological parameter and realizes.Special needs to be pointed out is, all similar replacements
Apparent to those skilled in the art with changing, they are considered as being included in the present invention.The method of the present invention
And application is described by preferred embodiment, related personnel substantially can be without departing from present invention, spirit and model
Enclose and interior method described herein and application be modified or suitably change and combine, realize and apply the technology of the present invention.
The present invention point following steps are carried out:
The first step, sets up a kind of nucleic acid hybridization primer design method, the method include by the nucleic acid purpose fragment of whole section by
According to functional attributes be divided into capturing function guiding region, mark function guiding region, hide region, three, function guiding region separately design
Specific complementary primer, detects probe primer and includes the primer sequence group of 3 kinds of Various Functions in QuantiMAT technology.
Second step, sets up a kind of liquid phase magnetic bead nucleic acid method for coating, and the method is that the specific groups utilizing magnetic bead surfaces is repaiied
Decorations, react under certain condition with the universal primer (be used for and capturing function district primer hybridizes) of amido modified mistake, are formed
Stable NHS (sulfonic group), this magnetic bead being coated with nucleic acid captures target substance for specificity.In QuantiMAT technology
The universal sequence of capture magnetic bead is 22bp.
5’-NH2-AACGACTAAGCCCAAGCTAGTG-3’
3rd step, sets up a kind of nucleic acid branched chain signal amplifying system, and this system includes primary signal amplifier chain with secondary
Level signal amplifier chain, in QuantiMAT technology, the length of primary signal amplifier chain is between 500～560bp, puts including primary
The big preposition homing sequence of chain and the preposition boot section of secondary signal amplifier chain repeated for 20 times.The length of secondary signal amplifier chain is
Between 420～480bp, the reporter probe duplicate block repeated including the secondary preposition homing sequence of amplifier chain and 20 times.Wherein primary
Signal amplifier chain contains a preposition homing sequence, and this preposition homing sequence is responsible for miscellaneous with the mark function guiding region in the first step
Knot is closed, and secondary signal amplifier chain also contains a preposition homing sequence, and this homing sequence is responsible for and primary signal amplifier chain
Duplicate block hybridization combines.
4th step, sets up the reporter probe preparation method of a kind of AP coupling, and the method content is by the report of amido modified mistake
Accuse probe primer to react under certain condition with alkali phosphatase (AP), form a nucleic acid/protein complexes, this complex
Effect is with the reporter probe duplicate block in secondary signal amplifier chain carries out hybridization, in order to expand the signal of captured target
Value.
5’-AATCACAGGATTCAATGGATTC-AP-3’
Nucleotide sequence inventory:
The general purpose core acid sequence (5 '～3 ') of capturing function guiding region:
NNNNNNNNNNNNNNNNNNNNNNNNNTTTTTAGCTTGGGCTTAGTCGTT
The general purpose core acid sequence (5 '-3 ') of mark function guiding region:
GAATCATTCGAATTTTACCGTTTTTNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN
NTTTTTACGTATTCGGATTCGGCTT
The nucleotide sequence (the secondary amplifier chain preposition homing sequence district of preposition boot section+repetition) of primary amplifier chain:
AAGCCGAATCCGAATACGTCGGTAAAATTCGAATGATTC repetitive sequence district
-AGGATCGTAAGTAACTAAGGACTACCAGGATCGTAAGTAACTAAGGACTACCAGGATCGTAAGTAACT
AAGGACTACCAGGATCGTAAGTAACTAAGGACTACCAGGATCGTAAGTAACTAAGGACTACCAGGATCGTAAGTAAC
TAAGGACTACCAGGATCGTAAGTAACTAAGGACTACCAGGATCGTAAGTAACTAAGGACTACCAGGATCGTAAGTAA
CTAAGGACTACCAGGATCGTAAGTAACTAAGGACTACCAGGATCGTAAGTAACTAAGGACTACCAGGATCGTAAGTA
ACTAAGGACTACCAGGATCGTAAGTAACTAAGGACTACCAGGATCGTAAGTAACTAAGGACTACCAGGATCGTAAGT
AACTAAGGACTACCAGGATCGTAAGTAACTAAGGACTACCAGGATCGTAAGTAACTAAGGACTACCAGGATCGTAAG
TAACTAAGGACTACCAGGATCGTAAGTAACTAAGGACTACCAGGATCGTAAGTAACTAAGGACTACC
The nucleotide sequence (preposition boot section+duplicate reports probe sequence district) of secondary amplifier chain:
GGTAGTCCTTAGTTACTTACGATCCT repetitive sequence district
GAATCCATTGAATCCTGTGATTGAATCCATTGAATCCTGTGATTGAATCCATTGAATCCTGTGATTGAATCCATTGA
ATCCTGTGATTGAATCCATTGAATCCTGTGATTGAATCCATTGAATCCTGTGATTGAATCCATTGAATCCTGTGATT
GAATCCATTGAATCCTGTGATTGAATCCATTGAATCCTGTGATTGAATCCATTGAATCCTGTGATTGAATCCATTGA
ATCCTGTGATTGAATCCATTGAATCCTGTGATTGAATCCATTGAATCCTGTGATTGAATCCATTGAATCCTGTGATT
GAATCCATTGAATCCTGTGATTGAATCCATTGAATCCTGTGATTGAATCCATTGAATCCTGTGATTGAATCCATTGA
ATCCTGTGATTGAATCCATTGAATCCTGTGATTGAATCCATTGAATCCTGTGATT
AP labelling reporter probe sequence:
AATCACAGGATTCAATGGATTC-AP
The present invention improves branch's chain DNA method for amplifying signal detection sensitivity, and minimum detection sensitivity can reach
200～300 transcript/time sensitivity improve 4 times.；Shorten the detection time greatly, (the most anti-by OVEN before
Should) foreshorten to 5～7 hours in complete the detection time；QuantiMAT technical costs is lower compared with Siemens bDNA technology
Honest and clean, convenience is more preferable.By investigating the HIV quality-control product concentration of 250fg/ml, different hybridization time is set and investigates different magnetic
The recall rate difference of pearl, the technology path of QuantiMAT can well detect target substance, guarantee specificity simultaneously.
In the detection method that the present invention provides a kind of fast Acquisition hybridizing targets thing branch chain DNA signal amplifies used former
Material and reagent all can be buied by market.
Below in conjunction with embodiment, the present invention it is expanded on further:
Embodiment 1
Design QuantiMAT primer sequence such as table 1 institute in one group of high-risk human mammilla papillomavirus (HPV) 16 type E6E7 region
Show:
Table 1
Synthesizing above-mentioned primer, LEs:CEs:BLs mixes according to the ratio of 4:2:1, it is ensured that final mixed primer
Concentration is the every microlitre of 100pmol.
Compare the old process protocol carrying out branch's chain DNA according to the protocol of QuantiMAT simultaneously:
The first step, mixes the magnetic bead being coated with capture probe according to the ratio of 5:1 with primer mixture, then is separately added into
The each 50ul of hela100, Hela50, Hela25, Hela13, Hela6, Hela3, Hela1, Hela0.5 cell specimen after cracking,
It is built into the reaction system of 100ul, hatches 2～4 hours 50～55 degree of sealings；
Second step, washs third-order reaction cup with the PBS eluent of 1X, adds the primary amplifier chain mixing of 100ul after drying
Liquid, hatches 30～50 minutes 50～55 degree of sealings；
3rd step, washs third-order reaction cup with the PBS eluent of 1X, adds the secondary amplifier chain mixing of 100ul after drying
Liquid, hatches 30～50 minutes 50～55 degree of sealings；
4th step, washs third-order reaction cup with the PBS eluent of 1X, adds the reporter probe mixed liquor of 100ul after drying,
30～50 minutes are hatched 45～50 degree of sealings；
5th step, washs third-order reaction cup with the PBS eluent of 1X, adds the chemical luminous substrate of 100ul after drying,
35～40 degree of sealings hatch 15～20 minutes；
6th step, reads signal on chemiluminescent analyzer, and judges yin and yang attribute result.
Experimental group QuantiMAT group data are shown in Table 2.
Table 2
The minimum copy number (about 220 copies) that can detect that 0.5 cell；
Matched group bDNA group data are shown in Table 3.
Table 3
The minimum copy number (about 800 copies) that can detect that 3 cells.
From table 2, table 3 data, the method compared with prior art sensitivity that the present invention provides have submitted about 4 times.
Embodiment 2 range of linearity and be coated primer elaboration detection
Designing a primer, one section of capture primer on magnetic bead is combined, and another section is tied with reporter probe primer sequence district
Close the sequence of this primer as shown in SEQ ID No.44:
GAATCCATTGAATCCTGTGATTAACGACTAAGCCCAAGCT
Synthesize this primer, and this primer is diluted to the every microlitre of 100pmol, be coated inspection procedure according to QuantiMAT
Protocol operates.
The first step, mixes this primer according to the ratio of 1:5 with the magnetic bead being coated with capture primer, is built into 100ul's
Reaction system, hatches 2～4 hours 50～55 degree of sealings；
Second step, washs third-order reaction cup with the PBS eluent of 1X, adds the reporter probe mixed liquor of 100ul after drying,
30～50 minutes are hatched 45～50 degree of sealings；
3rd step, washs third-order reaction cup with the PBS eluent of 1X, adds the chemical luminous substrate of 100ul after drying,
35～40 degree of sealings hatch 15～20 minutes；
4th step, reads signal on chemiluminescent analyzer, and judges to be coated the elaboration of primer.
Gradient dilution primer sequence (GAATCCATTGAATCCTGTGATTAACGACTAAGCCCAAGCT).
Gradient 1:100pmol/ul；
Gradient 2:10pmol/ul；
Gradient 3:1pmol/ul；
Gradient 4:100fmol/ul；
Gradient 5:10fmol/ul；
Gradient 6:1fmol/ul；
Fabric swatch is as shown in table 4:
Table 4
| Blank | Gradient 1 | Gradient 2 | Gradient 3 | Gradient 4 | Gradient 5 | Gradient 6 |
| Blank | Gradient 1 | Gradient 2 | Gradient 3 | Gradient 4 | Gradient 5 | Gradient 6 |
| Blank | Gradient 1 | Gradient 2 | Gradient 3 | Gradient 4 | Gradient 5 | Gradient 6 |
| Blank | Gradient 1 | Gradient 2 | Gradient 3 | Gradient 4 | Gradient 5 | Gradient 6 |
| Blank | Gradient 1 | Gradient 2 | Gradient 3 | Gradient 4 | Gradient 5 | Gradient 6 |
| Blank | Gradient 1 | Gradient 2 | Gradient 3 | Gradient 4 | Gradient 5 | Gradient 6 |
| Blank | Gradient 1 | Gradient 2 | Gradient 3 | Gradient 4 | Gradient 5 | Gradient 6 |
| Blank | Gradient 1 | Gradient 2 | Gradient 3 | Gradient 4 | Gradient 5 | Gradient 6 |
Photonic data is as shown in table 5:
Table 5
| 485 | 1822630 | 173635 | 20375 | 1985 | 1140 | 655 |
| 525 | 1972955 | 187950 | 22055 | 2155 | 1235 | 700 |
| 530 | 1991745 | 189745 | 22260 | 2175 | 1245 | 715 |
| 455 | 1709890 | 162895 | 19115 | 1860 | 1060 | 615 |
| 430 | 1615945 | 153940 | 18060 | 1760 | 1015 | 585 |
| 495 | 1860210 | 177215 | 20795 | 2025 | 1165 | 660 |
| 510 | 1916585 | 182580 | 21425 | 2090 | 1190 | 685 |
| 550 | 2066900 | 196900 | 23105 | 2255 | 1295 | 745 |
Signal-to-noise ratio data is as shown in table 6:
Table 6
| 0 | 3663.58 | 349.02 | 40.95 | 3.99 | 2.29 | 1.32 |
| 0 | 3965.74 | 377.79 | 44.33 | 4.33 | 2.48 | 1.41 |
| 0 | 4003.51 | 381.40 | 44.74 | 4.37 | 2.50 | 1.44 |
| 0 | 3436.96 | 327.43 | 38.42 | 3.74 | 2.13 | 1.24 |
| 0 | 3248.13 | 309.43 | 36.30 | 3.54 | 2.04 | 1.18 |
| 0 | 3739.12 | 356.21 | 41.80 | 4.07 | 2.34 | 1.33 |
| 0 | 3852.43 | 366.99 | 43.07 | 4.20 | 2.39 | 1.38 |
| 0 | 4154.57 | 395.78 | 46.44 | 4.53 | 2.60 | 1.50 |
According to data analysis, magnetic bead be coated effect, when in gradient 1, be now coated primer and drawing of being added
Substrate concentration is proportional, and addition primer concentration is the highest, and obtained luminous signal value is the highest, and signal to noise ratio is the best；
Multiple proportions folding variation relation, 10 times of relations of theoretical value again between gradient and gradient；
Embody the good linear detection range of QuantiMAT technology (1nmol/ml～1pmol/ml)；Repeatability is good.
Embodiment 3 feasibility Experiment+accuracy experiment+repeatability experiment
Experimental designs,
Design HCV virus 1b complementary specificity primer sequence is as shown in table 7:
Table 7
CE, LE, BL after synthesis is mixed according to the ratio of 1:4:2, is diluted to 100fmol/ul as reaction primer, inspection
Surveying the standard substance (article No. GBW (E) 090142) of State center for standard matter, concentration is 4.4*105IU/ml.With this standard substance
For detection target, named to primer mixture " HCV1b probe ".Sensitivity and specificity to technology are verified.
First day experimental design:
Standard substance are diluted to 5 gradients, and StdA, StdB, StdC, StdD, StdE are 4.4*10 respectively5IU/ml、4.4*
104IU/ml、4.4*103IU/ml、4.4*102IU/ml、4.4*101IU/ml。
Preparation detection system:
Total system is 100ul, and every hole adds above-mentioned standard substance 2ul
Design fabric swatch is as shown in table 8:
Table 8
Detection method:
The microwell plate of above-mentioned mixed system is inserted between hybridizing 1.0～1.5 hours in 55 degree of calorstats by step 1, takes out
Microwell plate recovers to room temperature, prepares the eluent of 1X, is ready for washing plate；
96 hole micropore magnetic frames are placed at the bottom of plate by step 2, adsorb magnetic bead about 1 minute, hand rest liquid in firmly grillage throws away plate
Body, adds the 1X eluent of 200ul, three times the most repeatedly；
Step 3 takes off 96 hole microwell plate magnetic frames, and every hole adds primary amplification molecule solution 100ul, is placed in 55 degree of constant temperature
Case is placed 40 minutes；
96 hole micropore magnetic frames are placed at the bottom of plate by step 4, adsorb magnetic bead about 1 minute, hand rest liquid in firmly grillage throws away plate
Body, adds the 1X eluent of 200ul, three times the most repeatedly；
Step 5 takes off 96 hole microwell plate magnetic frames, and every hole adds secondary amplification molecule solution 100ul, is placed in 55 degree of constant temperature
Case is placed 40 minutes；
96 hole micropore magnetic frames are placed at the bottom of plate by step 6, adsorb magnetic bead about 1 minute, hand rest liquid in firmly grillage throws away plate
Body, adds the 1X eluent of 200ul, three times the most repeatedly；
Step 7 takes off 96 hole microwell plate magnetic frames, and every hole adds label probe solution 100ul, is placed in 50 degree of calorstats
Place 40 minutes；
96 hole micropore magnetic frames are placed at the bottom of plate by step 8, adsorb magnetic bead about 1 minute, hand rest liquid in firmly grillage throws away plate
Body, adds the 1X eluent of 200ul, three times the most repeatedly；
Step 9 takes off 96 hole microwell plate magnetic frames, and every hole adds substrate solution 100ul, is placed in 35 degree of calorstats placement
10 minutes；
Microwell plate is put in microplate reader or Chemiluminescence Apparatus and is carried out reading judgement by step 10.
RLU the results are shown in Table 9:
Table 9
Background correction RLU the results are shown in Table 10:
Table 10
| 0 | 0 | 434184 | 328344 | 1175351 | 1012360 | 13157 | 11262 | 923 | 724 | -41 | -31 |
| 0 | 0 | 494664 | 411504 | 1396554 | 1268489 | 15729 | 14240 | 1194 | 1037 | -126 | -116 |
| 0 | 0 | 366144 | 403944 | 1186994 | 1245205 | 13292 | 13969 | 938 | 1009 | -131 | -96 |
| 0 | 0 | 419064 | 358584 | 1198636 | 1105498 | 13428 | 12345 | 952 | 838 | -136 | -66 |
| 0 | 0 | 407724 | 298104 | 1088034 | 919222 | 12142 | 10179 | 816 | 610 | -131 | -111 |
| 0 | 0 | 335904 | 339684 | 1041465 | 1047287 | 11600 | 11668 | 759 | 767 | 9 | -91 |
| 0 | 0 | 426624 | 419064 | 1303416 | 1291774 | 14646 | 14511 | 1080 | 1066 | -131 | -21 |
| 0 | 0 | 366144 | 422844 | 1216099 | 1303416 | 13631 | 14646 | 973 | 1080 | -141 | -131 |
HCV-RNA second filial generation nucleic acid standards, uses QuantiMAT technology for detection to have the good range of linearity, about
Detection limit is at 440 IU/ml；When copy number is more than 4.4*105During IU/ml, signal is saturation, illustrates to arrive in detection
Limit value；Whereupon it may be inferred that go out the QuantiMAT technology detection range to HCV1b type virus at 440IU/ml～
Between 440000IU/ml；Other types HCV Viral diagnosis limit in the range of be suitable for.
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For Yuan, under the premise without departing from the principles of the invention, it is also possible to make some improvements and modifications, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (9)
1. the detection method that a fast Acquisition hybridizing targets thing branch chain DNA signal amplifies, it is characterised in that include walking as follows
Rapid:
Step 1: obtain nucleic acid hybridization primer；Nucleic acid purpose fragment is divided into capturing function guiding region, mark according to functional attributes
Cite sb. for meritorious service can guiding region, hide function guiding region, respectively according to described capturing function guiding region, described mark function guiding region, institute
State covering function guiding region and obtain complementary primer；
The primer in described capturing function district is capturing function district primer；
Step 2: take amido modified universal primer and be combined with the specific groups of magnetic bead surfaces, forms the magnetic bead being coated with nucleic acid；
Described amido modified universal primer is combined with described capturing function district primer hybridization；
The described magnetic bead being coated with nucleic acid can specificity capture target substance；
Step 3: obtain nucleic acid branched chain signal amplifying system；System includes primary signal amplifier chain and secondary signal amplifies
Chain；
The length of described primary signal amplifier chain is 500～560bp, repeats including the primary preposition homing sequence of amplifier chain and 20 times
The preposition boot section of secondary signal amplifier chain；
The length of described secondary signal amplifier chain is 420～480bp, repeats including the secondary preposition homing sequence of amplifier chain and 20 times
Reporter probe duplicate block；
The described primary preposition homing sequence of amplifier chain combines with the hybridization of mark function guiding region described in step 1；
The secondary signal amplifier chain of 20 repetitions of the described secondary preposition homing sequence of amplifier chain and described primary signal amplifier chain
The hybridization of preposition boot section combines；
Step 4: obtain the reporter probe of AP coupling；Amido modified reporter probe primer is combined with alkali phosphatase formation core
Acid/protein complexes, described nucleic acid/protein complexes hybridizes knot with the reporter probe duplicate block of 20 repetitions described in step 3
Close, it is thus achieved that AP labelling reporter probe, expand the signal value of captured target substance, detection.
Detection method the most according to claim 1, it is characterised in that the core of the universal primer of described capturing function guiding region
Acid sequence (5 '～3 ') is as shown in SEQ ID No.1.
Detection method the most according to claim 1 and 2, it is characterised in that the universal primer of described mark function guiding region
Nucleotide sequence (5 '-3 ') as shown in SEQ ID No.2.
4. according to the detection method described in any one of claims 1 to 3, it is characterised in that the described primary preposition guiding of amplifier chain
Sequence is as shown in SEQ ID No.3.
5. according to the detection method described in any one of Claims 1-4, it is characterised in that the secondary signal that described 20 times are repeated
The nucleotide sequence of the preposition boot section of amplifier chain is as shown in SEQ ID No.4.
6. according to the detection method described in any one of claim 1 to 5, it is characterised in that the described secondary preposition guiding of amplifier chain
Sequence is as shown in SEQ ID No.5.
7. according to the detection method described in any one of claim 1 to 6, it is characterised in that the reporter probe that described 20 times are repeated
The nucleotide sequence of duplicate block is as shown in SEQ ID No.6.
8. according to the detection method described in any one of claim 1 to 7, it is characterised in that the sequence of described AP labelling reporter probe
Row sequence-AP as shown in SEQ ID No.7.
9. according to the detection method described in any one of claim 1 to 8, it is characterised in that amido modified leading to described in step 2
It is NH2-sequence as shown in SEQ ID No.8 by the sequence of primer.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106701984A (en) * | 2017-02-07 | 2017-05-24 | 华南师范大学 | Electrochemical luminescence nucleic acid detection method and kit based on branched DNA (Deoxyribonucleic Acid) amplification signal |
| CN108504738A (en) * | 2018-04-27 | 2018-09-07 | 郑州科蒂亚生物技术有限公司 | A kind of prostate cancer detection probe sequence and its application |
| CN108504777A (en) * | 2018-04-03 | 2018-09-07 | 新乡医学院第三附属医院 | A method of based on signal amplification detection HCV virus carrying capacity |
| CN108796134A (en) * | 2018-07-12 | 2018-11-13 | 郑州科蒂亚生物技术有限公司 | A kind of cervical carcinoma detection probe sequence and its application |
| CN109943665A (en) * | 2019-04-15 | 2019-06-28 | 郑州科蒂亚生物技术有限公司 | A kind of probe sequence and its kit, application detected for detecting head and neck scale carcinoma oncogene |
| CN111378723A (en) * | 2020-04-24 | 2020-07-07 | 郑州科蒂亚生物技术有限公司 | Kit and method for detecting PD-L1mRNA on tissue |
| CN112239776A (en) * | 2020-10-23 | 2021-01-19 | 伯克利南京医学研究有限责任公司 | Multiple nucleic acid detection method and kit based on hybridization and cascade signal amplification principle |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103429755A (en) * | 2010-10-21 | 2013-12-04 | 领先细胞医疗诊断有限公司 | An ultra sensitive method for in situ detection of nucleic acids |
| EP2539355B1 (en) * | 2010-02-26 | 2016-10-05 | Ventana Medical Systems, Inc. | In-situ hybridization with polytag probes |
-
2016
- 2016-04-22 CN CN201610254920.5A patent/CN105861669B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2539355B1 (en) * | 2010-02-26 | 2016-10-05 | Ventana Medical Systems, Inc. | In-situ hybridization with polytag probes |
| CN103429755A (en) * | 2010-10-21 | 2013-12-04 | 领先细胞医疗诊断有限公司 | An ultra sensitive method for in situ detection of nucleic acids |
| CN104849472A (en) * | 2010-10-21 | 2015-08-19 | 领先细胞医疗诊断有限公司 | Ultra sensitive method for in situ detection of nucleic acids |
Non-Patent Citations (1)
| Title |
|---|
| 彭晓谋 等: "串联重复核酸序列构建及其杂交信号放大作用", 《生物医学工程学杂志》 * |
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