CN108414735A - A kind of large biological molecule immunoassay method mediated based on extension three times-RNA amplification - Google Patents
A kind of large biological molecule immunoassay method mediated based on extension three times-RNA amplification Download PDFInfo
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- 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
Abstract
The invention discloses a kind of based on the large biological molecule immunoassay method for extending RNA amplification mediation three times, belongs to field of immunology.This method contacts large biological molecule with the oligo1 and oligo2 of the substance coupling of identification large biological molecule, and the one end oligo1 is sequence label, another terminal sequence（It is about 6nt）It can be matched with oligo2 termini-complementaries；Oligo2, one end are T7 promoter sequences, another terminal sequence（It is about 6nt）It can be matched with oligo1 termini-complementaries；Extended under the action of Klenow enzymes, forms dsDNA, small RNA molecular is transcribed out under the action of T7 polymerases；It is put up a bridge using tiny RNA and two-way extension detection tiny RNA is carried out to the amplimer of two non-overlapping copies, thus judge the presence or absence of large biological molecule to be checked.The method high sensitivity, the requirement to instrument are low, can be used for the super quick detection of biomedical indicators.
Description
Technical field
The invention belongs to field of immunology, and in particular to a kind of large biological molecule mediated based on extension three times-RNA amplification
Immunoassay method.
Background technology
Recent decades, immunological method such as enzyme-linked immunization (ELISA) have been widely used in life science and have faced
In bed diagnosis.Product based on ELISA has the characteristics that using simple, versatility height and advantage of lower cost.But as
It is exactly the susceptibility of its detection that these the same common immunological methods of any other technology, which have limitation, most important problem,
It is not high, it is difficult to detect the protein of ultra-low volume, and these ultra-low volume protein for as coronary artery disease, ovarian neoplasm, with
And the early diagnosis of disease as senile dementia is most important.Therefore, a kind of large biological molecule inspection of superelevation sensitivity is established
Survey technology seems particularly necessary.
Immuno PCR (Immuno-PCR, iPCR) has become a strong technology, in detection target egg
Remolding sensitivity conventional ELISA method is 100~10000 times high when white.The raising of this methodology sensitivity is by antibody idol
Connection nucleotide carries out PCR amplification and realizes.But this design is there are many defects, that is, it is designing with antibody coupling nucleosides
Acid is all longer, such as in proximity ligation assay (Proximity ligation assay, PLA) and ortho position elongation technology
A pair of of the nucleotide fragments used in (Proximity extension assay, PEA) are all longer, about 40-60 nucleotide, this
The PCR molecules amplification of next step is easy in two kinds of technologies compared with the design of longer nucleotide.This defect compared with long probe design exists
Compound after nucleotide coupled antibody is difficult to detach with free nucleotide, and the such as HPLC separation of conventional separation method is pure
Change, needs certain amount of antibody that can just be effectively separated, it is still difficult to realize when the amount of antibody of label is seldom to efficiently separate.
Invention content
In view of the above problems, the present invention provides, a kind of large biological molecule height mediated based on extension three times-RNA amplification is quick to exempt from
Epidemic disease analysis method, i.e., immune-TEBRA (triple extension-based RNAamplification).This method has height
Sensitivity, the feature that antibody dosage is small, requirement to instrument is relatively low, can be used for the super quick detection of biomedical indicators, also can be used
In the foundation of the new medicament screen platform based on monitoring biomarker concentration.
To solve problems of the prior art, the invention is realized by the following technical scheme：
First aspect present invention provides a kind of large biological molecule immunoassay side mediated based on extension three times-RNA amplification
Method includes the following steps：
1) design of unrelated probe and primer：
(1) first and second adjacent to probe design：Oligo1, overall length are the DNA of 20-30nt, and one end is sequence label
ZC, another terminal sequence (a length of 5~6nt) can match with oligo2 termini-complementaries；Oligo2 is always about the DNA of 20-30nt,
One end is T7 promoter sequences, another terminal sequence (a length of 5~6nt) and the pairing of oligo1 termini-complementaries；
Substances of the Oligo1 and oligo2 respectively with identification large biological molecule is coupled, and forms the object of identification large biological molecule
Matter-oligo1, the substance-oligo2 for identifying large biological molecule, the substance of label oligo1 and oligo2 are to combine big point of biology
The antibody of son or the biotin with affinity；Due to this (the about 20-30nt) shorter to oligo length of design, after coupling
Can be the oligo and free oligo for realizing the substance for being coupled identification large biological molecule by simple spin-column chromatography method
It separates；
(2) amplimer R (being named as oligo3)：There are two part, the sequences at 3 ' ends and the tiny RNA half of transcription by oligo3
Row complementary pairing is hybridized by 10 or 11 length of nucleotides with half of molecule at 3 ' ends of the tiny RNA of transcription；The other end is mark
Sign sequence (being named as ZC1, ZC2) and T7 promoter sequences；Design capture probe and detection probe according to sequence label, for pair
The qualitative analysis of following amplification product, sequence label are designed as needed, are 1-2 items, and length is 16~20nt；T7 starts
Subsequence is T7 polymerase recognition sites, is used for subsequent T7 polymerase transcriptions；
(3) critical sequences 1：In order to increase oligo3 and transcription tiny RNA hybrid stability, introduce critical sequences 1,
It is combined with the sequence label of oligo3, T7 promoter complementary pairings；
(4) amplimer F (being named as oligo4)：It is similar to oligo3 features, there are two part, one end and transcription it is small
Half of molecular sequences at the 5 ' ends of RNA are identical (for 10~11 nucleotide), can be complementary with the cDNA sequence of the tiny RNA overall length of transcription
Pairing combines；Other end sequence label (being named as ZC3) containing there are one, it is qualitative which can be used for following amplification product
The design of dressing probe or detection probe when detection；
(5) critical sequences 2：In order to increase the stability that oligo4 and newly synthesized oligo3-RNA~cDNA is combined, draw
Enter critical sequences 2, it is characterized in that can be combined with sequence label ZC3 complementary pairings；
2) biomacromolecule detection
(1) substance-oligo1 of identification large biological molecule (marks the substance of oligo1 in the test tube of individual event reaction
Can be the antibody in conjunction with large biological molecule or biotin etc. with affinity) and identify the substance-of large biological molecule
Oligo2 (substance of label oligo2 can be in conjunction with the antibody of large biological molecule or the biotin etc. with affinity) is same
When combine target organisms macromolecular, due to ortho position (proximity), 6 nucleotide of the tail portions oligo1 and oligo2
Molecule meeting complementary pairing combines, and is then extended under the action of Klenow enzymes, forms a dsDNA, this is the first order
Row extend；
(2) dsDNA that step (1) is formed transcribes out tiny RNA point due to containing T7 promoters under the action of T7 polymerases
Son, size are 20~25nt；
(3) oligo3 and the tiny RNA 3 ' of step (2) transcription hold the sequence of half to combine, and it is compound to form tiny RNA-oligo3
Object, to increase the stability of the compound, critical sequences 1 by the principle of base pair complementarity be attached on the compound come,
Final oligo3, critical sequences 1 and tiny RNA by merge effect (stacking effect) formed one it is stable
Oligo3/ critical sequences 1-RNA compounds, the compound obtain the cDNA of small RNA molecular overall length under the action of reverse transcriptase,
As oligo3-RNA~cDNA：RNA compounds, this extends for second of sequence；
(4) step (3) oligo3-RNA~cDNA：RNA compounds are under the action of RNaseH, the RNA meetings in compound
It is degraded, forms oligo3-RNA~cDNA；
(5) oligo4 and oligo3-RNA~cDNA complementary pairings combine, and it is multiple to form oligo3-RNA~cDNA-oligo4
Close object；To increase the stability of the compound, critical sequences 2 are attached to by the principle of base pair complementarity on the compound
Come, final Oligo4, critical sequences 2, critical sequences 1 and oligo3-RNA~cDNA of oligo3 synthesis are by merging effect
(stacking effect) forms a stable compound, and as oligo3-RNA~cDNA-oligo4/ critical sequences 2 are multiple
Close object.The compound is obtained under the action of reverse transcriptase containing T7 promoter sequences, sequence label and small RNA molecular overall length
Double-stranded DNA, i.e. oligo3-RNA~cDNA-oligo4 double-stranded DNAs, this is that third time sequence extends；
(6) oligo3-RNA~cDNA-oligo4 double chain DNA sequence structures formed in (5) are T7 promoters-ZC2-
ZC1-RNA~cDNA-ZC3, the double-stranded DNA can be expanded by TMA or NASBA methods, and it is ZC2-ZC1- to obtain sequence
The RNA molecule of RNA~cDNA-ZC3；
(7) presence or absence and/or the quantity for detecting the amplified production of the RNA, thus judge large biological molecule to be checked
Presence or absence and/or quantity.
In another preferred example, above-mentioned steps 2) testing result described in (7) is qualitative or quantitative result.
In another preferred example, the method for the amplified production of the detection RNA includes molecular beacon, puts down in step 2) (7)
Plate hybridization-signal amplification or the detection of nucleic acid colloidal gold.
In another preferred example, the method for the amplified production of the detection RNA is tablet hybridization-signal in step 2) (7)
Amplification detection specially designs capture probe according to sequence label (such as ZC3), which can be with ZC3 complementary pairing knots
Close, so the RNA molecule of amplification can be fixed in microwell plate or other solid phase carriers on；(such as further according to sequence label
ZC1, ZC2) detection probe is designed, which can combine with ZC1 and ZC2 base pair complementarities, and detection probe can be marked
Biotin in note, and then by biotin labeling to the RNA molecule amplified, Streptavidin-HRP enzymes can be subsequently added successively
(Strep-HRP), chemiluminescent substrate detects the presence or absence of amplified production or quantity, in turn eventually by chemiluminescent method
Judge the detection situation of large biological molecule to be checked.
Above-described large biological molecule refers to the substance that can be mutually distinguishable by specific effect, such as antibody with it is anti-
Original, receptor and ligand, enzyme-to-substrate, agglutinin and polysaccharide.
The principle that the present invention detects large biological molecule is as shown in Figure 1.
Compared with prior art, the invention has the advantages that and advantageous effect：
1, the length of oligo1 and oligo2 designs is shorter, is easy to and dissociates after the substance of marker recognition large biological molecule
Oligo detached.
2, since into sequence extension three times is crossed, detection sensitivity is higher, by the sensitivity of testing inspection large biological molecule
The rank of 0.75pg/ml can be reached.
3, whole amplification is all constant-temperature amplification, and the requirement to instrument is relatively low, and a thermostat can realize detection,
Convenient for the popularization of technology.
Description of the drawings
Fig. 1 shows the flow diagram of the method for the present invention.
Specific implementation mode
By following detailed description combination attached drawing it will be further appreciated that the features and advantages of the invention.The implementation provided
Example is only the explanation to the method for the present invention, remaining content without limiting the invention in any way announcement.
In the following examples, the experimental methods for specific conditions are not specified, usually according to normal condition, such as Sa m b r o
O k et al., molecular cloning：Laboratory manual (N e w Y o r k:C o l d S p r i n g H a r b o r
Laboratory Press, 1989) condition described in, or according to the normal condition proposed by manufacturer.Unless otherwise stated, no
Then percentage and number are weight percent and parts by weight.
【Embodiment 1】The activation of antibody and neighbouring probe (oligo1 and oligo2)
For antibody before marking neighbouring probe, antibody and neighbouring probe will carry out corresponding activation process, specific as follows：
1) activation process of antibody
(1) antibody is dissolved in PBS buffer solution (pH8), antibody concentration is diluted to 1mg/ml.
(2)+5 μ l TCO-PEG4-NHS (10mM) of antibody (1mg/ml) of 100 μ l, mixing is taken to react at room temperature 30min.
(3) Tris-HCl buffer solutions (1M, Ph8.0) are added into system, terminate reaction, the amount of addition is whole to Tris-HCl
A concentration of 50~100mM.
(4) it is incubated at room temperature 5min.
(5) product is crossed into column (desalting column in the apertures 7K), removes free TCO-PEG4-NHS and non-activated antibody, greatly
It is eluted collection in the meeting of 7K, the meeting less than 7K remains in chromatographic column.Products therefrom is the antibody activated.
2) activation process of neighbouring probe
(1) neighbouring probe is dissolved into 100 μM with PBS buffer solution (pH8).
(2)+5 μ l PEG4-NHS (10mM) of oligo (100 μM) of 50 μ l, mixing is taken to react at room temperature 30min.
(3) Tris-HCl buffer solutions (1M, pH8.0) are added into system, terminate reaction, the amount of addition is whole to Tris-HCl
A concentration of 50~100mM.
(4) it is incubated at room temperature 5min.
(5) product is crossed into column (BioRad P-30Columns, 40,000MW limit, eluent PBS), removing is not lived
The oligo and Free PEG 4-NHS of change.Meeting more than 40K is eluted collection, and the meeting less than 40K remains in chromatographic column.Gained
Product is the oligo activated.
【Embodiment 2】Neighbouring probe after activation is linked with antibody
1) oligo1 after 15 μ l activation, the antibody after 50 μ l activation are taken, is dissolved in PBS buffer solution (pH8) to 200 μ l,
Room temperature reaction 1 hour.
2) antibody after purification tag：It is purified with Bio-Spin 30Tris Columns (buffer solution changes PBS into),
Crossing the oligo after column purification less than 30bp can all be trapped in pillar, to which the oligo on will be unmarked is removed.
3) the Milipore filter purification markers again of 50K are used.
4) last products therefrom is the antibody for marking upper neighbouring probe (oligo1, oligo2).
【Embodiment 3】The detection of vascular endothelial growth factor (VEGF)
1) by oligo1 (sequence ACCCGATGGATAGGTCGGTGAA-ACGCAT) and oligo2 (sequences
TAATACGACTCACTATAGGGAGA-ATGCGT) label VEGF polyclonal antibodies (purchase to Thermo Fisher, Lot
Number:P802), wherein oligo2 carries T7 promoter sequences, and oligo1 and oligo2 can pass through 6 alkali at respective 3 ' end
Base complementary pairing links.
2) detection of VEGF
(1) formation of antibody oligo1 and antibody oligo2 double-strands
By VEGF (purchases to Thermo Fisher, Lot Number:PHC9391 2 times of concentration gradient dilutions) are carried out, according to
Following system carries out first time sequence extension, obtains the duplex of oligo1 and oligo2：
37 DEG C of incubation 40min.
(2) rna transcription
(transcription system is purchased to Thermo Fisher) is transcribed according to following system：
37 DEG C 1 hour.
(3) RNA amplification
The tiny RNA transcribed in (2) is expanded, realizes that the sequence of second, third time extends, relevant primer and probe are set
Meter such as table 1.
The sequence of second, third time of table 1 extends, relevant primer and probe design
Tiny RNA is expanded according to following system：
(2) the tiny RNA product transcribed in：2μl
Amplification buffer (contain oligo3 and its critical sequences,
Oligo4 and its critical sequences)：17μl
Expand enzyme mixation (AMV＆T7 polymerases) 1 μ l
It is to be checked after reacting 90min at 42 DEG C.
(4) method for using plate hybridization is detected analysis to the product in (3)
It is set in detection architecture according to the ZC sequence informations of RNA with the tiny RNA product of micropore plate hybridization method detection transcription
Capture probe, detection probe (detection probe 1 and detection probe 2) are counted, it is compound to form detection probe-RNA products-capture probe
Object, the compound can be captured by coated one section of probe in micropore and be fixed in micropore.One section is sequentially added into micropore
Probe, Streptavidin-HRP enzymes can be combined with detection probe and indicating biotin, chemiluminescent substrate are final to shine
Detection.Specifically,
A, amplified production fixes hybridization
42 DEG C of incubation 60min.
B, it discards liquid in hole and is patted dry on clean blotting paper.The hybridisation wash liquid of 200 μ L preheatings is added per hole, altogether
Washing 3 times.
C, 200 μ L confining liquids, shaken at room temperature 1 minute are added per hole.
D, liquid in hole is discarded, the 100 enzyme-linked objects of μ L Streptavidins-HRP are added per hole, room temperature is shaken 30 minutes.
E, it discards liquid in hole and is patted dry on clean blotting paper.The hybridisation wash liquid of 200 μ L preheatings is added per hole, altogether
Washing 3 times.
F, substrate solution, substrate A, substrate B, substrate dilution (Tris buffer solutions, pH8.5) in proportion 1 are prepared：1：8 is mixed
It is even.
G, 95 μ L substrate solutions are added per hole, is incubated after five minutes, is placed on chemiluminescence detector and carries out relative light unit
(RLU) it measures.
H, experimental result is as follows：
| The content of VEGF | Dilution ratio | RLU values |
| 125pg/ml | 1:1600 | 3506542 |
| 62.5pg/ml | 1:3200 | 1813211 |
| 31pg/ml | 1:6400 | 1023212 |
| 15.5pg/ml | 1:12800 | 609832 |
| 7.5pg/ml | 1:25600 | 423216 |
| 3.5pg/ml | 1:51200 | 205432 |
| 1.5pg/ml | 1:102400 | 108844 |
| 0.75pg/ml | 1:204800 | 66543 |
| 0.36pg/ml | 1:409600 | 44332 |
| Blank | / | 10355 |
It is to detect the positive that detection value is more than 5 with Blank ratios, and in terms of testing result, the detection method is in detection VEGF
When, detection sensitivity can reach the rank of 0.75pg/ml.
Sequence table
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<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 8
acgtaacccg atggataggt tttgctcgac ttgccaccga ata 43
<210> 9
<211> 38
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 9
atggataggt cggtgaattt tatggatagg tcggtgaa 38
<210> 10
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 10
tattcggtgg caagtcgagc 20
Claims (5)
1. a kind of large biological molecule immunoassay method mediated based on extension three times-RNA amplification, which is characterized in that including with
Lower step：
1) design of unrelated probe and primer：
(1) first and second adjacent to probe design：Oligo1, overall length are the DNA of 20-30nt, and one end is sequence label ZC, separately
A length of 5~6nt the sequences in one end can be matched with oligo2 termini-complementaries；Oligo2 is always about the DNA of 20-30nt, one end T7
Promoter sequence, a length of 5~6nt sequences of the other end and the pairing of oligo1 termini-complementaries；
Substances of the Oligo1 and oligo2 respectively with identification large biological molecule is coupled, and forms the substance-of identification large biological molecule
Oligo1, the substance-oligo2 for identifying large biological molecule mark the substance combination large biological molecule of oligo1 and oligo2
Antibody or biotin with affinity；It is 20-30nt since this of design is shorter to oligo length, it can be with after coupling
It is to realize that oligo and free oligo points of the substance for being coupled identification large biological molecule are opened by simple spin-column chromatography method；
(2) amplimer R is named as oligo3：For oligo3 there are two part, 3 ' ends and the sequence of the tiny RNA half of transcription are mutual
It recruits pair, i.e., is hybridized with half of molecule at 3 ' ends of the tiny RNA of transcription by 10 or 11 length of nucleotides；The other end is label sequence
Arrange ZC1 and/or ZC2 and T7 promoter sequences；Capture probe and detection probe are designed according to sequence label, for following amplification
The qualitative analysis of product, sequence label are designed as needed, are 1-2 items, and length is 16~20nt；T7 promoter sequences are
T7 polymerase recognition sites are used for subsequent T7 polymerase transcriptions；
(3) critical sequences 1：In order to increase oligo3 and transcription tiny RNA hybrid stability, introduce critical sequences 1, with
Sequence label ZC1 and/or ZC2, T7 promoter complementary pairing combine；
(4) amplimer F is named as oligo4：It is similar to oligo3 features, there are two part, the tiny RNA of one end and transcription
Half of molecular sequences at 5 ' ends are identical, and length is 10~11 nucleotide, can mutually be recruited with the cDNA sequence of the tiny RNA overall length of transcription
To combining；The other end contains there are one sequence label ZC3, which can be used for being coated with when following amplification product qualitative detection
The design of probe or detection probe；
(5) critical sequences 2：In order to increase the stability that oligo4 and newly synthesized oligo3-RNA~cDNA is combined, introduce steady
Sequencing row 2, it is characterized in that can be combined with sequence label ZC3 complementary pairings；
2) biomacromolecule detection
(1) substance-oligo1 of identification large biological molecule and the object for identifying large biological molecule in the test tube of individual event reaction
Matter-oligo2 is in combination with target organisms macromolecular, due to ortho position, 6 nucleotide of the tail portions oligo1 and oligo2
Molecule meeting complementary pairing combines, and is then extended under the action of Klenow enzymes, forms a dsDNA, this is the first order
Row extend；
(2) dsDNA that step (1) is formed transcribes out small RNA molecular due to containing T7 promoters under the action of T7 polymerases,
Size is 20~25nt；
(3) oligo3 and the tiny RNA 3 ' of step (2) transcription hold the sequence of half to combine, and form tiny RNA-oligo3 compounds, are
Increase the stability of the compound, critical sequences 1 are attached on the compound by the principle of base pair complementarity come finally
Oligo3, critical sequences 1 and tiny RNA are compound by merging one stable oligo3/ critical sequences 1-RNA of effect formation
Object, the compound obtain the cDNA of small RNA molecular overall length, as oligo3-RNA~cDNA under the action of reverse transcriptase：RNA
Compound, this extends for second of sequence；
(4) step (3) oligo3-RNA~cDNA：Under the action of RNaseH, the RNA in compound can be dropped RNA compounds
Solution forms oligo3-RNA~cDNA；
(5) oligo4 can be combined with oligo3-RNA~cDNA complementary pairings, and it is compound to form oligo3-RNA~cDNA-oligo4
Object；To increase the stability of the compound, critical sequences 2 by the principle of base pair complementarity be attached on the compound come,
Final Oligo4, critical sequences 2, critical sequences 1 and oligo3-RNA~cDNA of oligo3 synthesis are by merging effect shape
At a stable compound, as 2 compound of oligo3-RNA~cDNA-oligo4/ critical sequences；The compound is reversing
The double-stranded DNA containing T7 promoter sequences, sequence label and small RNA molecular overall length, i.e. oligo3- are obtained under the action of record enzyme
RNA~cDNA-oligo4 double-stranded DNAs, this extends for third time sequence；
(6) oligo3-RNA~cDNA-oligo4 double chain DNA sequence structures formed in (5) are T7 promoters-ZC2-ZC1-
RNA~cDNA-ZC3, the double-stranded DNA can be expanded by TMA or NASBA methods, and it is ZC2-ZC1- to obtain one section of sequence
The RNA molecule of RNA~cDNA-ZC3；
(7) presence or absence and/or the quantity for detecting the amplified production of the RNA, thus judge the presence of large biological molecule to be checked
Whether and/or quantity.
2. the method as described in claim 1, which is characterized in that the testing result described in step 2) (7) is qualitative or quantitative
As a result.
3. the method as described in claim 1, which is characterized in that the method for the amplified production of the detection RNA in step 2) (7)
Including molecular beacon, tablet hybridization-signal amplification or the detection of nucleic acid colloidal gold.
4. method as claimed in claim 3, which is characterized in that the side of the amplified production of the detection RNA in step (2) (7)
Method is tablet hybridization-signal amplification detection, specially designs capture probe according to sequence label ZC3, which can be with
ZC3 complementary pairings combine, and then the RNA molecule of amplification can be fixed in microwell plate or other solid phase carriers on；Further according to
Sequence label ZC1 and/or ZC2 design detection probe, which can combine with ZC1 and/or ZC2 base pair complementarities,
Detection probe can mark biotin, and then by biotin labeling to the RNA molecule amplified, can subsequently add chain successively
Mould Avidin-HRP enzymes, chemiluminescent substrate detect the presence or absence of amplified production or quantity eventually by chemiluminescent method, into
And judge the detection situation of large biological molecule to be checked.
5. method according to any one of claims 1-4, which is characterized in that the large biological molecule refers to that can pass through spy
Opposite sex effect and the substance that is mutually distinguishable, such as antibody and antigen, receptor and ligand, enzyme-to-substrate, agglutinin and polysaccharide.
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