CN110208538A - A kind of detection kit of prostate specific antigen, detection method and application - Google Patents
A kind of detection kit of prostate specific antigen, detection method and application Download PDFInfo
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- CN110208538A CN110208538A CN201910566621.9A CN201910566621A CN110208538A CN 110208538 A CN110208538 A CN 110208538A CN 201910566621 A CN201910566621 A CN 201910566621A CN 110208538 A CN110208538 A CN 110208538A
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- G—PHYSICS
- G01—MEASURING; TESTING
- 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
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- G—PHYSICS
- G01—MEASURING; TESTING
- 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/34—Genitourinary disorders
- G01N2800/342—Prostate diseases, e.g. BPH, prostatitis
Abstract
The present invention provides a kind of detection kit of prostate specific antigen, detection method and applications, are related to Antigen Detection Techniques field.Kit of the present invention and detection method utilize secondary antibody modifying DNA, and the Ag-Ab-DNA hybridization chain to form Y type is connect with antigen ortho position, and antigen input is converted to the output of DNA signal as signal adapter；Simultaneously by chain alternative reaction and hybridization chain reaction joint, the two-stage amplification of signal is realized；Magnesium ion DNA enzymatic is formed per adjacent two terminal sequences (hair clip DNAH3 and hair clip DNA H4) assembling on long chain DNA, the molecular beacon in solution is cut in the digestion in the presence of magnesium ion, the molecular beacon of disconnection releases and issues fluorescence, new molecular beacon again in conjunction with magnesium ion DNA enzymatic, cut, circulation amplification fluorescence signal, the signal amplification for realizing third round, substantially increases detection efficiency and sensitivity, and detection limit is made to reach 0.73pg mL‑1。
Description
Technical field
The invention belongs to Antigen Detection Techniques fields, and in particular to a kind of detection kit of prostate specific antigen, inspection
Survey method and application.
Background technique
Antigen detection has important reference value in clinical diagnosis.The surface molecular of humans and animals cell: including thin
The various differentiation antigens of cellular surface (such as T cell differentiation antigen), allogenic antigen (blood group antigens or MHC antigen), virus-associated antigen and swollen
Tumor correlation disposition antigen etc..Detect these antigens to the classification of various cells, atomization and functional study, to it is various with it is immune
The diagnosis of related disease and the research of pathogenesis, it is significant.
The method of antigen detection at present mainly has traditional Enzyme-Linked Immunospot (ELISA), radiommunoassay
(RIA), fluoroimmunoassay (FIA), chemiluminescence immunoassay technology (CLIA), automation immunoassay etc..Fluorescence immunoassay
Detection technique has many advantages, such as that specificity is strong, high sensitivity, practicability are good, therefore it be used to measure the very low biology of content and lives
Property substance, such as protein (enzyme, antibody), hormone (steroid, thyroid hormone) etc..The Protein Detection skill of DNA auxiliary
The performance of detection technique, such as the amplification of polymerase chain reaction, rolling ring can be improved in art, but cumbersome step and polymerase expands
Increase the factors such as possible false positive to limit its application.
Summary of the invention
In view of this, the purpose of the present invention is to provide a kind of detection kits of prostate specific antigen, detection method
And application, the method are based on ortho position connection, chain substitution, hybridization chain reaction and magnesium ion DNA enzymatic and detect antigen, pass through modification
The antibody and target antigen of DNA is specifically bound, and antigen input is converted to the output of DNA signal, marriage chain substitution, hybridization chain
The signal amplification technique of formula reaction and magnesium ion DNA enzymatic, realizes highly sensitive, the specific detection of target antigen.
In order to achieve the above-mentioned object of the invention, the present invention the following technical schemes are provided:
The present invention provides a kind of detection kits of prostate specific antigen, including following components: PSA antibody, amber
Acid imide -4- (N- maleimide) thiacyclohexane -1-1 carboxylic esters, three (2- carboxyethyl) phosphines, mercapto-modified DNA1, sulfydryl are repaired
The DNA2 of decorations, hair clip DNA H1, hair clip DNA H2, hair clip DNA H3, hair clip DNA H4, beacon MB and Mg2+；The sulfydryl modification
DNA1 nucleotide sequence as shown in SEQ ID NO.1；The nucleotide sequence of the mercapto-modified DNA2 such as SEQ ID
Shown in NO.2；The nucleotide sequence of the hair clip DNA H1 is as shown in SEQ ID NO.3；The nucleotides sequence of the hair clip DNA H2
Column are as shown in SEQ ID NO.4；6 acid sequence of nucleosides of the hair clip DNA H3 is as shown in SEQ ID NO.5；The hair clip DNA
The nucleotide sequence of H4 is as shown in SEQ ID NO.6；The beacon MB includes the rA base of an identification ion enzyme and is modified with
FAM and BHQ group.
Preferably, the nucleotide sequence of the beacon MB is as shown in SEQ ID NO.7.
The present invention also provides a kind of detection methods of prostate specific antigen, comprising the following steps: (1) by PSA antibody
After carrying out coupling reaction 2h with succinimide -4- (N- maleimide) thiacyclohexane -1-1 carboxylic esters, resist after filtering to handle
Body；The molar ratio of the PSA antibody and succinimide -4- (N- maleimide) thiacyclohexane -1-1 carboxylic esters be 1:10~
20；
(2) mercapto-modified DNA1 and mercapto-modified DNA2 are carried out with three (2- carboxyethyl) phosphines respectively hydroxy activated anti-
1h is answered, DNA1 and DNA2 after processing after must handling；The mercapto-modified DNA1 and mercapto-modified DNA2 and three (2- carboxylic second
Base) phosphine molar ratio be 1:104；
(3) antibody after the processing is obtained into Ab1- respectively with DNA2 after DNA1 after processing and processing in 4 DEG C of incubation 12h
DNA1 and Ab2-DNA2；The volume ratio of antibody and DNA2 after DNA1 after processing and processing is 1:1 after the processing；
(4) 37 DEG C of reactions after mixing the Ab1-DNA1, Ab2-DNA2, PSA, hair clip DNA H1 and hair clip DNA H2
2h obtains chain alternative reaction product；The molar ratio of the Ab1-DNA1, Ab2-DNA2, PSA, hair clip DNA H1 and hair clip DNA H2
For 100:100:100:10-3:10-3；
(5) 1h is reacted for 37 DEG C after mixing the chain alternative reaction product with hair clip DNA H3 and hair clip DNA H4, is obtained miscellaneous
Interlinkage；The molar ratio of the hair clip DNA H3 and hair clip DNA H4 are 300:10-3:10-3；
(6) by the hybridization chain and molecular beacon MB, Mg2+37 DEG C of reaction 1h, fluorescence detection after mixing；
There is no chronological orders to limit between step (1) and step (2).
Preferably, step (1) reaction carries out in PBS buffer solution, and the temperature of the reaction is 18~25 DEG C.
Preferably, the molecular cut off of step (1) filtering filter membrane is 10000MW.
Preferably, the temperature of step (2) described reaction is 37 DEG C.
Preferably, after step (3) described incubation, further include the membrane filtration for being 100000MW using molecular cut off, protect
Substance on film is stayed, and three times using PBS buffer solution washing.
It preferably, further include UV absorbance detection after step (3) obtains the Ab1-DNA1 and Ab2-DNA2.
Preferably, step (6) fluorescence detection is the signal detection peak under 520nm visible light.
The present invention also provides the application of the detection kit or the detection method in the amplification of target antigen signal.
The present invention provides a kind of detection kits of prostate specific antigen, including following components: PSA antibody, amber
Acid imide -4- (N- maleimide) thiacyclohexane -1-1 carboxylic esters, three (2- carboxyethyl) phosphines, mercapto-modified DNA1, sulfydryl are repaired
The DNA2 of decorations, hair clip DNA H1, hair clip DNA H2, hair clip DNA H3, hair clip DNA H4, beacon MB and Mg2+；The sulfydryl modification
DNA1 nucleotide sequence as shown in SEQ ID NO.1；The nucleotide sequence of the mercapto-modified DNA2 such as SEQ ID
Shown in NO.2；The nucleotide sequence of the hair clip DNA H1 is as shown in SEQ ID NO.3；The nucleotides sequence of the hair clip DNA H2
Column are as shown in SEQ ID NO.4；6 acid sequence of nucleosides of the hair clip DNA H3 is as shown in SEQ ID NO.5；The hair clip DNA
The nucleotide sequence of H4 is as shown in SEQ ID NO.6；The beacon MB includes the rA base of an identification ion enzyme and is modified with
FAM and BHQ group.
Further, when carrying out the detection of prostate specific antigen using the kit, two anti-human forefront of mouse are utilized
The DNA of the antibody modification of gland specific antigen (PSA) connects the Ag-Ab-DNA hybridization chain to form Y type by antigen ortho position,
The free end DNA can trigger chain alternative reaction, successively open two hair clip DNA, and hybridize second hair clip up
DNA can push up lower Ag-Ab-DNA hybridization chain, realize the circulation of target antigen.Chain alternative reaction circulation generates a large amount of complementary
Hybridization chain causes again hybridizes the hybridization chain reaction formed by other two hair clip DNA circulation, forms a large amount of long chain DNA, long
It can assemble to form magnesium ion DNA enzymatic per two adjacent terminal sequences on chain DNA, finally the enzyme can be cut in the presence of magnesium ion
Molecular beacon in solution issues fluorescence, realizes the multiple signal amplification of target antigen.Using kit of the present invention and
Detection method, has been greatly improved detection efficiency and sensitivity, and detection limit reaches 0.73pgmL-1, and to alpha-fetoprotein (AFP),
Bovine serum albumin(BSA) (BSA) and carcinomebryonic antigen (CEA) non-target detectable substance are without response.
Detailed description of the invention
Fig. 1 is detection method schematic diagram；
Fig. 2 is the UV absorption figure of antibody modification DNA in the embodiment of the present invention；
Fig. 3 is fluorescence detection figure in the embodiment of the present invention；
Fig. 4 is the response diagram in the embodiment of the present invention to multiple protein.
Specific embodiment
The present invention provides a kind of detection kits of prostate specific antigen, including following components: PSA antibody, amber
Acid imide -4- (N- maleimide) thiacyclohexane -1-1 carboxylic esters, three (2- carboxyethyl) phosphines, mercapto-modified DNA1, sulfydryl are repaired
The DNA2 of decorations, hair clip DNA H1, hair clip DNA H2, hair clip DNA H3, hair clip DNA H4, beacon MB and Mg2+；The sulfydryl modification
DNA1 nucleotide sequence as shown in SEQ ID NO.1；The nucleotide sequence of the mercapto-modified DNA2 such as SEQ ID
Shown in NO.2；The nucleotide sequence of the hair clip DNA H1 is as shown in SEQ ID NO.3；The nucleotides sequence of the hair clip DNA H2
Column are as shown in SEQ ID NO.4；6 acid sequence of nucleosides of the hair clip DNA H3 is as shown in SEQ ID NO.5；The hair clip DNA
The nucleotide sequence of H4 is as shown in SEQ ID NO.6；The beacon MB includes the rA base of an identification ion enzyme and is modified with
FAM and BHQ group.
In detection kit of the present invention, the PSA antibody is preferably the antibody of mouse anti-human prostate specific antigen.
It include rA group in beacon MB of the present invention, the rA is the ribonucleic acid that a magnesium ion enzyme core sequence is identified and cut
Base, non-DNA base, nucleotide sequence is preferably as shown in SEQ ID NO.7.It is tried in detection of the present invention
In agent box, each sequence is as shown in table 1:
Sequence in 1 kit of table
The present invention also provides a kind of detection methods of prostate specific antigen, comprising the following steps: (1) by PSA antibody
After carrying out coupling reaction 2h with succinimide -4- (N- maleimide) thiacyclohexane -1-1 carboxylic esters, resist after filtering to handle
Body；The molar ratio of the PSA antibody and succinimide -4- (N- maleimide) thiacyclohexane -1-1 carboxylic esters be 1:10~
20；
(2) mercapto-modified DNA1 and mercapto-modified DNA2 are carried out with three (2- carboxyethyl) phosphines respectively hydroxy activated anti-
1h is answered, DNA1 and DNA2 after processing after must handling；The mercapto-modified DNA1 and mercapto-modified DNA2 and three (2- carboxylic second
Base) phosphine molar ratio be 1:104；
(3) antibody after the processing is obtained into Ab1- respectively with DNA2 after DNA1 after processing and processing in 4 DEG C of incubation 12h
DNA1 and Ab2-DNA2；The volume ratio of antibody and DNA2 after DNA1 after processing and processing is 1:1 after the processing；
(4) 37 DEG C of reactions after mixing the Ab1-DNA1, Ab2-DNA2, PSA, hair clip DNA H1 and hair clip DNA H2
2h obtains chain alternative reaction product；The molar ratio of the Ab1-DNA1, Ab2-DNA2, PSA, hair clip DNA H1 and hair clip DNA H2
For 100:100:100:10-3:10-3；
(5) 1h is reacted for 37 DEG C after mixing the chain alternative reaction product with hair clip DNA H3 and hair clip DNA H4, is obtained miscellaneous
Interlinkage；The molar ratio of the hair clip DNA H3 and hair clip DNA H4 are 300:10-3:10-3；
(6) by the hybridization chain and molecular beacon MB, Mg2+37 DEG C of reaction 1h, fluorescence detection after mixing；
There is no chronological orders to limit between step (1) and step (2).
In detection method of the present invention, by PSA antibody and succinimide -4- (N- maleimide) thiacyclohexane -
After 1-1 carboxylic esters carry out coupling reaction 2h, antibody after filtering to handle；The PSA antibody and the (Malaysia N- succinimide -4-
Acid imide) thiacyclohexane -1-1 carboxylic esters molar ratio be 1:10~20.It is of the present invention reaction preferably in PBS buffer solution into
Row, the reaction preferably carry out at room temperature, and more preferably 18~25 DEG C.The present invention filters after the reaction, preferably
It is purified with filter membrane (molecular cut off 10,000MW), PBS buffer solution is washed three times.Coupling reaction of the present invention, succinyl are sub-
Amine -4- (N- maleimide) thiacyclohexane -1-1 carboxylic esters (SMCC) be used as coupling agent, the NHS ester group of molecule one end with it is a certain
The primaquine of protein molecule reacts to form stable amido bond, and the other end (maleimide base group one end) can be with another protein
Specificity crosslinking occurs for the sulfydryl of molecule, can promote the formation of subsequent the multiple element compound.
Mercapto-modified DNA1 and mercapto-modified DNA2 is carried out hydroxyl with three (2- carboxyethyl) phosphines respectively and lived by the present invention
Change reaction 1h, DNA1 and DNA2 after processing after must handling；The mercapto-modified DNA1 and mercapto-modified DNA2 and three (2- carboxylics
Ethyl) phosphine molar ratio be 1:104.The temperature of reaction of the present invention is preferably 37 DEG C.In embodiments of the present invention, respectively to
2 μ L TCEP (100mM), 37 DEG C of reaction 1h are added in the 200 mercapto-modified DNA1 of μ L (10 μM) and mercapto-modified DNA2.
After must handling after antibody, processing after DNA1 and processing after DNA2, the present invention by antibody after the processing respectively with place
DNA1 and DNA2 after processing obtains Ab1-DNA1 and Ab2-DNA2 in 4 DEG C of incubation 12h after reason；Antibody and place after the processing
The volume ratio of DNA1 and DNA2 after processing are 1:1 after reason.After incubation of the present invention, it is also preferable to include utilize molecular cut off
For the membrane filtration of 100000MW, retain substance on film, and three times using PBS buffer solution washing.The present invention obtain it is described
After Ab1-DNA1 and Ab2-DNA2, it is also preferable to include UV absorbance detections, as can detect the characteristic peak of DNA and antibody protein,
Then proved response is good.
After Ab1-DNA1 and Ab2-DNA2, the present invention by the Ab1-DNA1, Ab2-DNA2, PSA, hair clip DNA H1 and
37 DEG C of reaction 2h after hair clip DNA H2 mixing, obtain chain alternative reaction product；The Ab1-DNA1, Ab2-DNA2, PSA, hair clip DNA
The molar ratio of H1 and hair clip DNA H2 are 100:100:100:10-3:10-3.In embodiments of the present invention, Ab1-DNA1, Ab2-
Each 1 μM of each 100nM of DNA2, PSA, hair clip DNA (H1, H2), 37 DEG C of reaction 2h after mixing.
After chain alternative reaction product, the present invention is by the chain alternative reaction product and hair clip DNA H3 and hair clip DNA H4
37 DEG C of reaction 1h, obtain hybridization chain after mixing；The molar ratio of the hair clip DNA H3 and hair clip DNA H4 are 300:10-3:10-3.?
In the embodiment of the present invention, each 1 μM of hair clip DNA (H3, H4) is added in Xiang Shangshu chain alternative reaction product, 37 DEG C of reactions after mixing
1h.The hybridization chain of the length of hair clip DNA (H1, H2, the H3, H4) assembling of generation.
After chain must being hybridized, the present invention is by the hybridization chain and molecular beacon MB, Mg2+37 DEG C of reaction 1h after mixing, fluorescence inspection
It surveys.In embodiments of the present invention, molecular beacon MB, Mg are added into hybridization chain reaction product obtained above2+Each 100nM,
37 DEG C of reaction 1h after mixing.The signal peak of the visible 520nm of fluorescence detection.
In detection method of the invention, testing principle passes through antigen as shown in Figure 1, using the antibody for having modified DNA
Ortho position connects the Ag-Ab-DNA hybridization chain to form Y type, and the free end DNA can trigger chain alternative reaction, successively open
Two hair clip DNA, and the second hair clip DNA of hybridization up can push up lower Ag-Ab-DNA hybridization chain, realize target antigen
Circulation.The a large amount of Complementary hybridization chain of chain alternative reaction circulation generation causes again to be made of the hybridization of other two hair clip DNA circulation
Hybridization chain reaction, form a large amount of long chain DNA, can assemble to form magnesium ion per two adjacent terminal sequences on long chain DNA
DNA enzymatic, finally the enzyme can cut the molecular beacon in solution and issue fluorescence in the presence of magnesium ion, realize target antigen
Multiple signal amplification.
The present invention also provides the application of the detection kit or the detection method in the amplification of target antigen signal.
In the present invention, the method for the target antigen signal amplification is identical as above-mentioned detection method, and details are not described herein.Institute of the present invention
It states in application, signal amplification includes three-level amplification, is modified using two mouse anti-human prostate specific antigen monoclonal antibodies
DNA connect the Ag-Ab-DNA hybridization chain to form Y type with antigen ortho position, is converted to antigen input as signal adapter
The output of DNA signal；By chain alternative reaction and hybridization chain reaction joint, the two-stage amplification of signal is realized.Reaction packet therein
It includes: (1) chain alternative reaction: the Ag-Ab-DNA hybridization chain and first hair clip DNA hybridization that Ab1-DNA1, Ab2-DNA2 are formed
(H1) the stem end for opening H1, the stem end of the H1 exposed hybridize with second hair clip DNA (H2), and hybridize the top H2 up
Lower Ag-Ab-DNA hybridizes chain, from the beginning target antigen, which returns to, to be recycled, and realizes the amplification of first round signal；(2) hybridize chain reaction: anti-
The chain alternative reaction that antigen-antibody-DNA hybridization chain causes generates hybridize chain of a large amount of H1 with H2, causes by other two hair clip
Hybridize chain reaction composed by DNA (H3 and H4) cyclical crosses, generate a large amount of long chain DNA, realizes that the signal of the second wheel is put
Greatly.Magnesium ion DNA is formed per adjacent two terminal sequences (H3 and H4) assembling on the long chain DNA formed after the completion of hybridization chain reaction
Enzyme, the molecular beacon in solution is cut in the digestion in the presence of magnesium ion, and the molecular beacon of disconnection releases and issue fluorescence,
New molecular beacon again with magnesium ion DNA enzymatic ining conjunction with, cut, circulation amplification fluorescence signal realizes that the signal of third round amplifies.
Below with reference to embodiment to a kind of detection kit of prostate specific antigen provided by the invention, detection method and
Using being described in detail, but they cannot be interpreted as limiting the scope of the present invention.
Embodiment 1
Apparatus: high speed freezing centrifuge (16R, Heima Medical Instrument Co., Ltd., Zhuhai City), ultrapure water machine
(SybergyUV, Merck Mi Libo), ultraviolet-uisible spectrophotometer (Cary60, Agilent Technologies, the U.S.), fluorescence spectrophotometer
Photometer (F-4600, Hitachi, Japan high and new technology company), (DYCP-31DN, Beijing 6 is one) for Agarose horizontal electrophoresis tank.
Agents useful for same: PSA antibody, SMCC (succinimide -4- (N- maleimide) thiacyclohexane -1-1 carboxylic esters),
(DNA includes by precious biosynthesis, MB by TCEP (three (2- carboxyethyl) phosphines), mercapto-modified DNA1, mercapto-modified DNA2, MB
One can by rA base that ion enzyme identifies and be modified with FAM, BHQ group).
1, antibody modification DNA
After antibody and SMCC (being suitable for molar ratio 1:10~20) react at room temperature 2h in PBS buffer solution, with filter membrane (retention point
Son amount 10,000MW) purifying, PBS buffer solution washing is three times；Respectively to the 200 mercapto-modified DNA1 of μ L (10 μM) and sulfydryl modification
DNA2 (10 μM) in be added 2 μ L TCEP (100mM), 37 DEG C of reaction 1h；Take each 200 μ L of treated antibody, DNA mixed
Even, 4 DEG C are incubated for 12 hours.Unreacted DNA is removed by filter membrane (molecular cut off 100,000MW), PBS buffer solution washing three
It is secondary.Obtain Ab1-DNA1, Ab2-DNA2.UV absorbance detection, it is seen that the characteristic peak (Fig. 2) of DNA and antibody protein.
2, sequence self assembly magnesium ion DNA enzymatic and detection fluorescence signal
Each 1 μM of each 100nM of Ab1-DNA1, Ab2-DNA2, PSA, hair clip DNA (H1, H2), 37 DEG C of reaction 2h after mixing.To
Each 1 μM of hair clip DNA (H3, H4) is added in above-mentioned chain alternative reaction product, 37 DEG C of reaction 1h after mixing.The hair clip DNA of generation
The hybridization chain of the length of (H1, H2, H3, H4) assembling.Hybridize and molecular beacon MB, Mg are added in chain reaction product2+Each 100nM is mixed
37 DEG C of reaction 1h after even.The signal peak of the visible 520nm of fluorescence detection, detection limit reach 0.73pg mL-1(Fig. 3).And to first tire egg
White (AFP), bovine serum albumin(BSA) (BSA) and carcinomebryonic antigen (CEA) non-target detectable substance are without response (Fig. 4).
The present invention provides a kind of detection kit of prostate specific antigen, detection method and applications, are repaired using secondary antibody
DNA is adornd, the Ag-Ab-DNA hybridization chain to form Y type is connect with antigen ortho position, antigen is inputted as signal adapter and is converted
For the output of DNA signal；Simultaneously by chain alternative reaction and hybridization chain reaction joint, the two-stage amplification of signal is realized；Long chain DNA
It is upper to form magnesium ion DNA enzymatic per adjacent two terminal sequences (hair clip DNA H3 and hair clip DNA H4) assembling, in the presence of magnesium ion
The molecular beacon in solution is cut in the digestion, and the molecular beacon of disconnection releases and issue fluorescence, new molecular beacon again with
Magnesium ion DNA enzymatic is combined, is cut, and circulation amplification fluorescence signal realizes the signal amplification of third round, substantially increases detection effect
Rate and sensitivity make detection limit reach 0.73pgmL-1。
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
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Claims (10)
1. a kind of detection kit of prostate specific antigen, which is characterized in that including following components: PSA antibody, succinyl are sub-
Amine -4- (N- maleimide) thiacyclohexane -1-1 carboxylic esters, three (2- carboxyethyl) phosphines, mercapto-modified DNA1 are mercapto-modified
DNA2, hair clip DNA H1, hair clip DNA H2, hair clip DNA H3, hair clip DNA H4, beacon MB and Mg2+；It is described mercapto-modified
The nucleotide sequence of DNA1 is as shown in SEQ ID NO.1；The nucleotide sequence of the mercapto-modified DNA2 such as SEQ ID NO.2
It is shown；The nucleotide sequence of the hair clip DNA H1 is as shown in SEQ ID NO.3；The nucleotide sequence of the hair clip DNA H2 is such as
Shown in SEQ ID NO.4；6 acid sequence of nucleosides of the hair clip DNA H3 is as shown in SEQ ID NO.5；The hair clip DNA H4's
Nucleotide sequence is as shown in SEQ ID NO.6；The beacon MB include one identification ion enzyme rA base and be modified with FAM and
BHQ group.
2. detection kit according to claim 1, which is characterized in that the nucleotide sequence of the beacon MB such as SEQ ID
Shown in NO.7.
3. a kind of detection method of prostate specific antigen, which comprises the following steps: (1) by PSA antibody and amber
After acid imide -4- (N- maleimide) thiacyclohexane -1-1 carboxylic esters carry out coupling reaction 2h, antibody after filtering to handle；It is described
The molar ratio of PSA antibody and succinimide -4- (N- maleimide) thiacyclohexane -1-1 carboxylic esters is 1:10~20；
(2) mercapto-modified DNA1 and mercapto-modified DNA2 are subjected to hydroxy activated react with three (2- carboxyethyl) phosphines respectively
1h, DNA1 and DNA2 after processing after must handling；The mercapto-modified DNA1 and mercapto-modified DNA2 and three (2- carboxyethyls)
The molar ratio of phosphine is 1:104；
(3) by antibody after the processing respectively with DNA2 after DNA1 after processing and processing in 4 DEG C of incubations 12h, obtain Ab1-DNA1 with
Ab2-DNA2；The volume ratio of antibody and DNA2 after DNA1 after processing and processing is 1:1 after the processing；
(4) 37 DEG C of reaction 2h after mixing the Ab1-DNA1, Ab2-DNA2, PSA, hair clip DNA H1 and hair clip DNA H2, obtain
Chain alternative reaction product；The Ab1-DNA1, Ab2-DNA2, PSA, hair clip DNA H1 and hair clip DNA H2 molar ratio be 100:
100:100:10-3:10-3；
(5) 1h is reacted for 37 DEG C after mixing the chain alternative reaction product with hair clip DNA H3 and hair clip DNA H4, obtains hybridization chain；
The molar ratio of the hair clip DNA H3 and hair clip DNA H4 are 300:10-3:10-3；
(6) by the hybridization chain and molecular beacon MB, Mg2+37 DEG C of reaction 1h, fluorescence detection after mixing；
There is no chronological orders to limit between step (1) and step (2).
4. detection method according to claim 3, which is characterized in that step (1) it is described reaction in PBS buffer solution into
Row, the temperature of the reaction are 18~25 DEG C.
5. detection method according to claim 3, which is characterized in that step (1) filtering molecular cut off of filter membrane
For 10000MW.
6. detection method according to claim 3, which is characterized in that the temperature of step (2) described reaction is 37 DEG C.
7. detection method according to claim 3, which is characterized in that further include utilizing retention point after step (3) described incubation
The membrane filtration that son amount is 100000MW retains substance on film, and three times using PBS buffer solution washing.
8. detection method according to claim 3, which is characterized in that after step (3) obtains the Ab1-DNA1 and Ab2-DNA2,
It further include UV absorbance detection.
9. detection method according to claim 3, which is characterized in that step (6) fluorescence detection is in 520nm visible light
Lower signal detection peak.
10. any one of detection kit as claimed in claim 1 or 2 or claim 3~9 detection method are believed in target antigen
Number amplification in application.
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