CN108660216B - Kit and its detection method based on peripheral blood free nucleotide miRNAs super sensitivity detection device - Google Patents
Kit and its detection method based on peripheral blood free nucleotide miRNAs super sensitivity detection device Download PDFInfo
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Abstract
The present invention relates to kit and its detection method based on peripheral blood free nucleotide miRNAs super sensitivity detection device, kit contains 1) microarray detection cell；2) external magnetic field；3) premixing reaction liquid A and 4) premixing reaction liquid B, when detecting multiple targets to be measured, configured in parallel, which respectively corresponds, detects a kind of target premixing reaction liquid A to be measured, is denoted as premixing reaction liquid A1, premixing reaction liquid A2 respectively；The bright binding biomolecules diagnosis of the kit, magnetic microsphere of receiving enrichment and biochip technology develop the novel technical method for being not necessarily to amplification, highly sensitive, the high-throughput direct accurately detection peripheral blood miRNAs highly relevant with the generation, development and drug resistance of malignant tumour of one kind.The present invention will have important value in terms of medicine tumour Non-invasive detection, personalized medicine and chemotherapy prognosis.
Description
Technical field
The invention belongs to field of biological detection, it is related to based on peripheral blood free nucleotide miRNAs super sensitivity detection device
Kit further relates to the detection method using the kit.
Background technique
Microrna (miRNA) is the single-stranded short microRNA that a kind of endogenous is about 21-25bp, passes through controlling gene tune
Ganglion cell's growth, apoptosis and signal transduction.MiRNA polymorphism and drug metabolism and drug resistance are closely related, and miRNA pairs of unconventionality expression
Generation, development and the drug susceptibility of prediction tumour play an important role；There is stable miRNA in peripheral blood at present, can make
For the biomarker of high sensitivity and high specificity.
Currently, the main detection method of peripheral blood miRNA has cloning and sequencing, Real-Time PCR, RNA blot hybridization, core
Piece etc.；Micro peripheral blood miRNA usually requires to be detected by amplification, but since miRNA sequence is shorter, regular-PCR is difficult to
It carries out, although having at present based on stem-loop (stem-loop) and the Real-Time PCR based on polyA tailing etc., these
Special amplification technique difficulty height is not easy popularization and application.
Summary of the invention
In view of this, the purpose of the present invention is to provide one kind to be based on peripheral blood free nucleotide miRNAs super sensitivity detection
The kit of device；Further relate to the detection method using the kit.
In order to achieve the above objectives, the invention provides the following technical scheme:
1. a kind of kit based on peripheral blood free nucleotide miRNAs super sensitivity detection device, including 1) microarray is examined
Survey pond, 2) with the matched external magnetic board of detection cell bottom structure, 3) premixing reaction liquid A and 4) premixing reaction liquid B.
Preferably, the microarray is N*N arrangement, and wherein N is greater than or equal to 2.
It is furthermore preferred that the detection bottom of pond portion is " V " shape.
Preferably, the external magnetic board is connect with microarray detection cell disengaging type.
It is furthermore preferred that the premix A is to link fluorescence hairpin probe by the polycyclic of group coupling with nanometer magnetic bead；
The premixing reaction liquid B is TNE buffer, is made of Tris, EDTA, NaCl, pH of buffer 8.0.
Preferably, the premixing reaction liquid A contains a kind of probe for detecting target, detects multiple targets to be measured
When, configured in parallel, which respectively corresponds, detects a kind of target premixing reaction liquid to be measured, is denoted as premixing reaction liquid A1, premix respectively
Close reaction solution A2.
The fluorescence hairpin probe is the probe with the miRNA-150 biarc connecting specifically bound, and probe sequence is as follows:
D1:FAM-cgcgattctcccaacccttgtaccagtgatcgcgtgagcacatgaaata cactggagaat
cg-BHQ1(SEQ ID NO.1)
D2:FAM-atgcgctctcccaacccttgtaccagtggcgcatcgattctccagtgta caatcccacag
tg-NH2(SEQ ID NO.2)
D3:cactgtgggattgtatttcatgtgctca-BHQ1 (SEQ ID NO.3).
2. being included the following steps using the method for the kit detection peripheral blood free nucleotide miRNAs
(1) premix is instilled in the detection cell of detection device；
(2) biological sample to be measured is clicked and entered in detection cell, is uniformly mixed with premixing reaction liquid A and premixing reaction liquid B,
3 repetitions are arranged in each sample；
(3) point sample microarray detection cell is placed in hybridizing box, is hybridized, whole process keeps strictly being protected from light state；
(4) by step (3), treated that microarray detection cell is placed on external magnetic board, reaches and receives magnetic cup aggregation effect
Fruit；
(5) fluorescence recognition compares.
Preferably, the biological sample wherein to be measured includes that patient peripheral's blood miRNAs extracts sample, healthy blood
MiRNAs extracts at least one of sample, artificial synthesized miRNA target sequence and artificial synthesized single-stranded NC sequence.It is furthermore preferred that
The hybridization is takes out after 95 DEG C of water-bath 10min, 0 DEG C of ice bath 30min, 18~25 DEG C of placement 30min.
The beneficial effects of the present invention are:
MiRNAs is stable in the presence of in peripheral blood, can be used as the biomarker of high sensitivity, high specificity, but it is usually
It needs to be detected by amplification, and there are amplification technique difficulty height to be not easy popularization and application；The present invention can directly detect peripheral blood
The expression quantity of middle miRNAs, without amplification.
Kit of the present invention, by the " V " shape bottom microassay substrate of unique design and external magnetic board to receiving magnetic
The absorption of microballoon has reached double fluorescent concentration effect, greatly improves the detection sensitivity of miRNAs.
Kit of the present invention, by design multiple groups premixing reaction liquid, (every group of premixing reaction liquid contains for one kind
The polycyclic link hairpin probe of specific ring sequence), realize the multiple biological markers of simultaneous reactions on one piece of microassay substrate
The purpose of object expression quantity has direct, quick command function to tumor disease diagnosis, curative effect and prognosis etc..
Detailed description of the invention
In order to keep the purpose of the present invention, technical scheme and beneficial effects clearer, the present invention provides following attached drawing and carries out
Illustrate:
Probe target sequences detection principle diagram (A: probe structure and schematic diagram is pressed from both sides in Fig. 1 dual loop chain sending and receiving；B: probe difference section
Detect solubility curve；C:PAGE electrophoretogram).
Fig. 2 connects the nanometer magnetic bead signal amplification micro-array chip testing principle that hairpin probe makees identification molecule based on dual loop chain
Figure.
Fig. 3 is fluorescence enriching apparatus and effect schematic diagram (A: structure drawing of device；B: effect schematic diagram).
Fig. 4 is miRNA-150 fluorogram in Peripheral Blood of Patients with Non-small Cell Lung.
Fig. 5 is that fluorogram is verified in miRNAs sensitivity.
Fig. 6 is that fluorescence curve figure is verified in miRNAs sensitivity.
Specific embodiment
Below in conjunction with attached drawing, a preferred embodiment of the present invention will be described in detail.
Embodiment 1, a kind of peripheral blood free nucleotide miRNAs super sensitivity detection device
A kind of peripheral blood free nucleotide miRNAs super sensitivity detection device, structure as shown in A in fig. 3, including microarray
Detection cell 1 and with the external magnetic board 2 of the matched disengaging type of detection cell bottom structure；Wherein, microarray is that N*N is arranged, such as 6 × 6,
N is greater than or equal to 2；Detection cell is gradually reduced by top to base diameter, and the preferred bottom of pond portion that detects is " V " shape, the micro- battle array of " V " shape
Column detection cell is more conducive to fluorescence enrichment, improves probe in detecting sensitivity.
When detection, on microassay substrate after the reaction was completed by premixing reaction liquid and biological sample to be measured, by external
Magnetic board reaches the fluorescence concentration effect to reaction system to the suction-operated of magnetic microsphere is received, thus improve detection efficiency (Fig. 3,
B).When detection detects multiple targets to be measured, configured in parallel, which respectively corresponds, detects a kind of target premixing reaction liquid to be measured,
It is denoted as premixing reaction liquid A1, premixing reaction liquid A2 respectively.Different colours indicate that the different nano magnetics for being directed to different miRNA are more
Loop chain connects fluorescence hairpin probe, and each detection detection cell only puts a Species specific probes, and porous does not interfere with each other, and sentences conducive to result
It is disconnected.
Embodiment 2, the method for detecting peripheral blood free nucleotide miRNAs
Patients with Non-small-cell Lung (NSCLC) peripheral blood free nucleotide is detected using the detection device in embodiment 1
The method of miRNA-150, includes the following steps:
(1) premix A and premix B are instilled in the detection cell of detection device, premix A is and miRNA-
The bicyclic link fluorescence hairpin probe of 150 specific bindings, probe sequence are as follows:
D1:FAM-cgcgattctcccaacccttgtaccagtgatcgcgtgagcacatgaaata cactggagaat
cg-BHQ1(SEQ ID NO.1)；
D2:FAM-atgcgctctcccaacccttgtaccagtggcgcatcgattctccagtgta caatcccacag
tg-NH2(SEQ ID NO.2)；
D3:cactgtgggattgtatttcatgtgctca-BHQ1 (SEQ ID NO.3)；
The mixed reaction solution B is TNE buffer, is made of Tris, EDTA, NaCl, pH of buffer 8.0.
Above-mentioned probe sequence forms bicyclic link fluorescence hairpin probe by complementary pairing, as a result with testing principle such as Fig. 1
Shown in middle A and Fig. 2, the target sequence to be measured of bicyclic identification is identical in the present embodiment, can also design the double of the different target sequences of identification
Loop chain connects fluorescence hairpin probe, can same detection cell detect 2 kinds of target sequences, can also by loop design be identify it is identical to
It surveys target sequence or identifies the polycyclic of different target sequences, identify that the polycyclic of different target sequences can detect multiple targets in same detection cell
Sequence.In addition, for the bicyclic link fluorescence hairpin probe of verifying the present embodiment design, it is respectively that D1, D1+D2, D1+D2+D3 is miscellaneous
Fluorescence intensity is handed over, then in 98 DEG C → 0 DEG C observation solubility curve, as a result as shown in figure 1 shown in B.It quenches the result shows that being added and containing
It goes out after the probe sequence D3 of group, dual loop chain sending and receiving folder probe structure is formed, and fluorescence signal is obviously reduced, and fluorescence resonance energy turns
Effect is moved to be achieved；As shown in figure 1 shown in C (PAGE electrophoretogram), while being added in the electrophoresis path of probe sequence D1, D2, D3,
There is the band that a base number significantly increases, which shows that a base number is greater than the new nucleic acid molecules of D1, D2, D3
It is formed, i.e., dual loop chain sending and receiving folder probe structure is formed, while the electrophoresis path of probe sequence D1, D2, D3 and miRNA-150 is added
It is interior, there is band and show that the more previous channel of base number increases, shows that bicyclic probe hybridizes with target sequence miRNA-150.
(2) biological sample to be measured is clicked and entered in detection cell, is uniformly mixed with premixed liquid reaction solution A and mixed liquid reaction solution B, often
3 repetitions are arranged in a sample；Wherein biological sample to be measured includes: miRNA-N1 (outside the non-patients undergoing chemotherapy of non-small cell lung cancer first visit
All blood miRNAs extracts sample), miRNA-N2 (Treatment of non-small-cell lung cancer with chemotherapy patient peripheral's blood miRNAs extracts sample),
MiRNA-H (healthy blood miRNAs extracts sample)；
(3) point sample microarray detection cell is placed in hybridizing box, is taken after carrying out 95 DEG C of water-bath 10min, 0 DEG C of ice bath 30min
Out, (18~25 DEG C) placement 30min of room temperature, whole process keep strictly being protected from light state；
(4) by step (3), treated that microarray detection cell is placed on external magnetic board, reaches and receives magnetic cup aggregation effect
Fruit；
(5) fluorescence recognition compares, and miRNAs unconventionality expression comparison result in blood of cancer patients can be obtained, as a result such as
Shown in Fig. 4 and table 1.
MiRNA-150 testing result in 1 Peripheral Blood of Patients with Non-small Cell Lung of table
Embodiment 3, the sensitivity verifying for detecting peripheral blood free nucleotide miRNAs
Using the miRNA-150 after gradient dilution as template, verify of the present invention based on peripheral blood free nucleotide
The kit of miRNAs super sensitivity detection device and its sensitivity of detection method, the specific steps are as follows:
(1) by Oligo miRNA-150 (being synthesized by TaKaRa company) carry out gradient dilution to 1 μM, 1nM, 10pM and
1pM, as sensitivity confirmatory experiment sample；
(2) it is detected using the device in embodiment 1, premix A and premix B is instilled to the inspection of detection device
Survey pond in, premix A be with miRNA-150 specifically bind it is bicyclic link fluorescence hairpin probe, probe sequence is as follows:
D1:FAM-cgcgattctcccaacccttgtaccagtgatcgcgtgagcacatgaaata cactggagaat
cg-BHQ1(SEQ ID NO.1)；
D2:FAM-atgcgctctcccaacccttgtaccagtggcgcatcgattctccagtgta caatcccacag
tg-NH2(SEQ ID NO.2)；
D3:cactgtgggattgtatttcatgtgctca-BHQ1 (SEQ ID NO.3)；
The mixed reaction solution B is TNE buffer, is made of Tris, EDTA, NaCl, pH of buffer 8.0.
(3) gradient dilution sample in (1) is clicked and entered in detection cell, is uniformly mixed with premixed liquid reaction solution A, each sample is set
Set 3 repetitions；It is compared simultaneously with probe groups and blank group (TNE buffer)；
(4) point sample microarray detection cell is placed in hybridizing box, is taken after carrying out 95 DEG C of water-bath 10min, 0 DEG C of ice bath 30min
Out, (18~25 DEG C) placement 30min of room temperature；Whole process keeps strictly being protected from light state；
(5) by step (4), treated that microarray detection cell is placed on external magnetic board, reaches and receives magnetic cup aggregation effect
Fruit；
(6) fluorescence recognition compares, and sensitivity verification result is obtained, as a result as shown in Fig. 5, Fig. 6 and table 2.
Table 2, miRNAs sensitivity verification result
The result shows that: kit of the present invention based on peripheral blood free nucleotide miRNAs super sensitivity detection device and
Its detection method is reached by the " V " shape bottom microassay substrate of unique design and external magnetic board to receiving the absorption of magnetic microsphere
Double fluorescent concentration effect, greatly improves the detection sensitivity to miRNAs, minimum detection limit can arrive 10pM rank.
Kit and its detection side of the present invention based on peripheral blood free nucleotide miRNAs super sensitivity detection device
Method, the premixing reaction liquid of unique design are a kind of polycyclic link hairpin probe containing specific ring sequence, every kind of probe
The miRNA targeting marker in peripheral blood can be specifically bound, it is multiple swollen to realize the simultaneous reactions on one piece of microassay substrate
The purpose of tumor markers variable expression；Experimental result also shows: based on high degree of specificity of the invention, passing through final fluorescence
Intensity contrast can significantly distinguish positive patient and healthy control group.
Finally, it is stated that preferred embodiment above is only used to illustrate the technical scheme of the present invention and not to limit it, although logical
It crosses above preferred embodiment the present invention is described in detail, however, those skilled in the art should understand that, can be
Various changes are made to it in form and in details, without departing from claims of the present invention limited range.
Sequence table
<110>the first affiliated hospital, army medical university, ground force, the Chinese People's Liberation Army
<120>kit and its detection method based on peripheral blood free nucleotide miRNAs super sensitivity detection device
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 62
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
cgcgattctc ccaacccttg taccagtgat cgcgtgagca catgaaatac actggagaat 60
cg 62
<210> 2
<211> 62
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
atgcgctctc ccaacccttg taccagtggc gcatcgattc tccagtgtac aatcccacag 60
tg 62
<210> 3
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
cactgtggga ttgtatttca tgtgctca 28
Claims (4)
1. a kind of kit based on peripheral blood free nucleotide miRNAs super sensitivity detection device characterized by comprising 1)
Microarray detection cell, 2) with the matched external magnetic board of detection cell bottom structure, 3) premixing reaction liquid A, 4) premixing reaction
Liquid B；
The premix A is to link fluorescence hairpin probe by the polycyclic of group coupling with nanometer magnetic bead；The premixing is anti-
Answering liquid B is TNE buffer, is made of Tris, EDTA, NaCl, pH of buffer 8.0；
The fluorescence hairpin probe is the probe with the miRNA-150 biarc connecting specifically bound, and probe sequence is as follows:
D1:FAM-cgcgattctcccaacccttgtaccagtgatcgcgtgagcacatgaaata cactggagaatcg-
BHQ1(SEQ ID NO.1)
D2:FAM-atgcgctctcccaacccttgtaccagtggcgcatcgattctccagtgta caatcccacagtg-NH2
(SEQ ID NO.2)
D3:cactgtgggattgtatttcatgtgctca-BHQ1(SEQ ID NO.3).
2. the kit according to claim 1 based on peripheral blood free nucleotide miRNAs super sensitivity detection device, special
Sign is: the microarray is N*N arrangement, and wherein N is greater than or equal to 2.
3. the kit according to claim 1 based on peripheral blood free nucleotide miRNAs super sensitivity detection device, special
Sign is: the detection bottom of pond portion is " V " shape.
4. the kit according to claim 1 based on peripheral blood free nucleotide miRNAs super sensitivity detection device, special
Sign is: the external magnetic board is connect with microarray detection cell disengaging type.
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