CN110484440A - A kind of nucleic acid fluorescent in situ detection device and method - Google Patents
A kind of nucleic acid fluorescent in situ detection device and method Download PDFInfo
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- CN110484440A CN110484440A CN201910818784.1A CN201910818784A CN110484440A CN 110484440 A CN110484440 A CN 110484440A CN 201910818784 A CN201910818784 A CN 201910818784A CN 110484440 A CN110484440 A CN 110484440A
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- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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- C12Q1/6841—In situ hybridisation
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Abstract
The invention discloses a kind of nucleic acid fluorescent in situ detection devices, including a pair of of c-type probe, triggering segment, the hair clip segment of several fluorescent markers, target sequence, the triggering segment and the target sequence are respectively cooperating with the two sides for being set to a pair of c-type probe, and the pair of c-type probe is combined with target sequence cooperation；Partial sequence, free sequence are provided in the hair clip segment, trigger sequence is provided in the triggering segment, the partial sequence of the hair clip segment is combined with the trigger sequence pairing of the triggering segment, and the free sequence of the hair clip segment is combined with the pairing of the partial sequence of hair clip skin section described in another.The present invention also proposes detection method, the present invention greatly improves the efficiency, accuracy and accuracy of detection with a pair of of two key technologies of c-type probe and Cascaded amplification signal system, the unicellular target sequence singly copied in minimum detectable sample, has expanded the application range of nucleic acid hybridization in situ detection technique.
Description
Technical field
The present invention relates to a kind of nucleic acid fluorescent in situ detection device and method, belong to technical field of biological, it can be achieved that
Specific nucleic acid sequence is accurately measured in tissue in situ.
Background technique
Existing nucleic acid fluorescent in situ hybridization detection technique is mainly to utilize the probe of 500 bases longs or so, in probe
Mixed with the nucleotide of digoxigenin labeled, after primer is in conjunction with target sequence, the DigiTAb and primer of fluorescent marker is added
On digoxin combine, generate fluorescence signal, to detect whether there is target sequence in sample.
Summary of the invention
Existing technical solution is primarily present following two problem.
First is that probe length is too long.This can bring two problems.It is reaction efficiency problem first, since probe needs to penetrate
Gap on cell membrane enters cell interior in conjunction with target sequence, and probe length is too long to will affect membrane efficiency, so that
The time of detection greatly prolongs.Secondly, probe is too long to will lead to non-specific binding, it is possible to create false positive influences detection
Accuracy.
Second is that signal amplifying system is weaker.Existing technical solution is the digoxigenin labeled and fluorescent marker by nucleotide
DigiTAb, the digoxigenin labeled that probe carries is limited, therefore signal amplifying power is general, can not detect low-abundance
Target sequence greatly limits the application range of the prior art.
The technical problem to be solved by the present invention is to overcome the deficiencies of existing technologies, a kind of nucleic acid fluorescent in situ detection is provided
Device and method.
In order to solve the above technical problems, the present invention provides a kind of nucleic acid fluorescent in situ detection device, including a pair of of c-type is visited
Needle, triggering segment, the hair clip segment of several fluorescent markers, target sequence, the triggering segment are matched respectively with the target sequence
The two sides for being set to a pair of c-type probe are closed, the pair of c-type probe is combined with target sequence cooperation；The hair clip
It is provided with partial sequence, free sequence in segment, is provided with trigger sequence, the part of the hair clip segment in the triggering segment
Sequence is combined with the trigger sequence pairing of the triggering segment, the free sequence of the hair clip segment and another described hair clip skin
The partial sequence of section, which is matched, to be combined.
As a kind of preferred embodiment, the shape of the triggering segment is L-type.
As a kind of preferred embodiment, the length of the c-type probe is 50 bases.
As a kind of preferred embodiment, the partial sequence a-b of the hair clip segment, the free sequence of the hair clip segment
For a*-b*, the trigger sequence of the triggering segment is a*-b*。
As a kind of preferred embodiment, the hair clip segment includes hair clip segment one, the part of the hair clip segment one
The sequence of sequence and free sequence is a-b-a*-b*。
As a kind of preferred embodiment, the hair clip segment includes hair clip segment two, and the hair clip segment two is dissociated
The sequence of sequence and partial sequence is b*-a*-b-a。
As a kind of preferred embodiment, the trigger sequence of the triggering segment is in response to the pair of c-type probe and institute
Target sequence cooperation is stated to combine and generate fluorescence signal.
The present invention also proposes a kind of detection method of nucleic acid fluorescent in situ detection device, includes the following steps:
Step SS1: pretreatment goal sample, negative control, land control；
Step SS2: being added c-type probe and hybridization solution carries out hybridization 1 hour, is washed 3 times with cleaning solution；
Step SS3: the triggering segment and hybridization solution that L-type is added carry out hybridization 1 hour, are washed 3 times with cleaning solution；
Step SS4: being added the hair clip segment of fluorescent marker, reacts 30 minutes, is washed 3 times with cleaning solution；
Step SS5: fluorescence microscope shot detection result is used.
As a kind of preferred embodiment, the hybridization solution is digoxin solution.
As a kind of preferred embodiment, the cleaning solution is the SDS solution that concentration is 0.1%.
SDS is a kind of anion scale remover, it is combined with the amino acid residue 1:1 on albumen keeps albumen negatively charged, in this way
The secondary structure for allowing for albumen fully opens and can all take negative electricity.
Advantageous effects of the invention: first, the present invention is visited with a pair of of c-type probe instead of the long of the prior art
Needle, about 50 base of the length of c-type probe, about 1/10th of prior art probe length, therefore greatly improve probe
Membrane efficiency is crossed, to improve the efficiency of reaction.In terms of accuracy, present invention ensure that a pair of of c-type probe with correct mesh
When marking sequence combination, segment identification can be triggered by L-type, to generate fluorescence signal, be eliminated caused by non-specific binding
False positive.Meanwhile the present invention utilizes Cascaded amplification signal system instead of the ground of digoxin in the prior art and fluorescent marker
Digoxin antibody.Cascaded amplification signal system of the invention is made of the hair clip segment that L-type triggers segment and fluorescent marker, works as L-type
After the hair clip segment of trigger sequence one fluorescent marker of opening for triggering segment, the partial sequence and L-type of the hair clip segment are triggered
After the trigger sequence of segment, which is matched, to be combined, while the hair clip segment is opened, another section of free sequence is also one section of trigger sequence,
Another hair clip segment will be then opened, repeatedly, Cascaded amplification, to generate very strong fluorescence signal.Third, the present invention
With two key technologies of a pair of of c-type probe and Cascaded amplification signal system instead of the related art scheme of the prior art, substantially
The efficiency, accuracy and accuracy of detection are improved, the unicellular target sequence singly copied in minimum detectable sample is opened up
The application range of nucleic acid hybridization in situ detection technique is opened up.4th, the advantages of c-type probe technique of the invention, is: improving
The efficiency of detection shortens the time that detection needs；Detection accuracy is improved, the probability of false positive results is greatly reduced.The
Five, the advantages of Cascaded amplification signal system of the invention, is: improving the accuracy of detection, can detecte slender in sample
Born of the same parents, the target sequence singly copied, greatly expand the application range of nucleic acid hybridization in situ detection technique.
Detailed description of the invention
Fig. 1 is a kind of structural schematic diagram of the preferred embodiment of nucleic acid fluorescent in situ detection device of the invention.
Fig. 2-1 is the structural schematic diagram of the preferred embodiment of hair clip segment one of the invention.
Fig. 2-2 is the structural schematic diagram of the preferred embodiment of hair clip segment two of the invention.
Fig. 2-3 is the structural schematic diagram of the preferred embodiment of triggering segment of the invention.
Fig. 3 is that hair clip segment one of the invention applies schematic diagram in detection device of the invention.
Fig. 4 is that hair clip segment one and hair clip segment two of the invention apply schematic diagram in detection device of the invention.
Meaning marked in the figure: 1- triggers segment, 2-C type probe, 3- target sequence, 4- hair clip segment, 41- hair clip segment
One, 42- hair clip segment two.
Specific embodiment
The invention will be further described below in conjunction with the accompanying drawings.Following embodiment is only used for clearly illustrating the present invention
Technical solution, and not intended to limit the protection scope of the present invention.
As shown in Figure 1, the present invention provides a kind of nucleic acid fluorescent in situ detection device, including a pair of of c-type probe 2, isolation
Section 1, the hair clip segment 4 of several fluorescent markers, target sequence 3, the triggering segment 1 is respectively cooperating with the target sequence 3 to be set
The two sides of a pair of c-type probe 2 are placed in, the pair of c-type probe 2 is combined with the target sequence 3 cooperation；The hairpin-chip
It is provided with partial sequence, free sequence in section 4, is provided with trigger sequence, the portion of the hair clip segment 4 in the triggering segment 1
Sub-sequence is combined with the trigger sequence pairing of the triggering segment 1, the free sequence of the hair clip segment 4 and another described hair
The partial sequence of bark pocket section 4, which is matched, to be combined.Target sequence 3 is 5 ' -3 '.
As a kind of preferred embodiment, the shape of the triggering segment 1 is L-type.
As a kind of preferred embodiment, the length of the c-type probe 2 is 50 bases.
As Figure 2-3, as a kind of preferred embodiment, the partial sequence a-b of the hair clip segment 4, the hair clip
The free sequence of segment 4 is a*-b*, the trigger sequence of the triggering segment 1 is a*-b*。
As shown in Fig. 2-1, as a kind of preferred embodiment, the hair clip segment 4 includes hair clip segment 1, the hair
The sequence of the partial sequence of intermediate plate section 1 and free sequence is a-b-a*-b*。
As shown in Fig. 2-2, as a kind of preferred embodiment, the hair clip segment 4 includes hair clip segment 2 42, the hair
The free sequence of intermediate plate section 2 42 and the sequence of partial sequence are b*-a*-b-a。
As a kind of preferred embodiment, the trigger sequence of the triggering segment 1 in response to the pair of c-type probe 2 with
The cooperation of target sequence 3 combines and generates fluorescence signal.
As shown in Fig. 3 hair clip segment one applies the inspection of the detection device in the schematic diagram of detection device of the invention
Survey method, includes the following steps:
Step SS1: pretreatment goal sample, negative control, land control；
Step SS2: being added c-type probe and hybridization solution carries out hybridization 1 hour, is washed 3 times with cleaning solution；
Step SS3: the triggering segment and hybridization solution that L-type is added carry out hybridization 1 hour, are washed 3 times with cleaning solution；
Step SS4: being added the hair clip segment 1 of fluorescent marker, reacts 30 minutes, is washed 3 times with cleaning solution；
Step SS5: fluorescence microscope shot detection result is used.
As a kind of preferred embodiment, the hybridization solution is digoxin solution.
As a kind of preferred embodiment, the cleaning solution is the SDS solution that concentration is 0.1%.
SDS is a kind of anion scale remover, it is combined with the amino acid residue 1:1 on albumen keeps albumen negatively charged, in this way
The secondary structure for allowing for albumen fully opens and can all take negative electricity.
As shown in Fig. 4 hair clip segment one and hair clip segment two apply the schematic diagram in detection device of the invention, should
The detection method of detection device, includes the following steps:
Step SS1: pretreatment goal sample, negative control, land control；
Step SS2: being added c-type probe and hybridization solution carries out hybridization 1 hour, is washed 3 times with cleaning solution；
Step SS3: the triggering segment and hybridization solution that L-type is added carry out hybridization 1 hour, are washed 3 times with cleaning solution；
Step SS4: being added the hair clip segment 1 and hair clip segment 2 42 of fluorescent marker, reacts 30 minutes, washs 3 with cleaning solution
It is secondary；
Step SS5: fluorescence microscope shot detection result is used.
As a kind of preferred embodiment, the hybridization solution is digoxin solution.
As a kind of preferred embodiment, the cleaning solution is the SDS solution that concentration is 0.1%.
SDS is a kind of anion scale remover, it is combined with the amino acid residue 1:1 on albumen keeps albumen negatively charged, in this way
The secondary structure for allowing for albumen fully opens and can all take negative electricity.
Innovative principle of the invention is: first, long probe of the present invention with a pair of of c-type probe instead of the prior art, and C
About 50 base of the length of type probe, about 1/10th of prior art probe length, therefore greatly improve the film excessively of probe
Efficiency, to improve the efficiency of reaction.In terms of accuracy, present invention ensure that a pair of of c-type probe with correct target sequence
When column combine, segment identification can be triggered by L-type, to generate fluorescence signal, eliminate the sun of vacation caused by non-specific binding
Property.Meanwhile the present invention utilizes Cascaded amplification signal system instead of the digoxin of digoxin in the prior art and fluorescent marker
Antibody.Cascaded amplification signal system of the invention is made of the hair clip segment that L-type triggers segment and fluorescent marker, when L-type triggers
After the trigger sequence of segment opens the hair clip segment of a fluorescent marker, the partial sequence and L-type of the hair clip segment trigger segment
Trigger sequence match combine, while the hair clip segment open after, another section of free sequence is also one section of trigger sequence, will be connect
Open another hair clip segment, repeatedly, Cascaded amplification, to generate very strong fluorescence signal.Third, this present invention use
Two key technologies of a pair of of c-type probe and Cascaded amplification signal system are substantially mentioned instead of the related art scheme of the prior art
Efficiency, accuracy and the accuracy of detection are risen, the unicellular target sequence singly copied in minimum detectable sample is expanded
The application range of nucleic acid hybridization in situ detection technique.4th, the advantages of c-type probe technique of the invention, is: improving inspection
The efficiency of survey shortens the time that detection needs；Detection accuracy is improved, the probability of false positive results is greatly reduced.5th,
The advantages of Cascaded amplification signal system of the invention, is: improving the accuracy of detection, can detecte unicellular, single in sample
The target sequence of copy greatly expands the application range of nucleic acid hybridization in situ detection technique.
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, without departing from the technical principles of the invention, several improvement and deformations can also be made, these improvement and deformations
Also it should be regarded as protection scope of the present invention.
Claims (10)
1. a kind of nucleic acid fluorescent in situ detection device, which is characterized in that including a pair of of c-type probe, triggering segment, several fluorescence marks
The hair clip segment of note, target sequence, the triggering segment and the target sequence, which are respectively cooperating with, is set to a pair of c-type probe
Two sides, the cooperation of the pair of c-type probe and the target sequence combined；Partial sequence, trip are provided in the hair clip segment
From sequence, trigger sequence, the touching of the partial sequence of the hair clip segment and the triggering segment are provided in the triggering segment
It sends out sequence pairing to combine, the free sequence of the hair clip segment is combined with the pairing of the partial sequence of hair clip skin section described in another.
2. a kind of nucleic acid fluorescent in situ detection device according to claim 1, which is characterized in that the shape of the triggering segment
Shape is L-type.
3. a kind of nucleic acid fluorescent in situ detection device according to claim 1, which is characterized in that the length of the c-type probe
Degree is 50 bases.
4. a kind of nucleic acid fluorescent in situ detection device according to claim 1, which is characterized in that the portion of the hair clip segment
Sub-sequence a-b, the free sequence of the hair clip segment are a*-b*, the trigger sequence of the triggering segment is a*-b*。
5. a kind of nucleic acid fluorescent in situ detection device according to claim 4, which is characterized in that the hair clip segment includes
Hair clip segment one, the partial sequence of the hair clip segment one and the sequence of free sequence are a-b-a*-b*。
6. a kind of nucleic acid fluorescent in situ detection device according to claim 4, which is characterized in that the hair clip segment includes
Hair clip segment two, the free sequence of the hair clip segment two and the sequence of partial sequence are b*-a*-b-a。
7. a kind of nucleic acid fluorescent in situ detection device according to claim 1, which is characterized in that the touching of the triggering segment
Hair sequence is combined with target sequence cooperation in response to the pair of c-type probe and generates fluorescence signal.
8. a kind of detection method based on nucleic acid fluorescent in situ detection device described in claim 1, which is characterized in that including such as
Lower step:
Step SS1: pretreatment goal sample, negative control, land control；
Step SS2: being added c-type probe and hybridization solution is hybridized, and is washed with cleaning solution；
Step SS3: the triggering segment and hybridization solution that L-type is added are hybridized, and are washed with cleaning solution；
Step SS4: being added the hair clip segment of fluorescent marker, reacts 30 minutes, is washed with cleaning solution；
Step SS5: fluorescence microscope shot detection result is used.
9. a kind of detection method of nucleic acid fluorescent in situ detection device according to claim 8, which is characterized in that described miscellaneous
Friendship liquid is digoxin solution.
10. a kind of detection method of nucleic acid fluorescent in situ detection device according to claim 8, which is characterized in that described
Cleaning solution is the SDS solution that concentration is 0.1%.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113025690A (en) * | 2021-05-27 | 2021-06-25 | 广东品博易视生物科技有限公司 | Nucleic acid probe group and application thereof |
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Cited By (2)
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| CN113025690A (en) * | 2021-05-27 | 2021-06-25 | 广东品博易视生物科技有限公司 | Nucleic acid probe group and application thereof |
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