Summary of the invention
The object of the present invention is to provide the method that detects the microRNA for diagnosing cerebral apoplexy from blood, to solve the above problems.
A kind of method that detects the microRNA for diagnosing cerebral apoplexy from blood is provided in an embodiment of the present invention, has comprised the following steps:
Add testing sample and multiple nucleic acids probe in the mixture formed by multiple fluorescence-encoded micro-beads, carry out hybridization;
Wherein, described fluorescence-encoded micro-beads is fixed with the first nucleic acid fragment, and the sequence of described the first nucleic acid fragment on fluorescence-encoded and this fluorescence-encoded micro-beads of described fluorescence-encoded micro-beads is relation one to one;
The conjugate that described nucleic acid probe is vitamin H and the second nucleic acid fragment, and described the second nucleic acid fragment comprises: with the first sub-fragment of described the first nucleic acid fragment complementation on described fluorescence-encoded micro-beads, and with a kind of the second sub-fragment of microRNA complementation; The described second sub-fragment is different from the sequence of described the first nucleic acid fragment;
The described second sub-fragment respectively with a kind of microRNA complementation with lower:
hsa-let-7a-5p，hsa-let-7c，hsa-let-7d-5p，hsa-let-7f-5p，hsa-let-7g-5p，hsa-let-7i-5p，hsa-miR-1182，hsa-miR-1225-3p，hsa-miR-1225-5p，hsa-miR-1228-3p，hsa-miR-1234，hsa-miR-1238，hsa-miR-126-3p，hsa-miR-126-5p，hsa-miR-1260b，hsa-miR-1275，hsa-miR-128，hsa-miR-1280，hsa-miR-1304-3p，hsa-miR-142-3p，hsa-miR-146a-5p，hsa-miR-148a-3p，hsa-miR-148b-3p，hsa-miR-151a-3p，hsa-miR-151a-5p，hsa-miR-151b，hsa-miR-152，hsa-miR-15a-5p，hsa-miR-181a-5p，hsa-miR-1825，hsa-miR-18a-5p，hsa-miR-191-3p，hsa-miR-197-3p，hsa-miR-197-5p，hsa-miR-199a-3p，hsa-miR-199a-5p，hsa-miR-21-5p，hsa-miR-211-3p，hsa-miR-221-3p，hsa-miR-223-3p，hsa-miR-2392，hsa-miR-23a-3p，hsa-miR-23b-3p，hsa-miR-26b-5p，hsa-miR-27b-3p，hsa-miR-30b-5p，hsa-miR-3162-3p，hsa-miR-3180-5p，hsa-miR-3196，hsa-miR-326，hsa-miR-328，hsa-miR-331-3p，hsa-miR-335-5p，hsa-miR-340-5p，hsa-miR-3676-3p，hsa-miR-376a-3p，hsa-miR-376c，hsa-miR-377-3p，hsa-miR-382-5p，hsa-miR-425-3p，hsa-miR-4270，hsa-miR-4271，hsa-miR-4298，hsa-miR-4313，hsa-miR-4433-5p，hsa-miR-4442，hsa-miR-4463，hsa-miR-4484，hsa-miR-4499，hsa-miR-4532，hsa-miR-4655-3p，hsa-miR-4665-3p，hsa-miR-4669，hsa-miR-4695-5p，hsa-miR-4725-5p，hsa-miR-4734，hsa-miR-4749-3p，hsa-miR-4763-3p，hsa-miR-4767，hsa-miR-4778-5p，hsa-miR-4800-5p，hsa-miR-483-5p，hsa-miR-486-3p，hsa-miR-494，hsa-miR-5195-3p，hsa-miR-574-3p，hsa-miR-590-5p，hsa-miR-642b-3p，hsa-miR-652-3p，hsa-miR-652-5p，hsa-miR-671-5p，hsa-miR-936；
Add multiple strand degrading enzyme in the mixture occurred after described hybridization, carry out DeR, make a kind of described strand degrading enzyme a kind of microRNA that degrades;
In the mixture occurred after described DeR, add fluorescently-labeled Streptavidin to carry out association reaction; Wherein, the wavelength of fluorescence that described Streptavidin sends is different from the wavelength of fluorescence that described fluorescence-encoded micro-beads sends;
Described fluorescence-encoded micro-beads after described association reaction occurs in cleaning;
Described fluorescence-encoded micro-beads after identification is cleaned fluorescence-encoded detects the fluorescence intensity of the Streptavidin on described fluorescence-encoded micro-beads simultaneously;
The fluorescence-encoded kind of determining the microRNA of its detection according to described fluorescence-encoded micro-beads;
According to the fluorescence intensity of the Streptavidin on described fluorescence-encoded micro-beads, determine the microRNA that whether is combined with respective type on this fluorescence-encoded micro-beads, thereby determine the microRNA that whether has respective type in described testing sample.
The method of the microRNA for diagnosing cerebral apoplexy that detects from blood of the above embodiment of the present invention, can complete by primary first-order equation the detection of multiple microRNA, therefore and without synthetic primer, saved the step of synthetic primer, made and detect that multiple microRNA saves time and laborsaving.
Embodiment
Below by specific embodiment, the present invention is described in further detail.
The invention provides detection fluorescence-encoded micro-beads and the nucleic acid probe used of microRNA, as embodiment mono-and embodiment bis-.
Embodiment mono-
This embodiment provides a kind of fluorescence-encoded micro-beads, and this fluorescence-encoded micro-beads is fixed with the first nucleic acid fragment, and the sequence of the first nucleic acid fragment on fluorescence-encoded and this fluorescence-encoded micro-beads of fluorescence-encoded micro-beads is relation one to one.
Because the fluorescence-encoded micro-beads of this embodiment is fixed with the first nucleic acid fragment, thereby can be used to directly or indirectly in conjunction with microRNA by this fluorescence-encoded micro-beads of nucleic acid hybridization, and then for detecting for diagnosing the microRNA of cerebral apoplexy from blood.And the sequence of fluorescence-encoded and first nucleic acid fragment of fluorescence-encoded micro-beads is relation one to one, therefore, a kind of fluorescence-encoded micro-beads can correspondingly detect a kind of microRNA, by the fluorescence-encoded kind of distinguishing microRNA.Therefore, the fluorescence-encoded micro-beads of this embodiment both can be realized the high throughput testing of microRNA, can guarantee again the specific detection of microRNA.
Above-mentioned fluorescence-encoded micro-beads can be improved: preferably, be fixed with a plurality of the first nucleic acid fragments on fluorescence-encoded micro-beads, and the sequence of the first nucleic acid fragment on each fluorescence-encoded micro-beads is identical.Can make like this on each fluorescence-encoded micro-beads to connect a plurality of microRNA, take full advantage of the space of fluorescence-encoded micro-beads, utilize less fluorescence-encoded micro-beads to detect the sample of greater concn, the consumption of having saved fluorescence-encoded micro-beads, reduce testing cost.
Embodiment bis-
This embodiment provides a kind of nucleic acid probe, the conjugate that this nucleic acid probe is vitamin H and the second nucleic acid fragment, and the second nucleic acid fragment comprises: the first sub-fragment of the first nucleic acid fragment complementation on the fluorescence-encoded micro-beads provided with a kind of embodiment mono-, and with a kind of the second sub-fragment of microRNA complementation; The second sub-fragment is different from the sequence of the first nucleic acid fragment.
And the second sub-fragment on above-mentioned nucleic acid probe respectively with a kind of microRNA complementation with lower:
hsa-let-7a-5p，hsa-let-7c，hsa-let-7d-5p，hsa-let-7f-5p，hsa-let-7g-5p，hsa-let-7i-5p，hsa-miR-1182，hsa-miR-1225-3p，hsa-miR-1225-5p，hsa-miR-1228-3p，hsa-miR-1234，hsa-miR-1238，hsa-miR-126-3p，hsa-miR-126-5p，hsa-miR-1260b，hsa-miR-1275，hsa-miR-128，hsa-miR-1280，hsa-miR-1304-3p，hsa-miR-142-3p，hsa-miR-146a-5p，hsa-miR-148a-3p，hsa-miR-148b-3p，hsa-miR-151a-3p，hsa-miR-151a-5p，hsa-miR-151b，hsa-miR-152，hsa-miR-15a-5p，hsa-miR-181a-5p，hsa-miR-1825，hsa-miR-18a-5p，hsa-miR-191-3p，hsa-miR-197-3p，hsa-miR-197-5p，hsa-miR-199a-3p，hsa-miR-199a-5p，hsa-miR-21-5p，hsa-miR-211-3p，hsa-miR-221-3p，hsa-miR-223-3p，hsa-miR-2392，hsa-miR-23a-3p，hsa-miR-23b-3p，hsa-miR-26b-5p，hsa-miR-27b-3p，hsa-miR-30b-5p，hsa-miR-3162-3p，hsa-miR-3180-5p，hsa-miR-3196，hsa-miR-326，hsa-miR-328，hsa-miR-331-3p，hsa-miR-335-5p，hsa-miR-340-5p，hsa-miR-3676-3p，hsa-miR-376a-3p，hsa-miR-376c，hsa-miR-377-3p，hsa-miR-382-5p，hsa-miR-425-3p，hsa-miR-4270，hsa-miR-4271，hsa-miR-4298，hsa-miR-4313，hsa-miR-4433-5p，hsa-miR-4442，hsa-miR-4463，hsa-miR-4484，hsa-miR-4499，hsa-miR-4532，hsa-miR-4655-3p，hsa-miR-4665-3p，hsa-miR-4669，hsa-miR-4695-5p，hsa-miR-4725-5p，hsa-miR-4734，hsa-miR-4749-3p，hsa-miR-4763-3p，hsa-miR-4767，hsa-miR-4778-5p，hsa-miR-4800-5p，hsa-miR-483-5p，hsa-miR-486-3p，hsa-miR-494，hsa-miR-5195-3p，hsa-miR-574-3p，hsa-miR-590-5p，hsa-miR-642b-3p，hsa-miR-652-3p，hsa-miR-652-5p，hsa-miR-671-5p，hsa-miR-936。
Be each nucleic acid probe and a kind of above-mentioned microRNA complementation.
The nucleic acid probe of this embodiment can be used for detecting for diagnosing the microRNA of cerebral apoplexy from blood, its can specific binding embodiment mono-fluorescence-encoded micro-beads, microRNA in also can the specific binding sample, thereby set up relation correspondingly between fluorescence-encoded micro-beads and microRNA, by this nucleic acid probe, fluorescence-encoded micro-beads and microRNA are combined indirectly, so that high throughput testing.The vitamin H of simultaneously on this nucleic acid probe, having gone back mark, be convenient to the Streptavidin of combined with fluorescent mark, thereby complete final detection.
Above-mentioned nucleic acid probe can be improved: preferably, vitamin H, the second sub-fragment, the first sub-fragment connect successively.After such design can make fluorescence-encoded micro-beads capture nucleic acid probe and microRNA, vitamin H on nucleic acid probe is away from fluorescence-encoded micro-beads, thereby avoid follow-up Streptavidin and biological association reaction to be subject to the steric hindrance of fluorescence-encoded micro-beads, reduce reaction efficiency.
Embodiment tri-
This embodiment provides a kind of method that detects the microRNA for diagnosing cerebral apoplexy from blood, and the method has been utilized above-mentioned fluorescence-encoded micro-beads and nucleic acid probe, and it comprises the following steps:
The first step: the nucleic acid probe that adds testing sample and various embodiments two to provide in the mixture formed to the fluorescence-encoded micro-beads provided by various embodiments one, carry out hybridization.
Second step: add multiple strand degrading enzyme in the mixture occurred after hybridization, carry out DeR, make a kind of strand degrading enzyme a kind of microRNA that degrades.
The 3rd step: add fluorescently-labeled Streptavidin in the mixture occurred after DeR, carry out association reaction; Wherein, the wavelength of fluorescence that the wavelength of fluorescence that Streptavidin sends sends from fluorescence-encoded micro-beads is different.
The 4th step: the fluorescence-encoded micro-beads after association reaction occurs in cleaning.
The 5th step: the fluorescence-encoded micro-beads after identification is cleaned fluorescence-encoded detects the fluorescence intensity of the Streptavidin on fluorescence-encoded micro-beads simultaneously.
The 6th step: according to the fluorescence-encoded kind of determining the microRNA of its detection of fluorescence-encoded micro-beads.
The 7th step: according to the fluorescence intensity of the Streptavidin on fluorescence-encoded micro-beads, determine the microRNA that is combined with respective type of the type that whether has its detection on this fluorescence-encoded micro-beads, thereby determine the microRNA that whether has respective type in testing sample.
The detection method of above-mentioned microRNA can complete by primary first-order equation the detection of multiple microRNA, and without synthetic primer, has therefore saved the step of synthetic primer, makes to detect that multiple microRNA saves time and laborsaving, and its concrete testing process is as follows:
Take fluorescence-encoded micro-beads as reaction carriers.
Except sample, reactant required in testing process comprises: the first nucleic acid fragment on fluorescence-encoded micro-beads, nucleic acid probe, strand degrading enzyme, fluorescently-labeled Streptavidin.
Reaction process is: in the first step, the complementary hybridization that occurs of the first nucleic acid fragment on the first sub-fragment of the second nucleic acid fragment on nucleic acid probe and fluorescence-encoded micro-beads, the complementary hybridization that occurs of microRNA in the second sub-fragment and sample, be fixed on fluorescence-encoded micro-beads thereby the microRNA in sample is combined simultaneously.In second step, due to the second sub-fragment of some microRNA and nucleic acid probe in sample only have partial sequence complementary and by Incomplete matching be fixed on fluorescence-encoded micro-beads; Also have simultaneously excessive nucleic acid probe only with the first nucleic acid fragment hybridization not with example reaction.Therefore, add the strand degrading enzyme in this step, be used for degrading nucleic acid together of Incomplete matching hybridization and only with the first nucleic acid fragment hybridization not with the nucleic acid probe of example reaction on the second sub-fragment, be that these materials are called unbound state, thereby the first nucleic acid fragment on nucleic acid probe fixing on fluorescence-encoded micro-beads and microRNA and this fluorescence-encoded micro-beads is all mated fully.In the 3rd step, add again fluorescently-labeled Streptavidin, by the association reaction between the vitamin H on Streptavidin and nucleic acid probe, the fluorescent substance of marked by streptavidin is fixed on fluorescence-encoded micro-beads, thus fluorescence-encoded micro-beads the is had fluorescent substance of report microRNA content.In the 4th step, remove chemical substance free in reaction solution by cleaning, thereby avoid the interference of these chemical substances to subsequent detection.In the 5th step, the fluorescence-encoded micro-beads after judge cleaning fluorescence-encoded detects the fluorescence intensity of the fluorescently-labeled Streptavidin on fluorescence-encoded micro-beads simultaneously.Finally judged: according to the fluorescence-encoded kind of determining the microRNA of its detection of fluorescence-encoded micro-beads; According to the fluorescence intensity of the Streptavidin on fluorescence-encoded micro-beads, determine the microRNA that is combined with respective type of the type that whether has its detection on this fluorescence-encoded micro-beads, thereby determine the microRNA that whether has respective type in testing sample.
From above, the kind of the kind of fluorescent microsphere, the kind of nucleic acid probe, microRNA is relation one to one, thereby in final result is passed judgment on, by the fluorescence-encoded kind that can determine the microRNA of fixing (catching) on this fluorescence-encoded micro-beads of identification fluorescence-encoded micro-beads, determine in testing sample whether have microRNA according to the fluorescence intensity of the Streptavidin on this fluorescence-encoded micro-beads more simultaneously.Finally by primary first-order equation, realized the qualitative detection of multiple microRNA.
Wherein, can be undoubtedly definitely: during the fluorescence that exists Streptavidin to send, show in testing sample to exist the corresponding microRNA of this microballoon on fluorescence-encoded micro-beads; If during the fluorescence that does not exist Streptavidin to send on fluorescence-encoded micro-beads, show in testing sample not exist the corresponding microRNA of this microballoon.
In addition, the wavelength of fluorescence that the wavelength of fluorescence sent due to fluorescently-labeled Streptavidin sends from fluorescence-encoded micro-beads is different, thereby source that can be by identification wavelength of fluorescence identification fluorescence (from the coding fluorescence of fluorescence-encoded micro-beads or from the fluorescence of mark on Streptavidin).
In addition, because the second sub-fragment is different from the sequence of the first nucleic acid fragment, thereby avoided microRNA and the complementary hybridization of the first nucleic acid fragment to make microRNA directly not be fixed on fluorescence-encoded micro-beads by nucleic acid probe, and then avoided further fixing fluorescently-labeled Streptavidin so that the problem detected.
Visible, above-described embodiment has not only been realized the purpose of high throughput testing, has avoided synthetic primer simultaneously, thereby time saving and energy saving.
Above-mentioned detection method has realized detecting the purpose of the microRNA for diagnosing cerebral apoplexy from blood, and this detected result can be used as other purposes, for example is used for making a definite diagnosis cerebral apoplexy or other purposes.Wherein, the concrete grammar of making a definite diagnosis cerebral apoplexy is: when detecting in blood by aforesaid method while containing in following 92 kinds of microRNA one or more, can determine that the detected person suffers from cerebral apoplexy.92 kinds of microRNA are as follows:
hsa-let-7a-5p，hsa-let-7c，hsa-let-7d-5p，hsa-let-7f-5p，hsa-let-7g-5p，hsa-let-7i-5p，hsa-miR-1182，hsa-miR-1225-3p，hsa-miR-1225-5p，hsa-miR-1228-3p，hsa-miR-1234，hsa-miR-1238，hsa-miR-126-3p，hsa-miR-126-5p，hsa-miR-1260b，hsa-miR-1275，hsa-miR-128，hsa-miR-1280，hsa-miR-1304-3p，hsa-miR-142-3p，hsa-miR-146a-5p，hsa-miR-148a-3p，hsa-miR-148b-3p，hsa-miR-151a-3p，hsa-miR-151a-5p，hsa-miR-151b，hsa-miR-152，hsa-miR-15a-5p，hsa-miR-181a-5p，hsa-miR-1825，hsa-miR-18a-5p，hsa-miR-191-3p，hsa-miR-197-3p，hsa-miR-197-5p，hsa-miR-199a-3p，hsa-miR-199a-5p，hsa-miR-21-5p，hsa-miR-211-3p，hsa-miR-221-3p，hsa-miR-223-3p，hsa-miR-2392，hsa-miR-23a-3p，hsa-miR-23b-3p，hsa-miR-26b-5p，hsa-miR-27b-3p，hsa-miR-30b-5p，hsa-miR-3162-3p，hsa-miR-3180-5p，hsa-miR-3196，hsa-miR-326，hsa-miR-328，hsa-miR-331-3p，hsa-miR-335-5p，hsa-miR-340-5p，hsa-miR-3676-3p，hsa-miR-376a-3p，hsa-miR-376c，hsa-miR-377-3p，hsa-miR-382-5p，hsa-miR-425-3p，hsa-miR-4270，hsa-miR-4271，hsa-miR-4298，hsa-miR-4313，hsa-miR-4433-5p，hsa-miR-4442，hsa-miR-4463，hsa-miR-4484，hsa-miR-4499，hsa-miR-4532，hsa-miR-4655-3p，hsa-miR-4665-3p，hsa-miR-4669，hsa-miR-4695-5p，hsa-miR-4725-5p，hsa-miR-4734，hsa-miR-4749-3p，hsa-miR-4763-3p，hsa-miR-4767，hsa-miR-4778-5p，hsa-miR-4800-5p，hsa-miR-483-5p，hsa-miR-486-3p，hsa-miR-494，hsa-miR-5195-3p，hsa-miR-574-3p，hsa-miR-590-5p，hsa-miR-642b-3p，hsa-miR-652-3p，hsa-miR-652-5p，hsa-miR-671-5p，hsa-miR-936。
In addition, aforesaid method also can further improve, and reaches more beneficial effect, for example:
Preferably, in above-described embodiment one, the hybridization of the first step can adopt the hybridizing method of progressively lowering the temperature, to improve the specificity of reacting between the first nucleic acid fragment, nucleic acid probe and microRNA three.
Preferably, in nucleic acid probe, vitamin H, the second sub-fragment, the first sub-fragment connect successively.Identical with the improvement of the nucleic acid probe of above describing, thereby can reach same technique effect.
Preferably, be fixed with a plurality of the first nucleic acid fragments on fluorescence-encoded micro-beads, and the sequence of the first nucleic acid fragment on each fluorescence-encoded micro-beads is identical.Identical with the improvement of the fluorescence-encoded micro-beads of above describing, thereby can reach same technique effect.
Preferably, while adding testing sample and multiple nucleic acids probe, the kind of the nucleic acid probe added is identical with the kind of microRNA to be measured in testing sample.The microRNA that had so both guaranteed kind to be measured has the nucleic acid probe matched, and can avoid again the waste of material that added the multiple types nucleic acid probe to cause.
On above-mentioned preferred basis, the kind of fluorescence-encoded micro-beads is identical with the kind of the nucleic acid probe added.Equally, this step both can guarantee that all nucleic acid probes had the fluorescence-encoded micro-beads matched, and then the microRNA that guarantees all kinds to be measured has the fluorescence-encoded micro-beads matched, can avoid again the waste of material that added the multiple types fluorescence-encoded micro-beads to cause.
The foregoing is only the preferred embodiments of the present invention, be not limited to the present invention, for a person skilled in the art, the present invention can have various modifications and variations.Within the spirit and principles in the present invention all, any modification of doing, be equal to replacement, improvement etc., within all should being included in protection scope of the present invention.