CN114645073A - Microsphere coated with multiple probes as well as preparation method and application thereof - Google Patents
Microsphere coated with multiple probes as well as preparation method and application thereof Download PDFInfo
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- 239000000523 sample Substances 0.000 title claims abstract description 90
- 239000004005 microsphere Substances 0.000 title claims abstract description 83
- 238000002360 preparation method Methods 0.000 title abstract description 5
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 14
- 239000011248 coating agent Substances 0.000 claims description 13
- 238000000576 coating method Methods 0.000 claims description 13
- 230000004913 activation Effects 0.000 claims description 8
- 239000000243 solution Substances 0.000 claims description 8
- 238000005406 washing Methods 0.000 claims description 8
- 238000000034 method Methods 0.000 claims description 7
- 239000007788 liquid Substances 0.000 claims description 5
- 230000003213 activating effect Effects 0.000 claims description 4
- 239000000872 buffer Substances 0.000 claims description 4
- 239000007853 buffer solution Substances 0.000 claims description 3
- 238000010382 chemical cross-linking Methods 0.000 claims description 3
- 239000012536 storage buffer Substances 0.000 claims description 3
- 239000012190 activator Substances 0.000 claims description 2
- 230000002194 synthesizing effect Effects 0.000 claims description 2
- 150000001408 amides Chemical class 0.000 claims 1
- 238000003556 assay Methods 0.000 claims 1
- 150000002632 lipids Chemical class 0.000 claims 1
- 238000012360 testing method Methods 0.000 abstract description 26
- 230000035945 sensitivity Effects 0.000 abstract description 8
- 238000001514 detection method Methods 0.000 description 20
- 230000000694 effects Effects 0.000 description 9
- 230000003321 amplification Effects 0.000 description 6
- 239000003795 chemical substances by application Substances 0.000 description 6
- 238000003199 nucleic acid amplification method Methods 0.000 description 6
- 238000009396 hybridization Methods 0.000 description 5
- 238000012408 PCR amplification Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 241000701806 Human papillomavirus Species 0.000 description 2
- 108020005187 Oligonucleotide Probes Proteins 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 239000002751 oligonucleotide probe Substances 0.000 description 2
- 244000052769 pathogen Species 0.000 description 2
- 230000001717 pathogenic effect Effects 0.000 description 2
- 239000002096 quantum dot Substances 0.000 description 2
- 238000000018 DNA microarray Methods 0.000 description 1
- 239000012807 PCR reagent Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 238000000504 luminescence detection Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
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- C12Q1/708—Specific hybridization probes for papilloma
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Abstract
The invention discloses a microsphere coated with a plurality of probes, which realizes that a plurality of probes are coated on one microsphere, and the plurality of probes can be specifically combined with a target gene, and has the characteristics of good specificity, high sensitivity and strong signal value when being applied to the target gene test; the invention also discloses a preparation method and application of the microsphere coated with the plurality of probes.
Description
Technical Field
The invention relates to the field of molecular biology, in particular to a microsphere coated with a plurality of probes, a preparation method and application thereof.
Background
A liquid chip (also called flow fluorescence technology) is a clinical application type high-flux luminescence detection technology based on a microsphere and flow technology, belongs to a novel biochip technology platform, and consists of four parts, namely microspheres, probe molecules, molecules to be detected and reporter molecules. The core technology is that the microspheres with different fluorescent codes are coated with probes corresponding to different targets, a sample to be detected is added, then the sample is combined with a reporter molecule with another type of fluorescence to form a microsphere-probe-target-reporter molecule compound, and a signal value is read in a flow type fluorescence instrument, so that multiple targets can be detected on one sample simultaneously.
In the prior art, when the liquid chip technology is applied to the field of nucleic acid detection, an oligonucleotide probe specifically bound to a target gene is usually designed for the target gene, and then the probe is bound to a microsphere with a fluorescent code by a chemical crosslinking method. However, in practical applications, the liquid chip technology is often used for multi-target detection, and multiple oligonucleotide probes corresponding to the multi-target need to be hybridized in the same reaction tube, so that the reaction conditions of the systems corresponding to the multi-target probes need to be consistent; in addition, if SNP (single nucleotide polymorphism) detection is required for different typing of the same pathogen, there is a limitation in the probe design region, and there is also a possibility of other non-specific mutations in the probe region; in the case of designing a probe based on the above limitations, there may be a case where the signal is low or the sensitivity is low, the specificity is poor, or one probe cannot satisfy the detection requirement, and there is no effective solution in the art.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a microsphere, wherein a plurality of probes are connected on the microsphere, and the probes are specifically combined with a target gene to be detected, so that the problems that a single probe possibly generates low signal, low sensitivity, poor specificity and the like, and the detection requirements cannot be met can be effectively overcome.
In order to solve the technical problems, the invention provides a microsphere coated with a plurality of probes, the probes are specific to a target gene to be detected, and the probes and the microsphere are connected through covalent bonds to complete coating. Preferably, the 5' ends of the plurality of probes are covalently linked to the microsphere. Preferably, the covalent bond is in the form of one or more of an amide bond or a lipid bond or a disulfide bond; preferably, the microspheres used for coating may be fluorescently encoded microspheres or quantum dot microspheres.
When the invention tests the same target gene, a plurality of probes are designed and synthesized based on the target gene, and a plurality of probes are coated on one microsphere, the plurality of probes are all likely to be specifically combined with the target, the signal is strong, the sensitivity is high, the specificity is strong, and the detection effect of the target is effectively improved; aiming at a target gene with more mutation, probes comprising different mutant bases can be designed to increase the coverage rate of a target so as to achieve strong signal and high sensitivity and improve the detection effect; or probes are designed aiming at a plurality of areas of the same pathogen, so that the detection specificity is increased, and the detection sensitivity is improved.
In order to solve the technical problems, the invention also provides a preparation method of the microsphere coated with the plurality of probes, which comprises the following steps:
s1, designing and synthesizing a plurality of probes aiming at a target gene to be detected, wherein the probes can be specifically combined with the target;
step S2, carrying out activation treatment on the microspheres;
step S3, carrying out chemical crosslinking reaction on the plurality of probes synthesized in the step S1 and the microspheres activated in the step S2, wherein after the reaction, the plurality of probes are all connected to the same microspheres through covalent bonds;
through the steps, microspheres coated with a plurality of probes are prepared, and the microspheres prepared in S3 are stored through the following steps:
step S4: and (4) washing the microspheres prepared in the step S3 by using a washing solution, and storing the microspheres in a storage buffer solution for later use, wherein the storage temperature is 1-10 ℃. The washing solution in step S4 is generally selected from a group of buffers that can keep the ionic strength and pH of the system stable, and the storage buffer in step S4 generally serves to stabilize the nucleic acid and maintain the pH and ionic strength of the system, therefore, any such buffer solution in the art can be used in the present application; preferably, the storage temperature is 2-5 ℃; more preferably, the storage temperature is 4 ℃.
In a specific embodiment, the step of activating the microspheres in step S2 is as follows:
s21, washing the microspheres by using an activation buffer solution;
s22, resuspending the microspheres washed in step S21 in an activation buffer, and then adding an activator to activate the microspheres.
In the above activation step, the purpose of the activation buffer in step S21 and step S22 is to keep the ionic strength and the pH of the system stable; the activating agent in the step S22 is used to act as an activating group to assist the effective covalent reaction, thereby improving the reaction efficiency; preferably, the number of washing in the step S21 is 2 to 5; more preferably, the number of washes is 2.
In order to solve the technical problems, the invention also provides application of the microspheres coated with the plurality of probes in a liquid chip detection method. Understandably, the microspheres coated with a plurality of probes are diluted as required, mixed with other probe-coated microspheres, hybridized with a target to be detected, added with a color developing agent, incubated at constant temperature, and detected by using a flow-type fluorescence instrument. Understandably, since a plurality of probes are designed based on the same target gene, the detection specificity can be increased and the detection sensitivity can be improved.
Drawings
In order to more clearly illustrate the technical solution of the present invention, the drawings needed to be used in the present invention are briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a schematic diagram of the distinction between the multi-probe coating and the single-probe coating, in which for the multi-probe coated microspheres, a plurality of probes can be specifically bound to the amplification product, and the specificity and signal intensity of the hybridization between the probe-microspheres and the target gene are increased.
Detailed Description
The technical solutions in the present invention will be described clearly and completely with reference to the accompanying drawings, and it should be understood that the described embodiments are some, but not all embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example one
This example designed two probes for one type of Human Papillomavirus (HPV) 45, tested using conventional single probe-coated microspheres and using the dual probe-coated microspheres of the invention, respectively. It should be noted that, the probe-microspheres of group one, group two and group three of the present example were all mixed with probe-microspheres of type 39, and type 39 and type 45 were tested, respectively, to obtain specificity and signal value data (examples two and three are here); when the type 39 is added into a system to be detected, whether the 45 probe-microsphere can interfere with other detection indexes when detecting the target gene type 45 can be shown, namely, the change condition of the specificity of the 45 probe-microsphere after double-probe coating is carried out is shown.
1) PCR amplification for HPV-45 type, resulting in amplification product:
selecting primers, designing amplification primers according to published sequences:
a forward primer F: GGACATTGTCATTACGATGG, respectively;
reverse primer R: TTAAATGTTAATCAGATTGGTCG, respectively;
the PCR reagent system is shown in table 1:
TABLE 1 PCR System formulation
| Components | 25 μ L System |
| 2x Taq Mix | 12.5μL |
| Form panel | 5μL |
| F + R primer | 5μL |
| ddH2O | The volume is constant to 25 mu L |
The PCR procedure is shown in table 2:
TABLE 2 PCR procedure
2) Preparing microspheres for coating the probe: two probes were designed in the range of the amplification products, P1 and P2, respectively, and the microspheres were numbered C21:
P1：GGCACATGAATTGTGTAGGC；
P2：ACTAACTCTTGGGCTTAGTACG；
p1 and P2 were linked to microspheres (both fluorescent-encoded microspheres or quantum dot microspheres) to form three experimental groups of microspheres with different coating modes:
group one (control group): C21-P1, only P1 probes were coated on C21 microspheres;
group two (control group): C21-P2, only P2 probes were coated on C21 microspheres;
group three (test group): C21-P1/P2, wherein a P1 probe and a P2 probe are coated on the C21 microsphere at the same time, and the P1 probe and the P2 probe are mixed according to the ratio of 2:1 and then are connected to the C21 microsphere;
3) adding the PCR amplification products obtained in the step 1) into the three groups of microspheres obtained in the step 2) respectively to perform hybridization reaction, wherein the reaction procedure is that the temperature is 90-98 ℃ for 1-10min, and the temperature is 40-60 ℃ for 10-60 min;
4) adding a color developing agent (SA-PE) into the step 3), incubating for 15min at a constant temperature of 50 ℃, and detecting by using a flow-type fluorometer, wherein the detection results are shown in a table 3:
table 3 example one test results
| Group one | Type 39 | 45 type |
| Single probe C21-P1 group 39 type | 1055 | 138 |
| Single probe C21-P1 group 45 type | 8 | 1035 |
| Group two | Type 39 | 45 type |
| Single probe C21-P2 group 39 type | 1011 | 139 |
| Single probe C21-P2 group 45 type | 23 | 1304 |
| Group III | Type 39 | 45 type |
| Double probe C21-P1/P2 group 39 type | 1064 | 5 |
| Double probe C21-P1/P2 group 45 type | 14 | 1414 |
The test result shows that the specificity of the test group coated by the double probes is better than that of the group I, the signal value of the test group is higher than that of the group I and the group II, and the reaction sensitivity of the test group III is higher; in the group I and the group II which are coated by the single probe, the weak positive non-specific signal of the HPV39 type which appears when the type 45 is detected does not appear in the group III which is coated by the double probe, so that the specificity of the detection of the HPV45 microsphere probe is increased.
Example two
In the embodiment, on the basis of the first embodiment, the influence of the coating proportion relation of P1 and P2 on C21 on HPV-45 type detection is researched; mixing P1 and P2 according to the proportion of 1:1, 2:1 and 3:1 respectively, connecting with C21 microspheres, hybridizing with the amplification product of HPV45 under the hybridization reaction conditions of 95 ℃ for 5min and 50 ℃ for 30min, then adding a color developing agent, incubating at 50 ℃ for 15min, and detecting by using a flow-type fluorescence instrument, wherein the test results are shown in Table 4:
table 4 test results of example two
| Group one | Type 39 | 45 type |
| C21-P1/P21: group 1 type 39 | 1064 | 5 |
| C21-P1/P21: 1 group 45 type | 14 | 1414 |
| Group two | Type 39 | 45 type |
| C21-P1/P22: group 1 type 39 | 1616 | 15 |
| C21-P1/P22: 1 group 45 type | 2 | 1977 |
| Group III | Type 39 | 45 type |
| C21-P1/P23: group 1 type 39 | 1086 | 12 |
| C21-P1/P23: 1 group 45 type | 18 | 1898 |
From the test results, the test effect is better when the mixing ratio of the P1 to the P1 is 2:1 for the 45-type detection; it can be concluded that when a target is tested by coating a microsphere with a plurality of probes, the mixing ratio of each probe will affect the testing effect, and specifically, the mixing ratio can be adjusted according to different targets, so as to obtain the optimal mixing ratio.
EXAMPLE III
In this embodiment, on the basis of the first embodiment, the influence of the total concentration of the probes on the subsequent test effect when the microspheres are coated is studied, that is, the test effect is the best when the C21 microspheres are coated with the total concentrations of P1 and P2, in this embodiment, the mixing concentration ratio of P1 to P2 is selected to be 2: 1; the total concentration of P1 and P2 was linked to C21 microspheres at 10nM and 30nM, respectively, and then hybridized with the amplified products of HPV45, respectively, under the same hybridization conditions as in example two; then, a color developing agent was added, the mixture was incubated at a constant temperature of 50 ℃ for 15min, and the detection was performed by a flow-type fluorometer, and the test results are shown in table 5:
table 5 results of the three tests of example
| Group one | Type 39 | 45 type |
| C21-P1/P210 nM group 39 | 1113 | 14 |
| Group 45 of C21-P1/P210 nM | 9 | 1240 |
| Group two | Type 39 | 45 type |
| Group 39 of C21-P1/P230 nM | 1083 | 6 |
| Group 45 of C21-P1/P230 nM | 11 | 1900 |
As can be seen from the test results, for the detection of type 45, when the C21 microspheres are coated with the P1 and the P2 at a mixing ratio of 2:1, the test effect is better when the total probe concentration is 30 nM; it can be concluded that when a target is tested by coating a microsphere with a plurality of probes, the total concentration of the probes has an influence on the test effect, and the optimal total concentration of the probes can be obtained according to the mixing ratio of different targets and probes.
Example four
This example tests against HPV68 type, which has two subtypes HPV68a and HPV68b, and designs one probe for each of the two subtypes to be connected to microsphere C22, forming a test group of C22-P68a/P68 b; as a control group, two probes are respectively coated on a C23 microsphere and a C24 microsphere to form two control groups of C23-P68a and C24-P68 b; after carrying out PCR amplification on HPV68a and HPV68b, hybridizing the test group and the control group under the same hybridization conditions as in example two, adding a color developing agent, incubating at the constant temperature of 50 ℃ for 15min, and detecting by using a flow-type fluorescence instrument, wherein the test results are shown in Table 6:
table 6 results of the four tests of the example
| Group one | Type 68a | Type 68b |
| Group 68a of C23-P68a | 1124 | 21 |
| Group 68b of C23-P68a | 9 | 15 |
| Group 68a of C24-P68b | 11 | 2 |
| Group 68b of C24-P68b | 4 | 922 |
| Group two | Type 68a | Type 68b |
| Group 68a of C22-P68b/P68a | 1616 | 13 |
| C22-P68b/P68a group 68b | 6 | 1124 |
As can be seen from the test results, compared with the microsphere coated with the single probe, the microsphere coated with the mixed probe has better specificity and higher signal value after being hybridized with the amplification product, namely, the detection effect of the mixed probe coating is better than that of the single probe coating aiming at different subtypes; and the positive signals of the target products with two different sequences can be detected by directly using the same microsphere.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (7)
1. The microsphere coated with the probes is characterized in that the probes are specific to a target gene to be detected, and the probes and the microsphere are connected through covalent bonds to complete coating.
2. The microsphere of claim 1, wherein the 5' ends of the plurality of probes are covalently linked to the microsphere.
3. The microsphere of claim 2, wherein the covalent bonds are in the form of one or more of amide or lipid or disulfide bonds.
4. The method for preparing the microsphere coated with a plurality of probes according to claim 1, comprising the following steps:
s1, designing and synthesizing a plurality of probes aiming at a target gene to be detected, wherein the probes can be specifically combined with the target;
step S2, carrying out activation treatment on the microspheres;
and S3, carrying out chemical crosslinking reaction on the plurality of probes synthesized in the S1 and the microspheres activated in the S2, wherein after the reaction, the plurality of probes are all connected to the same microspheres through covalent bonds.
5. The method of claim 4, further comprising the steps of:
s4: and (4) washing the microspheres prepared in the step S3 by using a washing solution, and storing the microspheres in a storage buffer solution for later use, wherein the storage temperature is 1-10 ℃.
6. The method of claim 4, wherein the step of activating the microspheres in step S2 is as follows:
s21, washing the microspheres with an activation buffer solution for 2-5 times;
s22, resuspending the microspheres washed in step S21 in an activation buffer, and then adding an activator to activate the microspheres.
7. Use of the microsphere coated with a plurality of probes according to claim 1 in a liquid chip assay.
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Citations (7)
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| CN1570140A (en) * | 2003-07-25 | 2005-01-26 | 宋克 | Double probe gene chip signal amplification method |
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2022
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