CN118086459A - Universal primer single fluorescent molecule detection method - Google Patents
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- 238000001514 detection method Methods 0.000 title claims abstract description 38
- 239000007850 fluorescent dye Substances 0.000 claims abstract description 18
- 230000035945 sensitivity Effects 0.000 claims abstract description 10
- 230000009261 transgenic effect Effects 0.000 claims abstract description 7
- 230000005284 excitation Effects 0.000 claims abstract description 4
- 238000001215 fluorescent labelling Methods 0.000 claims abstract description 4
- 238000002360 preparation method Methods 0.000 claims abstract description 4
- 238000000034 method Methods 0.000 claims description 33
- 238000006243 chemical reaction Methods 0.000 claims description 19
- 238000012360 testing method Methods 0.000 claims description 12
- 108700019146 Transgenes Proteins 0.000 claims description 11
- 239000013642 negative control Substances 0.000 claims description 8
- 239000013641 positive control Substances 0.000 claims description 8
- 230000003321 amplification Effects 0.000 claims description 7
- 230000000694 effects Effects 0.000 claims description 7
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 7
- 238000012408 PCR amplification Methods 0.000 claims description 6
- 238000002474 experimental method Methods 0.000 claims description 6
- 238000005457 optimization Methods 0.000 claims description 6
- 238000002866 fluorescence resonance energy transfer Methods 0.000 claims description 5
- 108090000623 proteins and genes Proteins 0.000 claims description 4
- 102000004169 proteins and genes Human genes 0.000 claims description 4
- 239000012634 fragment Substances 0.000 claims description 3
- 238000001502 gel electrophoresis Methods 0.000 claims description 3
- 238000012803 optimization experiment Methods 0.000 claims description 3
- 238000003759 clinical diagnosis Methods 0.000 abstract description 2
- 238000013461 design Methods 0.000 abstract description 2
- 238000012545 processing Methods 0.000 abstract description 2
- 238000011160 research Methods 0.000 abstract description 2
- 238000012216 screening Methods 0.000 abstract description 2
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 4
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000004075 alteration Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000005281 excited state Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
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Abstract
The invention relates to the technical field of molecular detection, and discloses a universal primer single fluorescent molecular detection method, which optimizes factors such as universal primer types, concentrations, modified specific primer concentrations and the like by establishing a high-flux and strong-specificity multiplex transgenic component detection system, and specifically comprises the following steps: s1, designing and preparing a universal primer; s2, fluorescent labeling of the universal primer and preparation of a fluorescent probe; s3, detecting target molecules; s4, fluorescence excitation and detection; s5, analyzing a detection result. Through the design of the universal primers, the types, the concentrations of modified specific primers and other factors of the universal primers are optimized, and a set of high-flux and high-specificity multiple transgenic component screening detection system is established, so that the detection method has the characteristics of high sensitivity, high selectivity and diversity, does not need expensive equipment and complicated processing steps, and has wide application potential in biological research and clinical diagnosis.
Description
Technical Field
The invention relates to the technical field of molecular detection, in particular to a universal primer single fluorescent molecule detection method.
Background
Fluorescent molecule detection methods use Fluorescence Resonance Energy Transfer (FRET) techniques. The method utilizes two interacting fluorescent molecules, one as the emitting phosphor (detector) and the other as the receiving phosphor (receiver). When the two molecules are close to each other and properly arranged, the excited state energy of the emitted phosphor can be transferred to the received phosphor by non-radiative energy, thereby causing fluorescence enhancement of the acceptor, and in the detection of the prior art, the detection result is poor due to the complex kind of the primer and the target molecule composition.
Disclosure of Invention
(One) solving the technical problems
Aiming at the defects of the prior art, the invention provides a universal primer single fluorescent molecule detection method.
(II) technical scheme
In order to achieve the above purpose, the present invention provides the following technical solutions: a method for detecting single fluorescent molecule of universal primer optimizes factors such as universal primer type, concentration and concentration of each modified specific primer by establishing a high-flux and strong-specificity multiplex transgenic component detection system, and specifically comprises the following steps:
S1, designing and preparing a universal primer;
s2, fluorescent labeling of the universal primer and preparation of a fluorescent probe;
S3, detecting target molecules;
s4, fluorescence excitation and detection;
S5, analyzing a detection result.
2. The method for detecting the single fluorescent molecule of the universal primer according to claim 1, wherein the method comprises the following steps: the step S1 specifically comprises the following steps: according to the characteristics of target molecules and the required specificity, proper primer types and universal primers are selected, and the universal primers have sequences or functional fragments capable of combining with various target molecules, wherein the target molecules are DNA, RNA and protein.
Preferably, the step S2 specifically includes: preparing a fluorescent molecule labeled with one end or a specific region of the primer; another fluorescent molecule, which binds to the other end of the primer or to a specific region of the target molecule, is prepared for forming a FRET pair with the labeled fluorescent molecule.
Preferably, the step S3 specifically includes: the universal primer reacts with the target molecule to enable the primer to be specifically combined with the target molecule, and the fluorescent label and the fluorescent probe are replaced to adapt to different target molecule detection requirements.
Preferably, the step S4 specifically includes: the emitted fluorescent body is excited to obtain a fluorescent signal, the intensity of the fluorescent signal of the received fluorescent body is measured, the signal is enhanced when the fluorescent signal is combined with the target molecule, whether the target molecule exists or not is determined by comparing the intensity of the fluorescent signal, and the content or the activity of the target molecule is judged.
Preferably, in step S1, the method further includes: the primer concentration is optimized, the specificity and the sensitivity of the universal primer are improved by optimizing the concentration of the universal primer, and the optimal primer concentration is determined, so that the target transgene sequence is accurately and efficiently amplified;
The reaction conditions are optimized, including the composition of a reaction system and the temperature cycle conditions, and the specificity and the sensitivity of the universal primer are improved and the generation of nonspecific amplification products after the reaction is reduced by optimizing the reaction conditions.
Preferably, the primer concentration optimization is specifically:
primer concentration gradient experiment: the concentration of the primer was gradually increased from a lower concentration to a higher concentration, and a gradient experiment was performed in a certain range. Then carrying out PCR amplification reaction, observing the amplification result, and finding out the optimal primer concentration;
primer concentration optimization experiment: performing independent PCR amplification reaction, carefully observing the quality of the amplified product and the definition of the band by gel electrophoresis and other methods, and selecting the optimal primer concentration;
preferably, the primer concentration optimization further comprises: repeating the test, confirming the reliability of the result, performing repeated tests for a plurality of times, and verifying the applicability of the optimal primer concentration in different batches of samples.
Preferably, the step S5 specifically includes: and (3) performing data comparison through multiple tests, firstly setting a control group, then performing data comparison on other test results and the control group, determining whether target molecules exist or not and determining the content or activity of a judging device, determining corresponding primers, and fluorescent markers and fluorescent probes matched with the corresponding primers, so as to ensure the accuracy of experimental results.
Preferably, the step S5 further includes: suitable positive and negative control samples were designed for verifying the specificity and reliability of the method, the positive control samples being samples containing known transgene components, the negative control samples being samples without any transgene components, excluding false positive and false negative results.
(III) beneficial effects
Compared with the prior art, the invention provides a universal primer single fluorescent molecule detection method, which has the following beneficial effects:
1. according to the universal primer single fluorescent molecule detection method, the factors such as the types and the concentrations of the universal primers and the concentrations of the modified specific primers are optimized through the design of the universal primers, and a set of high-flux and high-specificity multiplex transgenic component screening detection system is established, so that the detection method has the characteristics of high sensitivity, high selectivity and diversity, does not need expensive equipment and complex processing steps, and has wide application potential in biological research and clinical diagnosis.
2. According to the universal primer single fluorescent molecule detection method, different target molecules can be better detected by the universal primer through changing fluorescent marks and fluorescent probes, so that the target molecules can be better detected, and the application range of the primer for detection is improved.
3. The method for detecting the universal primer single fluorescent molecule selects the primer capable of reacting with DNA, RNA and protein, and can improve the detection effect by optimizing the type and concentration of the primer and the concentration of each modified specific primer when detecting the universal primer single fluorescent molecule.
4. According to the universal primer single fluorescent molecule detection method, the concentration of the primer is optimized, the specificity and the sensitivity are improved, and the optimal primer concentration is determined, so that the target transgenic sequence can be accurately and efficiently amplified, the reaction condition is optimized, the specificity and the sensitivity can be improved, and the generation of non-specific amplification products is reduced.
5. In the detection process, two control groups are designed and divided into positive control samples and negative control samples, wherein the positive control samples are samples containing known transgene components, and the negative control samples are samples without any transgene components. These control samples can help to exclude false positive and false negative results, improving the accuracy and reliability of the detection method.
Detailed Description
The technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
A method for detecting single fluorescent molecule of universal primer optimizes factors such as universal primer type, concentration and concentration of each modified specific primer by establishing a high-flux and strong-specificity multiplex transgenic component detection system, and specifically comprises the following steps:
S1, designing and preparing a universal primer;
s2, fluorescent labeling of the universal primer and preparation of a fluorescent probe;
S3, detecting target molecules;
s4, fluorescence excitation and detection;
S5, analyzing a detection result.
The step S1 specifically comprises the following steps: according to the characteristics of target molecules and the required specificity, proper primer types and universal primers are selected, and the universal primers have sequences or functional fragments capable of combining with various target molecules, wherein the target molecules are DNA, RNA and protein.
The step S2 specifically comprises the following steps: preparing a fluorescent molecule labeled with one end or a specific region of the primer; another fluorescent molecule, which binds to the other end of the primer or to a specific region of the target molecule, is prepared for forming a FRET pair with the labeled fluorescent molecule.
The step S3 specifically comprises the following steps: the universal primer reacts with the target molecule to enable the primer to be specifically combined with the target molecule, and the fluorescent label and the fluorescent probe are replaced to adapt to different target molecule detection requirements.
The step S4 specifically comprises the following steps: the emitted fluorescent body is excited to obtain a fluorescent signal, the intensity of the fluorescent signal of the received fluorescent body is measured, the signal is enhanced when the fluorescent signal is combined with the target molecule, whether the target molecule exists or not is determined by comparing the intensity of the fluorescent signal, and the content or the activity of the target molecule is judged.
The step S1 also comprises the following steps: the primer concentration is optimized, the specificity and the sensitivity of the universal primer are improved by optimizing the concentration of the universal primer, and the optimal primer concentration is determined, so that the target transgene sequence is accurately and efficiently amplified;
The reaction conditions are optimized, including the composition of a reaction system and the temperature cycle conditions, and the specificity and the sensitivity of the universal primer are improved and the generation of nonspecific amplification products after the reaction is reduced by optimizing the reaction conditions.
The primer concentration optimization specifically comprises the following steps:
primer concentration gradient experiment: the concentration of the primer was gradually increased from a lower concentration to a higher concentration, and a gradient experiment was performed in a certain range. Then carrying out PCR amplification reaction, observing the amplification result, and finding out the optimal primer concentration;
primer concentration optimization experiment: performing independent PCR amplification reaction, carefully observing the quality of the amplified product and the definition of the band by gel electrophoresis and other methods, and selecting the optimal primer concentration;
the primer concentration optimization further comprises the following steps: repeating the test, confirming the reliability of the result, performing repeated tests for a plurality of times, and verifying the applicability of the optimal primer concentration in different batches of samples.
The step S5 specifically comprises the following steps: and (3) performing data comparison through multiple tests, firstly setting a control group, then performing data comparison on other test results and the control group, determining whether target molecules exist or not and determining the content or activity of a judging device, determining corresponding primers, and fluorescent markers and fluorescent probes matched with the corresponding primers, so as to ensure the accuracy of experimental results.
The step S5 further comprises the following steps: suitable positive and negative control samples were designed for verifying the specificity and reliability of the method, the positive control samples being samples containing known transgene components, the negative control samples being samples without any transgene components, excluding false positive and false negative results.
Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (10)
1. A universal primer single fluorescent molecule detection method is characterized in that: by establishing a high-flux and high-specificity multiplex transgenic component detection system, the factors such as the type and concentration of the universal primers, the concentration of each modified specific primer and the like are optimized, and the method specifically comprises the following steps:
S1, designing and preparing a universal primer;
s2, fluorescent labeling of the universal primer and preparation of a fluorescent probe;
S3, detecting target molecules;
s4, fluorescence excitation and detection;
S5, analyzing a detection result.
2. The method for detecting the single fluorescent molecule of the universal primer according to claim 1, wherein the method comprises the following steps: the step S1 specifically comprises the following steps: according to the characteristics of target molecules and the required specificity, proper primer types and universal primers are selected, and the universal primers have sequences or functional fragments capable of combining with various target molecules, wherein the target molecules are DNA, RNA and protein.
3. The method for detecting the single fluorescent molecule of the universal primer according to claim 1, wherein the method comprises the following steps: the step S2 specifically comprises the following steps: preparing a fluorescent molecule labeled with one end or a specific region of the primer; another fluorescent molecule, which binds to the other end of the primer or to a specific region of the target molecule, is prepared for forming a FRET pair with the labeled fluorescent molecule.
4. The method for detecting the single fluorescent molecule of the universal primer according to claim 1, wherein the method comprises the following steps: the step S3 specifically comprises the following steps: the universal primer reacts with the target molecule to enable the primer to be specifically combined with the target molecule, and the fluorescent label and the fluorescent probe are replaced to adapt to different target molecule detection requirements.
5. The method for detecting the single fluorescent molecule of the universal primer according to claim 1, wherein the method comprises the following steps: the step S4 specifically comprises the following steps: the emitted fluorescent body is excited to obtain a fluorescent signal, the intensity of the fluorescent signal of the received fluorescent body is measured, the signal is enhanced when the fluorescent signal is combined with the target molecule, whether the target molecule exists or not is determined by comparing the intensity of the fluorescent signal, and the content or the activity of the target molecule is judged.
6. The method for detecting the single fluorescent molecule of the universal primer according to claim 1, wherein the method comprises the following steps: the step S1 also comprises the following steps: the primer concentration is optimized, the specificity and the sensitivity of the universal primer are improved by optimizing the concentration of the universal primer, and the optimal primer concentration is determined, so that the target transgene sequence is accurately and efficiently amplified;
The reaction conditions are optimized, including the composition of a reaction system and the temperature cycle conditions, and the specificity and the sensitivity of the universal primer are improved and the generation of nonspecific amplification products after the reaction is reduced by optimizing the reaction conditions.
7. The method for detecting the single fluorescent molecule of the universal primer according to claim 6, wherein the method comprises the following steps: the primer concentration optimization specifically comprises the following steps:
primer concentration gradient experiment: the concentration of the primer was gradually increased from a lower concentration to a higher concentration, and a gradient experiment was performed in a certain range. Then carrying out PCR amplification reaction, observing the amplification result, and finding out the optimal primer concentration;
primer concentration optimization experiment: the individual PCR amplification reactions were performed, and then the quality of the amplified products and the clarity of the bands were carefully observed by gel electrophoresis or the like, and the optimal primer concentrations were selected.
8. The method for detecting the single fluorescent molecule of the universal primer according to claim 7, wherein the method comprises the following steps: the primer concentration optimization further comprises the following steps: repeating the test, confirming the reliability of the result, performing repeated tests for a plurality of times, and verifying the applicability of the optimal primer concentration in different batches of samples.
9. The method for detecting the single fluorescent molecule of the universal primer according to claim 1, wherein the method comprises the following steps: the step S5 specifically comprises the following steps: and (3) performing data comparison through multiple tests, firstly setting a control group, then performing data comparison on other test results and the control group, determining whether target molecules exist or not and determining the content or activity of a judging device, determining corresponding primers, and fluorescent markers and fluorescent probes matched with the corresponding primers, so as to ensure the accuracy of experimental results.
10. The method for detecting the single fluorescent molecule of the universal primer according to claim 9, wherein the method comprises the following steps: the step S5 further comprises the following steps: suitable positive and negative control samples were designed for verifying the specificity and reliability of the method, the positive control samples being samples containing known transgene components, the negative control samples being samples without any transgene components, excluding false positive and false negative results.
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