CN111321203A - Nucleic acid detection kit and application thereof - Google Patents
Nucleic acid detection kit and application thereof Download PDFInfo
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C12Q1/6813—Hybridisation assays
- C12Q1/6816—Hybridisation assays characterised by the detection means
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Abstract
The application discloses nucleic acid detect reagent box includes: the kit comprises a high-molecular fluorescent coding microsphere, a fluorescent microsphere capable of being resolved in time, an anti-TAG sequence, an extension product accounting gene sequence and a signal amplification system. The invention also discloses a use method of the nucleic acid detection kit. The kit provided by the invention is based on a related idea of complementary combination of time-resolved fluorescence and high-flux flow fluorescence, can effectively eliminate non-specific fluorescence, reduces background interference, and greatly improves the sensitivity and linear range of analysis.
Description
Technical Field
The invention belongs to the field of molecular biology, and particularly relates to a nucleic acid detection kit, which can utilize time-resolved flow fluorescence detection and nucleic acid genes and mutations thereof.
Background
The existing technologies for detecting nucleic acid and mutation in the market mainly comprise fluorescence quantitative PCR and high-throughput sequencing, and the fluorescence quantitative PCR detection technology cannot simultaneously detect and type multiple genes in a single tube, and has relatively low flux; the high-throughput sequencing detection technology has high equipment investment requirement, is relatively complex to operate and has relatively difficult data processing. . The invention adopts the high-flux time-resolved fluorescence technology, can realize the combined detection of nucleic acid, can avoid the interference of non-specific fluorescence, reduce background signals and increase the detection sensitivity, and has the advantages of relatively low equipment cost, strong specificity, accurate detection result, good repeatability, high flux and the like. To realize the advantages of the technology, the selection of the time-resolved microspheres and the polymer fluorescent coding microspheres, the design of primers and probes and the optimization of a reaction system are all important.
In biological fluid or serum detection, many of the complexes and proteins themselves fluoresce, and the sensitivity of fluorescence detection using conventional chromophores is severely compromised. Most of the background fluorescence signal is present for a short time, the fluorescence lifetime is very short, the excitation light disappears, and the fluorescence also disappears. However, the fluorescence lifetime of rare earth metals (Eu, Tb, Sm and Dy) is very long and can reach 1-2 ms, and the measurement requirement can be met, so that a time-resolved fluorescence analysis method is generated. The time-resolved fluorescence technology is mainly used in clinic and scientific research at present, lanthanide is utilized to mark antigens or antibodies, fluorescence is measured by the time-resolved technology according to the characteristics of lanthanide luminescence, and two parameters of wavelength and time are detected simultaneously to carry out signal analysis, so that nonspecific fluorescence can be effectively eliminated, the sensitivity of analysis is greatly improved, background interference is reduced, and the repeatability is good. At present, the time-resolved fluorescence technology cannot detect multiple indexes at one time in a high flux manner, and batch processing of samples is realized.
The flow-type fluorescence technology is a high-throughput luminescence detection technology based on coding microspheres and flow-type technology, and is also called liquid chip, suspension array and
the platform comprises polymer fluorescent coding microspheres which are dyed by two fluorescent dyes with different spectral characteristics, a 100 fluorescent coding microsphere array can be theoretically obtained by accurately controlling the concentration ratio of the two fluorescent dyes (10 × 10), a reactant can be combined on the surface of each microsphere, the surface of each microsphere is provided with an activated carboxyl group which can be covalently crosslinked with amino of a protein antibody or amino of a nucleic acid probe, and the microspheres can be identified by the coded fluorescence, so that the detection of 100 reactions in a mixed reaction system can be realized simultaneouslyMicrospheres) are crosslinked with a reporter molecule for determining the amount of biological reaction on the surface of the microspheres. When in use, the coded microspheres of different detection objects are directly mixed, then a trace of sample to be detected is added, the target molecules in suspension and the molecules crosslinked on the surfaces of the microspheres are specifically combined, and dozens of or hundreds of different biological reactions can be simultaneously completed in one reaction hole without mutual interference. And finally, analyzing by a flow type fluorescence detection instrument, and analyzing the microspheres by two beams of laser one by one. One 635nm red laser beam excites the fluorescent material of the microsphere itself (polystyrene encoded microsphere), while the other 532nm green laser beam excites the reporter fluorescent material (phycoerythrin, Alexa 532 or Cy3, non-time resolved fluorescent microsphere, time resolved fluorescence is excited with 340nm or 365nm laser) bound to the microsphere surface. The high-speed laser signal processing reads the microsphere codes to classify the microsphere codes and quantify the reaction on the surfaces of the microsphere codes. The signal value of 100 microspheres is read for each detection object, and the median value is taken.
Although the current flow-type fluorescence detection has the characteristics of high flux, high speed, high precision and good accuracy, the signal influence factors are many, the interference of non-specific fluorescence is caused, the background value is high, the detection sensitivity is low, and the repeatability is not high.
Until now, no report has been found on the methods of detecting nucleic acids and mutations thereof using time-resolved fluorescence in complementary combination with flow cytometry. The time-resolved flow-type fluorescence nucleic acid detection technology can avoid the interference of non-specific fluorescence background, and can also treat samples in batches at high flux to detect a plurality of indexes at one time.
Disclosure of Invention
The main purpose of the present application is to provide a nucleic acid detection kit and an application thereof, which uses fluorescent microspheres capable of time resolution to replace bathochromic protein in flow fluorescence, and overcomes the disadvantages of flow fluorescence.
In order to achieve the above object, in a first aspect, the present application provides a nucleic acid detecting kit comprising: fluorescent coding microspheres, fluorescent microspheres capable of being resolved in time, anti-TAG sequences, extension product accounting gene sequences and a signal amplification system.
Preferably, the surface of the fluorescent microsphere capable of time resolution is connected with streptavidin or biotin, and the polymer fluorescent coding microsphere is connected with the anti-TAG sequence.
Preferably, the signal amplification system comprises biotin and streptavidin.
Preferably, the extension product nucleic acid gene sequence is obtained by: obtaining target nucleic acid gene fragment by multiple Polymerase Chain Reaction (PCR), enzyme cutting and hydrolyzing to remove redundant primer and deoxyribonucleotide triphosphate (dNTP), and extending with Allele Specific Primer Extension (ASPE) to form extension product nucleic acid gene sequence.
Preferably, the allele-specific primer is labeled with biotin during extension, and the primer contains a Tag sequence which can be complementary-paired with an anti-Tag sequence.
Preferably, the fluorescent material of the fluorescent microsphere capable of time resolution is a quantum dot fluorescent material or a liquid or solid microsphere of rare earth lanthanide or chelate thereof.
Preferably, the rare earth lanthanide is selected from at least one of europium (Eu), terbium (Tb), samarium (Sm).
Another aspect of the invention relates to the application of the nucleic acid detection kit for high-throughput time-resolved flow fluorescence analysis.
The technical scheme of the invention has the following advantages:
the kit provided by the invention is based on a related idea of complementary combination of time-resolved fluorescence and high-flux flow fluorescence, can effectively eliminate non-specific fluorescence, reduces background interference, greatly improves the sensitivity of analysis, is comparable to chemiluminescence, is simple to operate, and does not need dialysis in the production process. In addition, the kit has good repeatability and high reagent stability in the using process.
The detection method has simple steps, and various nucleic acids and mutation sites thereof can be amplified and detected in multiple ways, so that the detection efficiency is greatly improved.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this application, serve to provide a further understanding of the application and to enable other features, objects, and advantages of the application to be more apparent. The drawings and their description illustrate the embodiments of the invention and do not limit it. In the drawings:
FIG. 1 is a schematic diagram of the microstructure of components in a high throughput time-resolved flow fluorescence analysis nucleic acid detection kit according to an embodiment of the present disclosure.
Reference numerals
1-polymer fluorescent coding microspheres; 2-anti-TAG sequence;
3-extension product nucleic acid gene sequence (containing TAG sequence, complementary with anti-TAG);
4-biotin; 5-time-resolved fluorescent micro or quantum dot fluorescent microspheres; 6-streptavidin.
Detailed Description
In order to make the technical solutions better understood by those skilled in the art, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application, and it is obvious that the described embodiments are only partial embodiments of the present application, but not all embodiments. 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 application.
It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict. The present application will be described in detail below with reference to the embodiments with reference to the attached drawings.
In order to clearly understand the technical contents of the present invention, the following examples are given in detail. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
Example 1:
the nucleic acid detection kit of the embodiment mainly comprises:
1. ASPE primer
Specific primer sequences are respectively designed aiming at wild types and mutant types of different genes. The ASPE primer consists of a tag sequence and a specific primer sequence.
Each ASPE primer comprises two parts, the 5 'end is a specific tag sequence aiming at an anti-tag sequence on the corresponding microsphere, and the 3' end is a mutant type or wild type specific primer segment (as shown in the table 1). All ASPE primers can be synthesized by service Inc. Each primer after synthesis was formulated with 10mmol/LTris Buffer as a 100pmol/mL stock solution.
2. anti-tag sequence coated high-molecular fluorescent coding microsphere
According to the designed ASPE specific primer segment, tag sequences are selected, and secondary structures possibly formed between anti-tag sequences of the high-molecular fluorescent coding microspheres and between tags and the ASPE specific primer segment are reduced to the maximum extent. Selected 14 microspheres were purchased from Luminex, usa and were coated with the anti-tag sequence. 5-10T spacer arm sequences are connected between the anti-tag sequences and the microspheres, namely a 5-10T spacer arm sequence is added in front of each anti-tag sequence, and the anti-tag sequences are synthesized by service company Limited. The synthesized anti-tag sequence was made into 100nmol/ml stock using sterile ddH 2O. The spacer arm is a sequence for spacing the anti-tag from the surface of the microsphere or placing the anti-tag in a hydrophilic environment. By arranging a spacer arm sequence with proper length between the anti-tag sequence and the microsphere, the steric hindrance can be reduced, and the efficiency of hybridization reaction and the specificity of the hybridization reaction are improved.
The process of coating the polymer fluorescent coding microspheres is as follows:
respectively take 5 × 106Polymer fluorescent encoding microspheres with different numbers are suspended in 50ul 0.1mol/L MES solution (pH4.5), and 10ul of synthetic anti-tag molecules (100nmol/ml) are added. Preparing 10ng/ml of EDC (N- (3-methylenepropyl-N-ethylenecarboxyl) working solution, adding 2.5ul of EDC working solution into the microsphere suspension, incubating at constant temperature for 30 minutes, adding 2.5ul of EDC working solution, incubating at constant temperature for 30 minutes, washing with 0.02% Tween-20 after reaction, washing with 0.1% SDS solution again, and re-suspending the washed microspheres coated with the anti-tag sequence in 100ul of Tris-EDTA solution [10m mol/L Tris (pH8.0) ]]In 1mmol/L EDTA, storing at 2-8 deg.c in dark.
3. Primer for PCR amplification of target sequence containing mutation site
By the general methods
4. Site-specific primer extension reactions (ASPE)
The primer extension reaction was performed using the ASPE primer designed as described above, and biotin-labeled dCTP was incorporated during the reaction, thereby allowing the product after the reaction to carry a plurality of biotin labels. Firstly, preparing mixed ASPE primer working solution: respectively taking 10ul of wild type and mutant ASPE primer stock solution corresponding to the gene to be detected, adding 10mmol/L Tris Buffer to supplement to 200ul, and uniformly mixing to obtain the ASPE mixed primer working solution.
5. Hybridization reaction
According to the designed ASPE primers, selecting a plurality of corresponding coated high-molecular fluorescent coding microspheres in each group, wherein the concentration of each microsphere is 2.5 ×/ml, respectively taking 1ul of microspheres with each number in a 1.5ml microcentrifuge tube, centrifuging the microspheres for 1-2min at a concentration of more than or equal to 10000g, discarding supernatant, suspending the microspheres in 100ul of 2 × Tm hybridization buffer solution, uniformly mixing by vortex, taking 25ul of the microsphere suspension in a corresponding hole of a 96-hole filter plate, adding 25ul of ddH2O in a control hole, taking 5-25ul of ASPE reaction solution in a corresponding hole, supplementing ddH2O to 50ul, wrapping a 96-hole plate with tin foil paper to avoid light, incubating and hybridizing at 95 ℃ for 15min, incubating and hybridizing the hybridized microspheres at 37 ℃ for 2-5min at a concentration of more than or equal to 3000g, removing supernatant, incubating the microspheres in 75ul of 1 Tm buffer solution, incubating in 3000g of 2-5min, resuspending the microspheres in 3000g of 2-5min, adding the supernatant, resuspending in a streptavidin-15-affinity hybridization buffer solution, and detecting the hybridization concentration of the streptavidin-15 ml of the hybridization buffer solution in a streptavidin affinity detector for 10 ml.
Example 2
A high-molecular fluorescent coded microsphere (anti-TAG) with diameter of 3-5.6 microns is prepared through coupling the anti-TAG sequence, cross-linking the europium (Eu) or samarium (Sm) time-resolved fluorescent microsphere with streptavidin (80-300 nm), amplifying by use of amplification primer to obtain amplified product, enzyme digestion of amplified product by SAP enzyme and Exo-I enzyme, primer extension reaction of amplified product after enzyme digestion, the dCTP marked by biotin is doped in the reaction process, so that the extension product nucleic acid gene sequence after reaction is provided with a plurality of biotin marks, the extension product nucleic acid gene sequence and the corresponding anti-tag sequence coated polymer fluorescent coding microspheres with different colors are subjected to hybridization reaction, then adding streptavidin-time resolution fluorescent microspheres (or quantum dot fluorescent microspheres) for incubation, and detecting on a time resolution flow type fluorescence instrument. During detection, reacted microsphere mixed liquid and sheath liquid are conveyed to a flow chamber together and sequentially pass through a capillary detection area of the flow chamber, a first laser is excited, a fluorescence detector of the first laser collects fluorescence signals, a second laser is excited at the same time, the excitation wavelength is 340nm or 365nm, the delay time is adjusted to be 5 nanoseconds to 8000 milliseconds, a time-resolved fluorescence detection unit of the second laser is started to record and collect fluorescence signals, and the collected signals are analyzed and processed by a data collection system.
Example 3
Specific primer sequences are respectively designed aiming at wild types and mutant types of multiple gene types of nucleic acid gene mutation. The extension primer consists of a Tag sequence and a specific primer sequence. Each extension primer comprises two parts, the 5' end is a specific tag sequence corresponding to the anti-tag sequence on the polymer fluorescent coding microsphere, and the anti-tag sequence is coated on the polymer fluorescent coding microsphere. 5-10T spacer arm sequences are connected between the anti-tag sequence and the polymer fluorescent coding microspheres, a designed extension primer carries out primer extension reaction, dCTP marked by biotin is doped in the reaction process, so that a product after the reaction is marked with a plurality of biotin, a plurality of biotin marks are carried on a nucleic acid gene sequence of the extension product after the reaction, the nucleic acid gene sequence of the extension product and the polymer fluorescent coding microspheres coated by the corresponding anti-tag sequence and having different colors carry out hybridization reaction, then streptavidin-time resolution fluorescent microspheres (or quantum dot fluorescent microspheres) are added for incubation, and the detection is carried out on a time resolution flow type fluorescent instrument.
The description of the preferred embodiments is merely exemplary in nature and is in no way intended to limit the disclosure, which may be modified or varied by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.
Claims (9)
1. A nucleic acid detection kit, comprising: the fluorescent microsphere capable of being resolved in time is selected from at least one of the fluorescent microsphere capable of being resolved in time and the fluorescent microsphere with quantum dots.
2. The nucleic acid detection kit of claim 1, wherein the surface of the fluorescent microsphere capable of time resolution is linked with streptavidin or biotin, and the polymeric fluorescent-encoded microsphere is linked with the anti-TAG sequence.
3. The nucleic acid detection kit of claim 1, wherein the signal amplification system comprises biotin and streptavidin.
4. The nucleic acid detection kit of claim 1, wherein the extension primer sequence is an ASPE primer comprising a TAG sequence, and the TAG sequence is complementary to the anti-TAG sequence.
5. The nucleic acid detection kit of claim 1, wherein the fluorescent material of the fluorescent microsphere capable of time resolution is a quantum dot fluorescent microsphere or a liquid or solid microsphere of rare earth lanthanide or its chelate.
6. The nucleic acid detection kit of claim 5, wherein the rare earth lanthanide is selected from at least one of europium, terbium and samarium.
7. The high throughput time-resolved flow fluorescence analysis nucleic acid detection kit of any of claims 1-6, which is used in its entirety with the process steps of:
1) amplifying by using an amplification primer to obtain an amplification product;
2) carrying out enzyme digestion treatment on the amplification product by using SAP enzyme and Exo-I enzyme;
3) carrying out primer extension reaction on the amplified product after enzyme digestion through an extension primer;
4) and (3) carrying out hybridization reaction on the extension product nucleic acid gene sequence and the corresponding polymer fluorescent coding microspheres which are coated by the anti-tag sequence and have different colors, adding the streptavidin or biotin-connected fluorescent microspheres which can be subjected to time resolution for incubation, and detecting on a time-resolution flow type fluorescence instrument.
8. The method of using the nucleic acid detection kit according to claim 7, wherein the primer extension is carried out using a biotin label.
9. Use of the high throughput time-resolved flow fluorescence analysis nucleic acid detection kit according to any one of claims 1-6.
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