CN110577984A - Multiple visual nucleic acid detection method - Google Patents
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Abstract
The invention belongs to the field of gene detection, and discloses a multiple visual nucleic acid detection method, which comprises the following steps: 1) designing corresponding padlock probe sequences and universal primer sequences aiming at different target nucleic acid molecules respectively, wherein the padlock probe sequences contain gene specific sequences and universal primer recognition sequences; 2) adding a locking probe into a solution to be detected, simultaneously adding ligase and a reaction buffer solution, and carrying out a linking reaction in the same reaction system; 3) distributing the chained reaction products into different reaction tubes, wherein the reaction tubes respectively contain universal primers matched with the universal primer identification sequences on the padlock probes, and performing hyperbranched rolling circle amplification reaction in the reaction tubes to obtain amplification products; 4) visual reaction: adding micro-nano particles, and observing a detection result according to whether agglomeration occurs. The method has the advantages of low detection cost, simple operation steps and high sensitivity, and can realize high-throughput detection of various nucleic acids.
Description
Technical Field
The invention belongs to the technical field of gene detection, and particularly relates to a multiple nucleic acid detection technology based on universal primer hyperbranched rolling circle amplification.
background
The great advantages of PCR and fluorescent quantitative PCR technologies in the aspects of sensitivity, specificity, detection speed and the like make the PCR and fluorescent quantitative PCR technologies more and more widely applied to nucleic acid target molecule detection. However, the demands on hardware equipment and personnel are high in the implementation process of the nucleic acid detection technology of PCR and multiplex PCR. Moreover, the detection cost is limited by a detection platform, high-throughput detection is difficult to realize, and most products can only realize the detection of a single or limited target sequence at present. Although the multiplex PCR technology or the multiplex fluorescence PCR detection technology can realize the detection of multiple target molecules, due to the difficulties of different compatibility of a plurality of primers to polymerase, mutual competition of the primers and the like, once the experimental conditions are controlled improperly, the sensitivity and specificity of the primers can be reduced, and even the amplification failure or the generation of non-specific products can be caused. In order to accomplish multiple target molecule detection, such methods require the provision of expensive multiple fluorescence detection equipment. And the whole detection process requires operators to have certain molecular biological skills and experiences. The above limitations largely limit the application of such techniques to practical inspection work.
in order to simplify the precise temperature cycling process, a Nucleic Acid Isothermal Amplification (NAIA) technology is developed, and only a simple temperature control device is needed to realize rapid Amplification of a target molecule. This feature greatly simplifies the requirements of the apparatus required for amplification and greatly shortens the reaction time. Currently, the major Isothermal Amplification technologies include Loop-Mediated Isothermal Amplification (LAMP), Rolling Circle Amplification (RCA), external Cross Primer Amplification (CPA), transcriptase Amplification (TMA), Strand Displacement Amplification (SDA), and ligase Dependent Amplification (HDA). The isothermal amplification technology can greatly reduce the requirements of nucleic acid detection on hardware by combining with a visual detection technology, thereby having great application value. For example, a group of professors of Landers, university of virginia, usa, published a non-labeled nucleic acid detection technique using a rotating magnetic field to cause magnetic bead aggregation, and applied to non-specific nucleic acid analysis (for example, total DNA, genomic DNA content, etc.), but the inability to perform specific nucleic acid target molecule detection makes this technique unable to meet the requirements of most genetic analyses, and has no wide applicability.
Through search, related applications have been disclosed in the prior art, for example, chinese patent CN102827929B describes a nucleic acid detection method based on rolling circle amplification and magnetic bead aggregation, in which an oligonucleotide chain capable of specifically connecting and cyclizing is used as a probe to hybridize with a specific target gene to form a cyclized single-stranded probe under the action of DNA ligase, and the single-stranded probe obtains a long-chain product by the rolling circle amplification method, and visual detection is achieved by aggregation with magnetic particles. However, the scheme cannot realize parallel detection of multiple target molecules, and the rolling circle amplification time is too long (>60 minutes), so that the scheme cannot meet various rapid and on-site nucleic acid detection applications.
although the isothermal amplification technology has a good application prospect, the current isothermal amplification technology generally lacks high-throughput detection capability and can only detect a single target molecule or a very limited number of target molecules, and meanwhile, some methods also have the problems of complex primer design, high background of amplified products, high false positive result rate and the like, and in addition, the application of the isothermal amplification technology in individualized nucleic acid detection is limited. The development of a nucleic acid information acquisition method which has high throughput, high sensitivity, accuracy and simplicity and can be suitable for various medical service platforms is still a problem to be explored and solved urgently.
Disclosure of Invention
1. problems to be solved
aiming at the problems of high detection cost, high requirement on detection conditions and low sensitivity in nucleic acid detection in the prior art, the invention aims to provide a multiple nucleic acid visual detection method based on universal primer hyperbranched rolling circle amplification and micro-nano particle agglomeration effect.
2. Technical scheme
in order to solve the problems, the technical scheme adopted by the invention is as follows:
The multiplex visual nucleic acid detection method comprises the following steps:
1) designing corresponding padlock probe sequences and universal primer sequences aiming at different target nucleic acid molecules respectively, wherein the padlock probe sequences contain gene specific sequences and universal primer recognition sequences; the gene specific sequence can be matched with a target nucleic acid molecule sequence, and the universal primer recognition sequence can be matched with a universal primer sequence;
2) Multiplex padlock probe ligation reaction: adding the padlock probe sequence into a nucleic acid solution to be detected, simultaneously adding ligase and reaction buffer solution, and carrying out a linking reaction in the same reaction system to obtain a linking reaction product;
3) Hyperbranched rolling circle amplification reaction: distributing the chained reaction products obtained in the step 2) into different reaction tubes, wherein the reaction tubes respectively contain a universal primer sequence matched with the universal primer identification sequence, and performing hyperbranched rolling circle amplification reaction in the reaction tubes to obtain amplification products;
4) Visual reaction: adding the micro-nano particles into the solution after the reaction in the step 3), and observing a detection result according to whether the agglomeration occurs.
in the step 2), the gene specific sequence of the padlock probe is complementary with the corresponding target nucleic acid molecule sequence, and if the gene sequence of the target nucleic acid molecule exists in the reaction system, the corresponding padlock probe forms a single-chain annular probe under the action of ligase; in the step, a nucleic acid chain-linked reaction system (ligase, reaction buffer solution and the like) is utilized to realize the reaction of various target nucleic acid molecules and corresponding lock probes, the reactions are not interfered with each other, and compared with the single-linked reaction aiming at the target nucleic acid molecules in the prior art, the method greatly shortens the time and reduces the cost.
In the step 3), specific universal primers are paired with the universal primer identification sequences in the padlock probe sequences, and the products of the chain reaction are amplified.
as a further improvement of the present invention, the target nucleic acid molecule comprises a DNA molecule or an RNA molecule. If the detected sample is RNA molecule, the lock probe and cDNA reverse transcribed by RNA carry out linking reaction; if the sample is a DNA molecule, a ligation reaction can be carried out after thermal denaturation to obtain a ligation reaction product.
The universal primer sequence comprises a universal primer sequence 1 and a universal primer sequence 2, and the universal primer identification sequence comprises a universal primer identification sequence 1 and a universal primer identification sequence 2. The dual-use primer sequence is designed to further improve the sensitivity and the accuracy of detection.
As a further reaction of the present invention, the ligase in the step 2) includes Taq DNA ligase or Hifi Taq DNA ligase.
Using the thermal stability of Taq DNA ligase or Hifi Taq DNA ligase, double stranded DNA can be melted by thermal denaturation, and then the padlock probe is annealed for the ligation reaction.
As a further improvement of the invention, the micro-nano particles comprise magnetic particles or non-magnetic particles. The agglomeration reaction of the amplification product with the magnetic particles may be formed by magnetic field perturbation or by pipetting using a pipette.
as a further improvement of the invention, the hyperbranched rolling circle amplification reaction system in the step 3) is as follows: 10 XBuffer 4 uL, 10U Phi29 DNA polymerase, 1 uL of 250nM universal primer, 0.8 uL of BSA 10mg/mL, 1.6 uL of 10 uMdNTP, 2 uL of the ligation product, and adding nuclease-free water to make up to 40 uL.
As a further improvement of the invention, the reaction conditions of the hyperbranched rolling circle amplification in the step 3) are as follows: the reaction is carried out for 45min at 30 ℃ and for 5min at 65 ℃.
As a further improvement of the invention, the multiplex padlock probe linking reaction system in the step 2) is as follows: 10 × HiFi Taq DNA Ligase Buffer 2 μ L, Taq DNA Ligase 1 μ L, 10 μ M padlock probes 2 μ L, 10 μ M target molecules 2 μ L each, add ddH2o was supplemented to 14. mu.L.
as a further improvement of the invention, the multiple padlock probe linking reaction conditions in the step 2) are as follows: the reaction was carried out at 37 ℃ for 30 minutes.
As a further improvement of the present invention, said step 4) is specifically operated as: adding magnetic beads into the solution reacted in the step 3), uniformly mixing the magnetic beads with the solution, standing, gathering the magnetic beads by using a magnet, slightly blowing the gathered magnetic beads, repeating the gathering and blowing operation for 2-3 times, and observing whether a molecular aggregation phenomenon is generated at the bottom of the reaction tube.
as a further improvement of the invention, the magnetic beads are added at a concentration of 10mg/mL and in a volume of 4. mu.L.
3. Advantageous effects
Compared with the prior art, the invention has the beneficial effects that:
(1) the multiple visualization nucleic acid detection method of the invention aims at different target nucleic acid molecules, multiple connection reactions are carried out in the same reaction system, each target nucleic acid molecule and a target sequence on the corresponding padlock probe are subjected to complementary pairing to carry out a linking reaction, multiple reactions in the same system do not influence each other, realize a large number of linking reactions, subpackage the linking reactions after finishing to an amplification system containing a universal primer matched with a padlock probe sequence, perform hyperbranched rolling circle amplification reaction by taking a linking reaction product as a template, the amplification product is detected by independently adding the micro-nano particles, high-flux detection of various target nucleic acid molecules can be realized on the premise of not using any fluorescence equipment, the accuracy is high, the operation steps are simple, the operation cost is greatly reduced, and the detection time is shortened. Although the multiplex PCR technology or the multiplex fluorescence PCR detection technology in the prior art can realize the detection of a plurality of target molecules, the difficulties of different compatibility of a plurality of primers to polymerase, mutual competition of the primers and the like exist, such as improper control of experimental conditions, the sensitivity and specificity of the primers can be reduced, even amplification failure or generation of non-specific products is caused, and the requirement on the experimental conditions is high.
(2) The multiple visual nucleic acid detection method is a universal detection method, and can be used for not only detecting DNA molecules but also detecting RNA molecules. If the detected sample is RNA molecule, the lock probe and cDNA reverse transcribed by RNA carry out linking reaction; if the sample is a DNA molecule, a ligation reaction can be carried out after thermal denaturation to obtain a ligation reaction product. The invention adopts the thermal stability of Taq DNA ligase or Hifi Taq DNA ligase, can melt double-stranded DNA by thermal denaturation, and then anneals a padlock probe to carry out ligation reaction.
(3) According to the multiple visual nucleic acid detection method, two different universal primer sequences are designed on the padlock probes, and in the process of carrying out the hyperbranched rolling circle amplification reaction, the padlock probes designed aiming at target nucleic acid molecules are matched and combined with the two universal primer sequences to start the amplification reaction, so that detection errors are avoided, and the sensitivity and the accuracy of detection are further improved.
(4) according to the multiple visual nucleic acid detection method, the aggregation effect of the micro-nano particles and the long DNA chains after winding is utilized to form the irreversible nanoparticle clusters which can be seen under naked eyes, and the detection result is obtained through the observation of the naked eyes, so that the detection process can be finished without expensive amplification and signal detection equipment, and the multiple visual nucleic acid detection method can be widely applied to laggard medical institutions and basic quarantine departments. In the nucleic acid detection method based on PCR or isothermal amplification technology in the prior art, signal detection after amplification is usually obtained by means of bulky and expensive fluorescence detection equipment, so that the wide application of the nucleic acid detection method is limited.
Drawings
FIG. 1 is a flow chart of multiplex visualization of nucleic acid detection;
FIG. 2 is a schematic of a hyperbranched rolling circle amplification reaction;
FIG. 3 shows the results of nucleic acid detection in example 1.
Detailed Description
The invention is further described with reference to specific examples.
example 1
The multiplex visualization nucleic acid detection method of the embodiment specifically comprises the following steps:
1) corresponding padlock probe sequences and universal amplification primer sequences are respectively designed for each target nucleic acid molecule, the present example relates to the detection of 8 target molecules, padlock probe sequences and universal amplification primer sequences of target molecules 1-8 (marked as #1, #2, #3, #4, #5, #6, #7, #8) are respectively shown as follows, and the universal amplification primer sequences respectively include two sequences, marked as sequence-1 and sequence-2:
Target molecule #1 padlock probe sequence: 5' -PO3-CTTACTGCTTGGTTATCTCG
GCTGAGAGCATGCCGTCTCTGGCTGAACACATGCCGTGCGTCCTGCAG TTC TTA CGCTACTGTTCCAGTTCCATTAC-3’；
Target molecule #1 amplification universal primer sequence: sequence-1: 5'-CGACTCTCGTACGGCAGAG-3', respectively;
Sequence-2: 5'-GCAGGACGTCAAGAATGCG-3', respectively;
Target molecule #2 padlock probe sequence: 5' -PO3-AAACAGTAGGATATTGCGAATAGGCACG
GCCGAGCTCCATCTCGTGTTCCAACACGCAATAATCGCGCGGACATCGGCTCCGTATATCGTCGCGTCTAT-3’
target molecule #2 amplification universal primer sequence: sequence-1: 5'-ATCCGTGCCGGCTCGAGGT-3', respectively;
Sequence-2: 5'-ATTAGCGCGCCTGTAGCCGA-3', respectively;
Target molecule #3 padlock probe sequence: 5' -PO3-TTATTTCAACCTCGATACCGTCAGGCGCAACGGAGGCTAGCATTGTCAGACGGCATCCTTGCGTAGCGGTAGGAGGATAGGCAATAGTTCTGGATG-3’；
Target molecule #3 amplification universal primer sequence: sequence-1: 5'-AGTCCGCGTTGCCTCCGAT-3', respectively;
Sequence-2: 5'-GAACGCATCGCCATCCTCC-3', respectively;
Target molecule #4 padlock probe sequence: 5' -PO3-GTAATGACTATCGGTAGCTGTATCGAGCCGCACGTAAGGCGGTGGCAATCCAACGGAGTGACACTCACCGCGTGAAGAAGTTCAACGGGAAAGCTTT-3’；
target molecule #4 amplification universal primer sequence: sequence-1: 5'-CATAGCTCGGCGTGCATTC-3', respectively;
Sequence-2: 5'-ACTGTGAGTGGCGCACTTC-3', respectively;
Target molecule #5 padlock probe sequence: 5' -PO3-AAAGGAAAAGAACCCTCAGC
TCGGCAGTGTGAAGCGTAGCTTACTCAACCGCCGGTAGCTTGCGCCATGAG
CAGTCCAACCCTCAGCAGTAATGCCC-3’；
Target molecule #5 amplification universal primer sequence: sequence-1: 5'-AGCCGTCACACTTCGCATC-3', respectively;
Sequence-2: 5'-GAACGCGGTACTCGTCAGG-3', respectively;
Target molecule #6 padlock probe sequence: 5' -PO3-CATTTTGCCGCTTGTTTCTTCGCTTGCCACGTCTTAGATGACCGAATTCACCTCTACCGTCTGCGCTACCAAGCCTGACGAGTTTCTGCATTCCGT-3’；
Target molecule #6 amplification universal primer sequence: sequence-1: 5'-GCGAACGGTGCAGAATCTA-3', respectively;
Sequence-2: 5'-CAG ACG CGATGGTTCG GAC-3', respectively;
Target molecule #7 padlock probe sequence: 5' -PO3-TGGTGATCGCGTCCTGTATATCGA
ACATGGACAGGAGCGTGTGCCCATGCGCTGCGACCTCGACCACGACGCAGCCTTCTGTCTAGACCTCGATGCAGG-3’；
Target molecule #7 amplification universal primer sequence: sequence-1: 5'-AGCTTGTACCTGTCCTCGCA-3', respectively;
Sequence-2: 5'-GCT GGT GCTG CGT CGG AAG-3', respectively;
Target molecule #8 padlock probe sequence: 5' -PO3-GCACAGGATGTAGCCGTATAACTAAGCGGCGCACGTGGAGTGCCCATGCGCCAGCATCCACTATGCCGCCCACGAGGTCGGTCTAGACTCGAGCCAA-3’；
Target molecule #8 amplification universal primer sequence: sequence-1: 5'-TGATTCGCCGCGTGCACCT-3', respectively;
Sequence-2: 5'-GTGATACGGCGGGTGCTCC-3', respectively;
2) multiple padlock probe ligation reaction: and mixing the padlock probes, the target nucleic acid molecules, the ligase and the reaction buffer solution, and carrying out respective linking reaction to obtain a linking product.
In this step, the reaction system has the following formula: 20 μ L ligation: 10 × HiFi Taq DNA Ligase Buffer 2 μ L, Taq DNA Ligase 1 μ L, 10 μ M padlock probe 2 μ L, 10 μ M target molecules #4, #5, #6, #7 each 2 μ L, and ddH added last2O was supplemented to 14. mu.L. (target molecules #1, #2, #3, and #8 were not added to the system).
Reaction conditions are as follows: the reaction was carried out at 37 ℃ for 30 minutes.
3) And (3) hyperbranched rolling circle amplification: the reaction is schematically shown in figure 2, and the reaction product can be rapidly and accurately amplified by utilizing the characteristics of simplicity, rapidness, high sensitivity and good specificity of hyperbranched rolling circle amplification.
The amplification system and the amplification reaction conditions were as follows: mu.L of 10 XBuffer 4. mu.L, 10U of Phi29 DNA polymerase, 1. mu.L of 250nM universal primer, 0.8. mu.L of BSA at 10mg/mL, 1.6. mu.L of dNTP at 10. mu.M, and 2. mu.L of the ligation reaction product were added to 40. mu.L of the amplification system, and nuclease-free water was added to make up to 40. mu.L.
reaction conditions are as follows: reacting for 45 minutes at 30 ℃ and reacting for 5 minutes at 65 ℃ to obtain the hyperbranched rolling circle amplification reaction product.
The whole reaction process is shown in fig. 1, and if a certain target nucleic acid molecule exists in the ligation reaction system of step 2), the target nucleic acid molecule can sequentially undergo the ligation reaction of step 2) and the amplification reaction of step 3) to form a hyperbranched rolling circle amplification reaction product. If the target nucleic acid molecule does not exist, the linking and amplification reaction can not be carried out, and finally the hyperbranched rolling circle amplification reaction product can not be formed.
4) Visual reaction: adding 4 mu L of 10mg/mL magnetic beads (Dynabeads M280) into 40 mu L of the solution reacted in the step 3), uniformly mixing the magnetic beads with the amplification products in the reaction solution by using a liquid transfer machine, standing at room temperature for 2min, gathering the magnetic beads by using a magnet, lightly blowing the gathered magnetic beads by using the liquid transfer machine, repeatedly gathering and blowing for 2-3 times, observing the aggregation of the amplification products and the magnetic beads at the bottom of a centrifugal tube, generating irreversible winding, and oscillating the magnetic beads again to prevent the magnetic beads from being redispersed in the system.
FIG. 3 shows the detection results, and it can be seen from FIG. 3 that the #4, #5, #6, #7 seed particles are in an agglomerated state, indicating that the corresponding target molecules can be detected, while the #1, #2, #3, #8 particles are in a discrete state and no target molecules are detected.
while embodiments of the invention have been described above, it is not limited to the applications set forth in the description and the embodiments, which are fully applicable in various fields of endeavor to which the invention pertains, and further modifications may readily be made by those skilled in the art, it being understood that the invention is not limited to the details shown and described herein without departing from the general concept defined by the appended claims and their equivalents.
Claims (10)
1. A multiplex visualization nucleic acid detection method, characterized in that: the method comprises the following steps:
1) designing corresponding padlock probe sequences and universal primer sequences aiming at different target nucleic acid molecules respectively, wherein the padlock probe sequences contain gene specific sequences and universal primer recognition sequences;
2) multiplex padlock probe ligation reaction: adding the padlock probe sequence into a nucleic acid solution to be detected, simultaneously adding ligase and reaction buffer solution, and carrying out a linking reaction in the same reaction system to obtain a linking reaction product;
3) Hyperbranched rolling circle amplification reaction: distributing the linked reaction products obtained in the step 2) into different reaction tubes, wherein the reaction tubes respectively contain corresponding universal primer sequences, and performing hyperbranched rolling circle amplification reaction in the reaction tubes to obtain amplification products;
4) Visual reaction: adding the micro-nano particles into the solution after the reaction in the step 3), and observing a detection result according to whether the agglomeration occurs.
2. The multiplex visualization nucleic acid detection method according to claim 1, characterized in that: the target nucleic acid molecule includes a DNA molecule or an RNA molecule.
3. the multiplex visualization nucleic acid detection method according to claim 1 or 2, characterized in that: the universal primer sequence comprises a universal primer sequence 1 and a universal primer sequence 2, and the universal primer identification sequence comprises a universal primer identification sequence 1 and a universal primer identification sequence 2.
4. the multiplex visualization nucleic acid detection method according to claim 1 or 2, characterized in that: the ligase in the step 2) comprises Taq DNA ligase or Hifi Taq DNA ligase.
5. The multiplex visualization nucleic acid detection method according to claim 4, characterized in that: the micro-nano particles comprise magnetic particles or non-magnetic particles.
6. The multiplex visualization nucleic acid detection method according to claim 5, characterized in that: the hyperbranched rolling circle amplification reaction system in the step 3) is as follows: 10 XBuffer 4 uL, 10U Phi29 DNA polymerase, 1 uL of 250nM universal primer, 0.8 uL of BSA 10mg/mL, 1.6 uL of 10 uM dNTP, 2 uL of the ligation product, and adding nuclease-free water to make up to 40 uL.
7. the multiplex visualization nucleic acid detection method according to claim 6, characterized in that: the reaction conditions of the hyperbranched rolling circle amplification in the step 3) are as follows: the reaction is carried out for 45min at 30 ℃ and for 5min at 65 ℃.
8. the multiplex visualization nucleic acid detection method according to claim 7, characterized in that: the multiple padlock probe linking reaction system in the step 2) is as follows: 10 × HiFi Taq DNA Ligase Buffer 2 μ L, Taq DNA Ligase 1 μ L, 10 μ M padlock probes 2 μ L, 10 μ M target nucleic acid molecules 2 μ L each, add ddH2o was supplemented to 14. mu.L.
9. The multiplex visualization nucleic acid detection method according to claim 8, characterized in that: the reaction conditions of the multiple padlock probe chaining in the step 2) are as follows: the reaction was carried out at 37 ℃ for 30 minutes.
10. The multiplex visual nucleic acid detection method according to claim 9, wherein: the step 4) is specifically operated as follows: adding magnetic beads into the solution reacted in the step 3), uniformly mixing the magnetic beads with the solution, standing, gathering the magnetic beads by using a magnet, slightly blowing the gathered magnetic beads, repeatedly gathering and blowing, and observing whether a molecular agglomeration phenomenon is generated at the bottom of the reaction tube.
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| WO2021031226A1 (en) | 2021-02-25 |
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