CN110004214B - Method for detecting microRNA-21 by double DNA machine - Google Patents
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Abstract
The invention discloses a method for detecting microRNA-21 by a double DNA machine. The method comprises the following steps: in the presence of microRNA-21, connecting the padlock probes subjected to 5' end phosphorylation by adopting T4 ligase to form a circular DNA template; and the circular DNA machine is initiated to operate under the simultaneous action of DNA polymerase and restriction endonuclease to obtain a single-stranded product; hybridizing the single-stranded product with a palindromic hairpin probe to obtain a hybrid double strand, and realizing cyclic strand displacement amplification of a bidirectional DNA machine to obtain an amplification product; and detecting the fluorescence signal of the amplified product in the reaction system to obtain the concentration of the microRNA-21. The invention has high miRNA-21 detection sensitivity, the detection limit is 10fM, and the invention has good specificity and is a universal nucleic acid detection platform.
Description
Technical Field
The invention belongs to the field of life health, relates to a detection method of a tumor marker microRNA-21, and particularly relates to a fluorescence detection method for detecting the microRNA-21 by a dual DNA machine based on the combination of an RCA (circular deoxyribonucleic acid) circular DNA machine and a cyclic strand displacement amplification bidirectional DNA machine.
Background
microRNA is a short and small endogenous non-coding RNA which plays an important role in organisms and is closely related to various biological processes such as gene regulation, cell division, apoptosis and the like. Research shows that many diseases occur along with the change of the expression type or content of microRNA, and the change is particularly obvious in cancer. Therefore, microRNA is considered as a novel marker for cancer detection, and the establishment of a sensitive, reliable, effective and accurate microRNA detection method has extremely important research and practical significance in various fields such as early cancer diagnosis, treatment, cancer mechanism research and the like. The conventional detection methods such as reverse transcriptase-polymerase chain reaction (RT-PCR), fluorescent quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR), molecular imprinting, microarray analysis and other technologies at present are difficult to meet the clinical detection requirements of ultra-micro microRNA due to high cost, complex operation, complex process, low sensitivity, poor specificity and the like. Therefore, it is necessary to develop a new method and a new technology for detecting microRNA with excellent properties such as simple operation, low cost, good detection performance and the like.
Disclosure of Invention
In order to solve the problems of operational complexity, low detection limit sensitivity and the like of microRNA detection, the invention establishes a fluorescent detection method for detecting microRNA-21 by a double DNA machine combining a circular DNA machine based on Rolling Circle Amplification (RCA) and a bidirectional DNA machine based on cycle strand displacement amplification.
In order to achieve the purpose, the technical scheme adopted by the invention comprises the following steps:
the embodiment of the invention provides a method for detecting microRNA-21 by a double DNA machine, which comprises the following steps:
in the presence of microRNA-21, connecting the padlock probes subjected to 5' end phosphorylation by adopting T4 ligase to form a circular DNA template; and the circular DNA machine is triggered to run under the simultaneous action of DNA polymerase and restriction endonuclease to obtain single-stranded product;
hybridizing the single-stranded product with the palindromic hairpin probe to obtain a hybridized double strand, performing intermolecular complementary pairing on the 3' end of the palindromic hairpin probe in the hybridized double strand, and realizing the cyclic strand displacement amplification of a bidirectional DNA machine under the action of DNA polymerase and restriction endonuclease to obtain an amplified product;
and detecting the fluorescence signal of the amplified product in the reaction system to obtain the concentration of the microRNA-21.
In some embodiments, the method specifically comprises:
(1) Uniformly mixing a T4 ligase buffer solution, a 5' -end phosphorylation padlock probe, microRNA-21 and deionized water, heating the obtained mixture at 90-95 ℃ for 2-5 min, cooling to room temperature, adding 5-10U of T4 ligase, and reacting the obtained first reaction system at 16-25 ℃ for 30-60 min to form a circular DNA template;
(2) Incubating and reacting a second reaction system comprising the first reaction system obtained in the step (1), 1-2 mu L of DNA polymerase buffer solution, 1-2 mu L of palindromic hairpin probe, 1-2 mu L of dNTPs, 3-5 mu L of DNA polymerase and 5-10U of restriction endonuclease for 1-2.5 h at the temperature of 30-37 ℃ to obtain an amplification product;
(3) According to the steps (1) and (2), carrying out quantitative detection on a series of microRNA-21 standard solutions with different concentrations, and determining the fluorescence intensity value of the amplified product in the excitation light wave band to obtain a microRNA-21 concentration-fluorescence intensity value standard curve;
(4) And (3) detecting the biological sample to be detected containing the microRNA-21 according to the steps (1) and (2), determining the fluorescence intensity value of the amplification product in the excitation light wave band, and comparing the fluorescence intensity value with the standard curve, thereby measuring the concentration of the microRNA-21 in the biological sample to be detected.
The embodiment of the invention also provides a product, which is applied to the method for detecting the microRNA-21 by the double DNA machine, wherein the product comprises a padlock probe and a palindromic hairpin probe, the sequence of the padlock probe is shown as SEQ ID NO. 1, and the sequence of the palindromic hairpin probe is shown as SEQ ID NO. 2.
Compared with the prior art, the invention has the beneficial effects that:
1) Compared with the existing fluorescence detection method, the method for detecting microRNA-21 by using the double DNA machine provided by the invention has the advantages that the miRNA-21 detection sensitivity is very high, the detection limit is 10fM, and the linear range is wide (10 fM-50 nM);
2) The method for detecting microRNA-21 by using the double DNA machine has the advantages that the background signal is low, the target signal is strong, the obtained signal-to-noise ratio is high (S/N = 24), and the method is far beyond the existing fluorescence detection method;
3) The method for detecting the microRNA-21 by the double DNA machine has good specificity, and can easily distinguish a completely complementary target RNA and a non-target RNA with mismatched bases;
4) The invention can be used for direct detection of human serum samples, can be used for sensitive detection of other DNA or RNA through simple design, and is a universal nucleic acid detection platform.
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FIG. 1 is a standard curve diagram obtained by detecting a microRNA-21 standard solution in example 1 of the present invention.
Detailed Description
As described above, in view of the problems of complexity in operation and insufficient sensitivity of detection limit in microRNA detection in the prior art, the present inventors have made extensive studies and practice to propose a technical solution of the present invention, which is mainly a fluorescence detection method for detecting microRNA-21 by using a dual DNA machine combining a circular DNA machine based on RCA and a bidirectional DNA machine based on cyclic strand displacement amplification, wherein the fluorescence detection method comprises the following operation steps: (1) ligation of circular DNA templates; (2) fluorescence signal amplification based on a dual DNA machine; (3) Detecting the microRNA-21 standard solution, and establishing a standard curve; (4) And (3) carrying out quantitative detection on the actual sample containing the microRNA-21.
One aspect of the embodiments of the present invention provides a method for detecting microRNA-21 by a dual DNA machine, which includes:
in the presence of microRNA-21, connecting the padlock probes subjected to 5' end phosphorylation by adopting T4 ligase to form a circular DNA template; and the circular DNA machine is triggered to run under the simultaneous action of DNA polymerase and restriction endonuclease to obtain single-stranded product;
hybridizing the single-chain product with the palindromic hairpin probe to obtain a hybrid double chain, performing intermolecular complementary pairing on the 3' end of the palindromic hairpin probe in the hybrid double chain, and realizing cyclic strand displacement amplification of a bidirectional DNA machine under the action of DNA polymerase and restriction endonuclease to obtain an amplification product;
and detecting the fluorescent signal of the amplified product in the reaction system to obtain the concentration of the microRNA-21.
(1) Uniformly mixing a T4 ligase buffer solution, a 5' -end phosphorylation padlock probe, microRNA-21 and deionized water, heating the obtained mixture at 90-95 ℃ for 2-5 min, cooling to room temperature, adding 5-10U of T4 ligase, and reacting the obtained first reaction system at 16-25 ℃ for 30-60 min to form a circular DNA template;
(2) Incubating and reacting a second reaction system comprising the first reaction system obtained in the step (1), 1-2 mu L of DNA polymerase buffer solution, 1-2 mu L of palindromic hairpin probe, 1-2 mu L of dNTPs, 3-5 mu L of DNA polymerase and 5-10U of restriction endonuclease at 30-37 ℃ for 1-2.5 h to obtain an amplification product;
(3) According to the steps (1) and (2), carrying out quantitative detection on a series of microRNA-21 standard solutions with different concentrations, and determining the fluorescence intensity value of an amplification product in an excitation light wave band to obtain a microRNA-21 concentration-fluorescence intensity value standard curve;
(4) And (3) detecting the biological sample to be detected containing the microRNA-21 according to the steps (1) and (2), determining the fluorescence intensity value of the amplification product in the excitation light wave band, and comparing the fluorescence intensity value with the standard curve, thereby measuring the concentration of the microRNA-21 in the biological sample to be detected.
Further, the sequence of the Padlock Probe (PP) is shown in SEQ ID NO: 1, specifically 5 '-P-CTGATAAGCTATACGCGTACTTCAACAACAACAACCTCTCAGCTCAACATCAGT-3'.
Further, the sequence of the Palindromic Hairpin Probe (PHP) is shown as SEQ ID NO. 2, and specifically comprises the following components:
5’-DABCYL-tacgcgtacttcAACAACAACAACAAC CCTCAGCgaagtacgcgt(FAM)a-3’。
further, the DNA polymerase includes Klenow, but is not limited thereto.
Further, the restriction endonuclease includes nb.
In some more specific embodiments, the method of making comprises: in the presence of microRNA-21, PP is firstly connected by T4 ligase to form a circular DNA template, and the circular DNA machine is triggered to run under the simultaneous action of DNA polymerase and restriction endonuclease to generate a large amount of single-stranded products. The obtained single-chain product can be directly hybridized with PHP, so that the fluorescent group and the quenching group are forced to be separated, and a strong fluorescent signal is released. Meanwhile, the 3' end of PHP in different PHP/PP double chains can be subjected to intermolecular complementary pairing, and the cyclic strand displacement amplification of a bidirectional DNA machine is realized under the action of DNA polymerase and restriction endonuclease. And finally, establishing the fluorescence detection method of the microRNA-21 according to the linear change relationship between the intensity of the fluorescence signal of the reaction system and the concentration of the microRNA-21.
In some more preferred embodiments, the method for detecting microRNA-21 by using dual DNA machine fluorescence combining a circular DNA machine based on RCA and a bidirectional DNA machine based on cycle strand displacement amplification comprises the following specific operation steps:
(1) Ligation of circular DNA templates: (1) sequentially adding 1 mu L of 10 XT 4 ligase buffer solution, 1 mu L of 2 mu M Padlock Probes (PP) with 5' end phosphorylation treatment, 1 mu L of microRNA-21 and 6 mu L of deionized water into a sterile centrifuge tube, heating the obtained mixture at 90 ℃ for 2 min, and then gradually cooling to room temperature; (2) mu.L of T4 ligase (10U) was further added to the above solution, and reacted at 16 ℃ for 30 min to cyclize PP.
(2) One-pot fluorescence signal amplification based on a dual DNA machine: mu.L of 10 XKlenow (exo-) buffer, 2. Mu.L of 10. Mu.M Palindromic Hairpin Probe (PHP), 2. Mu.L of 25mM dNTPs, 3U of Klenow and 10U of Nb, bbvCI were added to the centrifuge tube containing the circular DNA template, and the reaction was incubated at 37 ℃ for 2.5 hours.
(3) Detecting the microRNA-21 standard solution, and establishing a standard curve: (1) the obtained standard microRNA-21 powder is dissolved into 100 mu M stock solution by DEPC treated water. Continuously diluting the storage solution with DEPC-treated water step by step to obtain microRNA-21 standard solutions with different concentrations; (2) carrying out quantitative detection on the microRNA-21 standard solutions with different concentrations according to the method in the steps (1) and (2), and measuring the fluorescence intensity value of the product at 490 nm when the exciting light is 490 nm; (3) establishing a microRNA-21 concentration-fluorescence intensity value standard curve for detecting microRNA-21 by taking microRNA-21 with different concentrations as a horizontal coordinate and taking a fluorescence intensity value at 490 nm as a vertical coordinate;
(4) Carrying out quantitative detection on an actual biological sample containing microRNA-21: and (3) taking an unknown biological sample containing the microRNA-21 as a detection target, detecting according to the same processing method in the steps (1) and (2), substituting the obtained fluorescence intensity value into a standard curve for detecting the microRNA-21, and calculating the concentration of the microRNA-21.
Further, the biological sample to be tested includes human serum, but the detection of human serum according to the present invention is not intended for the purpose of obtaining a health condition of a human, that is, for the purpose of diagnosing a disease or its symptoms.
The product comprises a padlock probe and a palindromic hairpin probe, wherein the sequence of the padlock probe is shown as SEQ ID NO. 1, and the sequence of the palindromic hairpin probe is shown as SEQ ID NO. 2.
In conclusion, the method for detecting microRNA-21 by using the dual DNA machine provided by the invention has the advantages that the miRNA-21 detection sensitivity is very high, the detection limit is 10fM, the linear range is wide (10 fM-50 nM), the background signal is low, the target signal is strong, the obtained signal-to-noise ratio is high (S/N = 24), and the method is far beyond the existing fluorescence detection method; meanwhile, the method has good specificity, can easily distinguish completely complementary target RNA and non-target RNA with mismatched bases, can be used for direct detection of serum samples, can be used for sensitive detection of other DNA or RNA through simple design, and is a universal nucleic acid detection platform.
It is to be noted, however, that the detection of human serum according to the invention is not intended for the purpose of obtaining a healthy condition of the human body, i.e. for the purpose of diagnosing a disease or its signs.
The technical solutions of the present invention are further explained below with reference to several preferred embodiments, but the experimental conditions and the setting parameters should not be construed as limitations of the basic technical solutions of the present invention. And the scope of the present invention is not limited to the following examples.
Example 1
(1) Ligation of circular DNA templates: (1) sequentially adding 1 mu L of 10 XT 4 ligase buffer solution, 1 mu L of 2 mu M Padlock Probes (PP) with 5' end phosphorylation, 1 mu L of microRNA-21 and 6 mu L of deionized water into a sterile centrifuge tube, heating the obtained mixture at 90 ℃ for 2 min, and then gradually cooling to room temperature; (2) mu.L of T4 ligase (10U) was further added to the above solution, and reacted at 16 ℃ for 30 min to cyclize PP.
(2) One-pot fluorescent signal amplification based on a dual DNA machine: to the tube containing the circular DNA template, 2. Mu.L of 10 XKlenow (exo) was added - ) Buffer, 2. Mu.L of 10. Mu.M Palindromic Hairpin Probe (PHP), 2. Mu.L of 25mM dNTPs, 3U of Klenow and 10U of Nb, bbvCI, and the reaction mixture was incubated at 37 ℃ for 2.5 hours.
(3) Detecting the microRNA-21 standard solution, and establishing a standard curve: (1) the obtained standard microRNA-21 powder is dissolved into 100 mu M stock solution by DEPC treated water. Continuously diluting the storage solution with DEPC-treated water step by step to obtain microRNA-21 standard solutions with different concentrations; (2) carrying out quantitative detection on the microRNA-21 standard solutions with different concentrations according to the methods in (2) and (3), and measuring the fluorescence intensity value of the product at 520 nm when the exciting light is 490 nm; (3) and establishing a standard curve for detecting the microRNA-21 by taking the microRNA-21 with different concentrations as a horizontal coordinate and taking a fluorescence intensity value at 520 nm as a vertical coordinate, as shown in figure 1.
(4) Extraction of total RNA of cancer cells: (1) adding 1mL Trizol reagent into cancer cells, standing for 5-10 min, transferring into a centrifuge tube, adding 200 μ L chloroform, shaking, standing for 5-10 min, and centrifuging at 4 deg.C and 12000g for 15-20 min; (2) sucking the uppermost layer of water layer, adding equal amount of isopropanol, standing for 10-15 min, centrifuging at 4 deg.C and 12000g for 10-25 min, and discarding the supernatant; (3) 1ml of 75% ethanol was added, centrifuged: after standing at 4 ℃ for 5-10 min at 12000g and allowing ethanol to air dry, 20. Mu.l of DEPC water was added to dissolve RNA.
(5) Carrying out quantitative detection on a microRNA-21 sample extracted from cancer cells: and (3) taking the total RNA extracting solution of the cancer cells as a detection target, detecting according to the same treatment method in the steps (1), (2) and (3), substituting the obtained fluorescence intensity value into the standard curve for detecting the miRNA-21 established in the step (3), and calculating the concentration of the microRNA-21 in the cancer cell sample.
The sequences of the probes used in the fluorescence detection process of this example are shown in Table 1.
Table 1: probe sequences for use in fluorescence detection processes
Example 2
(1) Ligation of circular DNA templates: (1) sequentially adding 1 mu L of 10 XT 4 ligase buffer solution, 1 mu L of 2 mu M Padlock Probes (PP) with 5' end phosphorylation treatment, 1 mu L of microRNA-21 and 6 mu L of deionized water into a sterile centrifuge tube, heating the obtained mixture at 90 ℃ for 3 min, and then gradually cooling to room temperature; (2) mu.L of T4 ligase (10U) was further added to the above solution, and reacted at 16 ℃ for 40 min to cyclize PP.
(2) One-pot fluorescent signal amplification based on a dual DNA machine: to the centrifuge tube containing circular DNA template was added 2. Mu.L of 10 XKlenow (exo) - ) Buffer solution,2. mu.L of 10. Mu.M Palindromic Hairpin Probe (PHP), 2. Mu.L of 25mM dNTPs, 3U of Klenow and 10U of Nb. BbvCI, and the reaction mixture was incubated at 35 ℃ for 2.5 hours.
(3) Detecting the microRNA-21 standard solution, and establishing a standard curve: (1) the obtained standard microRNA-21 powder is dissolved into 100 mu M stock solution by DEPC treated water. Continuously diluting the storage solution with DEPC-treated water step by step to obtain microRNA-21 standard solutions with different concentrations; (2) carrying out quantitative detection on the microRNA-21 standard solutions with different concentrations according to the methods in (2) and (3), and measuring the fluorescence intensity value of the product at 520 nm when the exciting light is 490 nm; (3) and establishing a standard curve for detecting the microRNA-21 by taking the microRNA-21 with different concentrations as a horizontal coordinate and taking a fluorescence intensity value at 520 nm as a vertical coordinate.
(4) Treatment of serum samples: serum samples from healthy persons were diluted 5-10 fold with PB (10 mM, pH 7.0), heated at 95 ℃ for 5min and then cooled in an ice bath. 130000 g of the heat-treated serum sample was centrifuged (4 ℃,20 min) and two fifths of the supernatant was stored for use.
(5) Carrying out quantitative detection on a serum sample containing microRNA-21: and (3) taking the healthy human serum sample added with the microRNA-21 with the concentration of 10fM as a detection target, detecting according to the same treatment method in the steps (1), (2) and (3), substituting the obtained fluorescence intensity value into the standard curve for detecting the miRNA-21 established in the step (3), and calculating the concentration of the microRNA-21 in the serum sample. The sequences of the probes used in the fluorescence detection procedure of this example are shown in Table 1.
Example 3
(1) Ligation of circular DNA templates: (1) sequentially adding 1 mu L of 10 XT 4 ligase buffer solution, 1 mu L of 2 mu M Padlock Probes (PP) with 5' end phosphorylation treatment, 1 mu L of microRNA-21 and 6 mu L of deionized water into a sterile centrifuge tube, heating the obtained mixture at 92 ℃ for 2 min, and then gradually cooling to room temperature; (2) mu.L of T4 ligase (10U) was further added to the above solution, and reacted at 20 ℃ for 60 min to cyclize PP.
(2) One-pot fluorescent signal amplification based on a dual DNA machine:to the tube containing the circular DNA template, 1. Mu.L of 10 XKlenow (exo) was added - ) Buffer, 1.5. Mu.L of 10. Mu.M Palindromic Hairpin Probe (PHP), 1. Mu.L of 25mM dNTPs, 4U Klenow and 5U Nb BbvCI, and the reaction mixture was incubated at 30 ℃ for 2.5h.
(3) Detecting the microRNA-21 standard solution, and establishing a standard curve: (1) the obtained standard microRNA-21 powder is dissolved into 100 mu M stock solution by DEPC treated water. Continuously diluting the storage solution with DEPC-treated water step by step to obtain microRNA-21 standard solutions with different concentrations; (2) carrying out quantitative detection on the microRNA-21 standard solutions with different concentrations according to the methods in (2) and (3), and measuring the fluorescence intensity value of the product at 520 nm when the exciting light is 490 nm; (3) and establishing a standard curve for detecting the microRNA-21 by taking the microRNA-21 with different concentrations as a horizontal coordinate and taking a fluorescence intensity value at 520 nm as a vertical coordinate.
(4) Treatment of serum samples: serum samples from healthy persons were diluted 5-10 fold with PB (10 mM, pH 7.0), heated at 95 ℃ for 5min and then cooled in an ice bath. 130000 g of the heat-treated serum sample was centrifuged (4 ℃,20 min), and two fifths of the supernatant was collected and stored for further use.
(5) Carrying out quantitative detection on a serum sample containing microRNA-21: and (3) taking the healthy human serum sample added with the microRNA-21 with the concentration of 10 pM as a detection target, detecting according to the same treatment method in the steps (1), (2) and (3), substituting the obtained fluorescence intensity value into the standard curve for detecting the miRNA-21 established in the step (3), and calculating the concentration of the microRNA-21 in the serum sample. The sequences of the probes used in the fluorescence detection process of this example are shown in Table 1.
Example 4
(1) Ligation of circular DNA templates: (1) sequentially adding 1 mu L of 10 XT 4 ligase buffer solution, 1 mu L of 2 mu M Padlock Probes (PP) with 5' end phosphorylation treatment, 1 mu L of microRNA-21 and 6 mu L of deionized water into a sterile centrifuge tube, heating the obtained mixture at 95 ℃ for 5min, and then gradually cooling to room temperature; (2) mu.L of T4 ligase (10U) was further added to the above solution, and the reaction was carried out at 25 ℃ for 30 min to cyclize PP.
(2) One-pot fluorescent signal amplification based on a dual DNA machine: to the tube containing the circular DNA template, 1.5. Mu.L of 10 XKlenow (exo) was added - ) Buffer, 1. Mu.L of 10. Mu.M Palindromic Hairpin Probe (PHP), 1. Mu.L of 25mM dNTPs, 5U of Klenow and 8U of Nb. BbvCI, and the reaction mixture was incubated at 37 ℃ for 1 hour.
(3) Detecting the microRNA-21 standard solution, and establishing a standard curve: (1) the obtained standard microRNA-21 powder is dissolved into 100 mu M stock solution by DEPC treated water. Continuously diluting the storage solution with DEPC-treated water step by step to obtain microRNA-21 standard solutions with different concentrations; (2) carrying out quantitative detection on the microRNA-21 standard solutions with different concentrations according to the methods in (2) and (3), and measuring the fluorescence intensity value of the product at 520 nm when the exciting light is 490 nm; (3) and establishing a standard curve for detecting the microRNA-21 by taking the microRNA-21 with different concentrations as a horizontal coordinate and taking a fluorescence intensity value at 520 nm as a vertical coordinate.
(4) Treatment of serum samples: serum samples from healthy persons were diluted 5-10 fold with PB (10 mM, pH 7.0), heated at 95 ℃ for 5min and then cooled in an ice bath. 130000 g of the heat-treated serum sample was centrifuged (4 ℃,20 min) and two fifths of the supernatant was stored for use.
(5) Carrying out quantitative detection on a serum sample containing microRNA-21: and (3) taking the healthy human serum sample added with the microRNA-21 with the concentration of 10 nM as a detection target, detecting according to the same treatment method in the steps (1), (2) and (3), substituting the obtained fluorescence intensity value into the standard curve for detecting the miRNA-21 established in the step (3), and calculating the concentration of the microRNA-21 in the serum sample. The sequences of the probes used in the fluorescence detection process of this example are shown in Table 1.
Example 5
(1) Ligation of circular DNA templates: (1) sequentially adding 1 mu L of 10 XT 4 ligase buffer solution, 1 mu L of 2 mu M Padlock Probes (PP) with 5' end phosphorylation treatment, 1 mu L of microRNA-21 and 6 mu L of deionized water into a sterile centrifuge tube, heating the obtained mixture at 90 ℃ for 2 min, and then gradually cooling to room temperature; (2) mu.L of T4 ligase (10U) was further added to the above solution, and reacted at 16 ℃ for 30 min to cyclize PP.
(2) One-pot fluorescent signal amplification based on a dual DNA machine: to the tube containing the circular DNA template, 2. Mu.L of 10 XKlenow (exo) was added - ) Buffer, 2. Mu.L of 10. Mu.M Palindromic Hairpin Probe (PHP), 1.5. Mu.L of 25mM dNTPs, 3U Klenow and 5U Nb. BbvCI, and the reaction was incubated at 37 ℃ for 2 h.
(3) Detecting the microRNA-21 standard solution, and establishing a standard curve: (1) the obtained standard microRNA-21 powder is dissolved into 100 mu M stock solution by DEPC treated water. Continuously diluting the storage solution with DEPC-treated water step by step to obtain microRNA-21 standard solutions with different concentrations; (2) carrying out quantitative detection on the microRNA-21 standard solutions with different concentrations according to the methods in (2) and (3), and measuring the fluorescence intensity value of the product at 520 nm when the exciting light is 490 nm; (3) and establishing a standard curve for detecting the microRNA-21 by taking the microRNA-21 with different concentrations as a horizontal coordinate and taking a fluorescence intensity value at 520 nm as a vertical coordinate.
(4) Treatment of serum samples: serum samples of healthy persons were taken, diluted 5-10 fold with PB (10 mM, pH 7.0), heated at 95 ℃ for 5min, and then cooled in an ice bath. 130000 g of the heat-treated serum sample was centrifuged (4 ℃,20 min) and two fifths of the supernatant was stored for use.
(5) Carrying out quantitative detection on a serum sample containing microRNA-21: and (3) taking the healthy human serum sample added with the microRNA-21 with the concentration of 50 nM as a detection target, detecting according to the same treatment method in the steps (1), (2) and (3), substituting the obtained fluorescence intensity value into the standard curve for detecting the miRNA-21 established in the step (3), and calculating the concentration of the microRNA-21 in the serum sample. The sequences of the probes used in the fluorescence detection procedure of this example are shown in Table 1.
By the technical scheme, the method for detecting the microRNA-21 by the double DNA machine has the advantages that the miRNA-21 detection sensitivity is high, the detection limit is 10fM, the linear range is wide (10 fM-50 nM), the background signal is low, the target signal is strong, the obtained signal-to-noise ratio is high (S/N = 24), and the method is far beyond the existing fluorescence detection method; meanwhile, the method has good specificity, can easily distinguish completely complementary target RNA and non-target RNA with mismatched bases, can be used for directly detecting human serum samples, can be used for sensitive detection of other DNA or RNA through simple design, and is a universal nucleic acid detection platform.
Finally, it should be further noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
It will be appreciated by persons skilled in the art that the above-described embodiments of the invention are not intended to limit the scope of the invention. Any other corresponding changes and modifications made according to the technical idea of the present invention should be included in the protection scope of the claims of the present invention.
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<110> university of combined fertilizer industry
<120> method for detecting microRNA-21 by dual DNA machine
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 56
<212> DNA/RNA
<213> Artificial sequence (Artificial sequence)
<400> 1
ctgataagct atacgcgtac ttcaacaaca acaacaaccc tcagctcaac atcagt 56
<210> 2
<211> 46
<212> DNA/RNA
<213> Artificial sequence (Artificial sequence)
<400> 2
tacgcgtact tcaacaacaa caacaaccct cagcgaagta cgcgta 46
Claims (7)
1. A method for detecting microRNA-21 by a dual DNA machine is characterized by comprising the following steps:
(1) Uniformly mixing a T4 ligase buffer solution, a 5' -end phosphorylation padlock probe, microRNA-21 and deionized water, heating the obtained mixture at 90-95 ℃ for 2-5 min, cooling to room temperature, adding 5-10U of T4 ligase, and reacting the obtained first reaction system at 16-25 ℃ for 30-60 min to form a circular DNA template;
(2) Incubating and reacting a second reaction system comprising the first reaction system obtained in the step (1), 1-2 mu L of DNA polymerase buffer solution, 1-2 mu L of palindromic hairpin probe, 1-2 mu L of dNTPs, 3-5 mu L of DNA polymerase and 5-10U of restriction endonuclease at 30-37 ℃ for 1-2.5 h to obtain an amplification product;
(3) According to the steps (1) and (2), carrying out quantitative detection on a series of microRNA-21 standard solutions with different concentrations, and determining the fluorescence intensity value of an amplification product in an excitation light wave band to obtain a microRNA-21 concentration-fluorescence intensity value standard curve;
(4) According to the steps (1) and (2), detecting a biological sample to be detected containing the microRNA-21, determining the fluorescence intensity value of the amplification product in an excitation light wave band, and comparing the fluorescence intensity value with the standard curve, so as to determine the concentration of the microRNA-21 in the biological sample to be detected;
wherein, the sequence of the padlock probe is shown as SEQ ID NO. 1, and the sequence of the palindromic hairpin probe is shown as SEQ ID NO. 2.
2. The method of claim 1, wherein: the DNA polymerase includes Klenow enzyme.
3. The method of claim 1, wherein: the restriction endonuclease includes nb.
4. The method of claim 1, comprising: dissolving the standard microRNA-21 powder with DEPC-treated water to obtain a stock solution, and continuously diluting the stock solution with DEPC-treated water step by step to obtain a series of microRNA-21 standard solutions with different concentrations.
5. The method of claim 1, wherein: the wavelength of the excitation light wave band is 490 nm.
6. The method of claim 1, wherein: the biological sample to be tested comprises human serum.
7. A product for use in the method of any one of claims 1 to 6, wherein: the product comprises a padlock probe and a palindromic hairpin probe, wherein the sequence of the padlock probe is shown as SEQ ID NO. 1, and the sequence of the palindromic hairpin probe is shown as SEQ ID NO. 2.
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