CN111763713B - Method and kit for detecting miRNA-21 based on target isothermal cycle amplification and nucleic acid test strip technology for non-diagnostic purposes - Google Patents
Method and kit for detecting miRNA-21 based on target isothermal cycle amplification and nucleic acid test strip technology for non-diagnostic purposes Download PDFInfo
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Abstract
The invention belongs to the field of miRNA detection, and particularly relates to a method for detecting miRNA-21 based on target isothermal circulation amplification and a nucleic acid test strip technology for non-diagnosis purposes. Comprises the steps of 1) preparing colloidal gold nanoparticles with uniform particle size by using a hydrothermal method; 2) Activating sulfhydryl h-DNA by TCEP, and then reacting with colloidal gold to form gold sulfide bond for preparing colloidal gold nucleic acid probe; 3) Complementary pairing is carried out on the colloidal gold nucleic acid probe and miRNA to be detected, double-strand specific nuclease is added, and target circulation recovery and signal amplification are realized; after the reaction is finished, adding a stopping solution to inactivate enzymes to obtain a reaction solution; 4) Assembling and detecting a nucleic acid test strip: and 3) adding the reaction solution generated in the step 3) to the assembled nucleic acid test strip, and observing the detection result. The invention can obviously improve the detection sensitivity of miRNA.
Description
Technical Field
The invention belongs to the field of miRNA detection, and particularly relates to a method for detecting miRNA-21 based on target isothermal cyclic amplification and a nucleic acid test strip technology for non-diagnosis purposes.
Background
microRNA-21 (miRNA-21) is a class of endogenous non-coding RNA small molecules, and the remarkable abnormal expression of miRNA-21 is closely related to the occurrence and development of various diseases, particularly human tumors. Numerous literature studies on the function of miRNA-21 in cancers have been carried out, and various studies show that the peripheral blood miRNA expression profile of cancer patients is specifically changed, and more evidence shows that miRNA-21 can be used as a novel molecular marker for cancer diagnosis and prognosis evaluation. Early and convenient detection of miRNA-21 has therefore become a current research focus.
The target isothermal cycle amplification technology generally refers to a nucleic acid in-vitro isothermal amplification technology based on enzymatic reaction, mainly utilizes the capability of nuclease to cut DNA recognition sites, and promotes the combination and release of a target object under isothermal conditions, so as to generate corresponding signals to realize signal accumulation and amplification. Among them, double-strand specific nuclease (DSN) is the most common nuclease suitable for miRNA detection, and DSN can efficiently recognize and cleave DNA strands in a completely complementary matched DNA double strand or a DNA/RNA hybrid double strand, while having little effect on single-stranded DNA and single/double-stranded RNA, and furthermore, this degradation is directed only to at least 12 completely matched nucleotide sequences, so that it has high specificity.
The colloidal gold nucleic acid test strip is a solid phase immunoassay method for detecting target substances in complex samples, and the main principle is as follows: a short DNA sequence is marked by using colloidal gold, and reacts with target substance nucleic acid to form a compound, and the compound is captured by the DNA sequence on a detection line due to the fact that capillary action surges on a solid-phase chromatographic membrane, so that a macroscopic T line is formed. Under certain conditions, the T line signal intensity is positively correlated with the target nucleic acid concentration, and the colloidal gold immunochromatography detection test strip is combined with a colloidal gold reader, so that related substances can be rapidly, accurately and simply detected quantitatively or semi-quantitatively according to the principle of a reflection photometry, and the colloidal gold nucleic acid test strip becomes a Point-of-care testing (POCT) method.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provides a method for detecting miRNA-21 based on target isothermal cycle amplification and a nucleic acid test strip technology for non-diagnosis purposes.
The invention adopts the following technical scheme to realize the aim:
a method for detecting miRNA-21 based on target isothermal circulation amplification and nucleic acid test strip technology for non-diagnostic purposes comprises the following steps:
1) Preparing colloidal gold nanoparticles with uniform particle size by using a hydrothermal method;
2) Activating sulfhydryl h-DNA by TCEP, and then reacting with colloidal gold to form gold sulfide bond for preparing colloidal gold nucleic acid probe; wherein, the sulfhydryl h-DNA is 5'-SH-TTTTTAGCTTATCAACTACTGATCAACATCAGTCTGATAAGCTA-3';
3) Complementary pairing of the colloidal gold nucleic acid probe and miRNA to be detected is carried out, double-strand specific nuclease is added, and target circulation recovery and signal amplification are realized; after the reaction is finished, adding a stopping solution to inactivate enzymes to obtain a reaction solution;
4) Assembling and detecting a nucleic acid test strip: adding the reaction solution generated in the step 3) to an assembled nucleic acid test strip, and observing a detection result; the nucleic acid test strip comprises a detection pad; the detection pad comprises a detection line, sd-DNA is protected on the detection line, and the concentration is 5-20 mu M; the base sequence number of sd-DNA is 5'-biotin-AAAACATGGTTACCGATCCAAGTTCAGTAGTTGATA-3'.
2. The method for detecting miRNA-21 based on target isothermal cycle amplification and nucleic acid test strip technology for non-diagnostic purposes according to claim 1, wherein the specific steps of the step 1) are as follows:
adding sodium citrate solution into a reactor, heating to boiling under the magnetic stirring condition, and adding chloroauric acid solution for the first time; the mol ratio of chloroauric acid to sodium citrate is 1:10-20 parts of a base; stopping heating after the solution turns to light pink, cooling the solution to 90-95 ℃, adding chloroauric acid solution with the same amount as the first added amount for reaction for 20-30min, and adding chloroauric acid solution with the same amount as the first added amount again for reaction to obtain gold seed solution;
mixing a gold seed solution and a sodium citrate solution which is prepared in addition to be used as a growth solution for the next reaction, heating the growth solution to 90-95 ℃, adding chloroauric acid solution to react for 20-30min, and adding an equivalent chloroauric acid solution again to obtain a large-particle-size colloidal gold nanoparticle solution for the next reaction; wherein, gold seed solution: chloroauric acid: the molar ratio of the sodium citrate is 1:1:13.75; the particle size of the prepared colloidal gold nanoparticle solution is 13-40nm.
The specific steps of the step 2) are that 10-50 mu L of mercapto stem loop h-DNA probe with the concentration of 10-50 mu M is added with 10-50 mu L of tris (2-carboxyethyl) phosphine TCEP solution with the concentration of 10-50mM, after 1-3h of activation, 1-5mL of nano particles with uniform particle size are added, and the mixture is subjected to rotary culture reaction for 16-24 hours; then adding 4-10 mu L of 1mg/mL of a murine monoclonal primary antibody Ab1 (Beijing Boaosen biotechnology, bsm-2027M) for reaction for 1-3h; then adding 0.1-0.5mL of 3M sodium chloride solution, standing at 4 ℃ for reaction overnight, centrifuging for 15-30min at 8000-15000 rotating speed, removing supernatant, and re-suspending and precipitating with 0.1-0.5mL of 0.01M phosphate buffer solution PBS to obtain the colloidal gold nucleic acid probe.
The specific steps of the step 3) are that 3-7 mu L of colloidal gold nucleic acid probe and miRNA to be detected are mixed in phosphate buffer according to the volume ratio of 1-3:1, 0.1-0.6U of double-chain specific nuclease and 10 x double-chain specific nuclease buffer are added, the mixture is uniformly mixed by low-speed rotation on a vortex meter, and then the mixed solution is placed in a metal bath for reaction for 20-60 minutes at 35-60 ℃ to realize target circulation recovery and signal amplification. After the reaction, a stop solution was added and the reaction was carried out at 45℃for 5 minutes to inactivate the enzyme.
The nucleic acid test strip in the step 4) comprises a test strip card shell, a sample pad, a gold colloid pad, a detection pad and an absorption pad which are sequentially arranged in the test strip card shell; wherein the sample pad has a length of between 1.1 and 2 cm; the length of the gold-coated pad is 0.3-0.8cm; the length of the detection pad is 2.5-4.5cm; the length of the absorbent pad is 1.1-2cm.
The detection pad comprises a detection line and a quality control line, wherein the quality control line comprises a second antibody Ab2 (Siemens technology, A32723) of Ab1 ((Beijing Boaosen biotechnology, bsm-2027M)) with the concentration of 0.8-2mg.
The specific step of the step 4) is that the reaction liquid obtained in the step 3) is added into a test strip, the signal collection time of the test strip is 5-15min, and a standard curve of the detection technology is established through the relation between the accumulation of colloidal gold signals on a raw detection line and the concentration of corresponding miRNA in the corresponding time.
The invention also discloses a kit for detecting miRNA-21, which comprises a colloidal gold nucleic acid probe, a nucleic acid test strip and double-strand specific nuclease.
Compared with the prior art, the invention has the beneficial effects that:
1. the detection sensitivity of miRNA is obviously improved. The detection signal is amplified to reduce the organic combination of background noise, namely, the DSN-based auxiliary target circulating recovery and signal enrichment amplification strategy is used for providing more d-DNA to be detected for detecting the nucleic acid test strip; the colloidal gold nucleic acid probe is gathered to be used as a detection signal, and the characteristic that the colloidal gold signal is not required to be excited by extra light overcomes the interference of background noise; thereby significantly improving the sensitivity of detection.
2. The method realizes the rapid and sensitive direct detection of miRNA in the sample, and for the sample, the detection time of the miRNA in the sample is obviously improved due to the college specificity of DSN enzyme and the rapidity of the nucleic acid test strip, and the detection time of the miRNA is shortened to about 1 hour.
3. The high-efficiency specific detection of miRNA in the sample is realized, and the target miRNA can be detected from the sample to be detected with high specificity through the synergistic effect of the molecular stem-loop probe and DSN enzyme, so that the high-specificity detection is realized.
Drawings
FIG. 1 is a schematic diagram of the detection principle of a method for detecting miRNA-21 based on target isothermal cycle amplification and nucleic acid test strip technology for non-diagnostic purposes;
FIG. 2 is a graph of agarose gel electrophoresis characterization results based on DSN-assisted target isothermal cycle amplification;
FIG. 3 shows the color development of the h-DNA under negative conditions using different h-DNAs for the selection of h-DNA in the present method;
FIG. 4 is a graph of sensitivity experiments for detection of miRNA based on target isothermal cyclic amplification and nucleic acid test strip technology for non-diagnostic purposes.
Detailed Description
The present invention will be further described in detail with reference to the drawings and preferred embodiments, so that those skilled in the art can better understand the technical aspects of the present invention.
The principle of the kit for detecting miRNA-21 is shown in figure 1, firstly, a hydrothermal method is used for preparing colloidal gold nanoparticles with uniform particle size, and then the colloidal gold nanoparticles react with a thiolated h-DNA molecular stem-loop probe to prepare a high-specificity colloidal gold nucleic acid probe. Under the condition that a target exists, firstly, the h-DNA on the colloidal gold nucleic acid probe can be combined with the target miRNA-21 in a clinical sample with high efficiency and specificity; then, the target miRNA-21 is released through the capability of the DSN that can efficiently recognize and cleave the DNA strand in the DNA double strand or the DNA/RNA hybridization double strand of the complete complementary pairing, and the h-DNA is cleaved to generate colloidal gold d-DNA; the detection line on the nucleic acid test strip is used for fixing sd-DNA through biotin streptavidin, and the goat anti-mouse antibody is fixed on the quality control line; the d-DNA generated when miRNA-21 exists is complementarily paired with sd-DNA on a detection line on a nucleic acid test strip to generate a detection signal, and meanwhile, a corresponding quality control signal is generated when a nucleic acid probe flows through a quality control line. The feasibility of this method is verified as shown in FIG. 2, when the target miRNA-21 and DSN exist simultaneously, it can be seen that h-DNA is cut and the band becomes weak. When the target is not present, the colloidal gold nucleic acid probe cannot bind to the target substance from the sample, so that the subsequent reaction cannot be performed, and no colloidal gold signal of the nucleic acid test strip is generated. The detection sensitivity curve of the method is shown in FIG. 4, and the sensitivity can reach the nM level. The primer and DNA and RNA sequences used in the kit are all synthesized by Shanghai.
Design of specific h-DNA: by sequence analysis of target miRNA, a specific h-DNA stem-loop probe is creatively designed according to the difference of the pairing Tm temperature value and the delta G energy value. The miRNA-21 pairing Tm value is 57 ℃ and the delta G energy value is-12.3, so that the designed hDNA probe Tm value is higher than 57 ℃, the delta G energy value is higher than-12.3, and the h-DNA probe can be efficiently and specifically combined with the target miRNA. Because of the selective cleavage of DSN, three stem-loop probes, h-DNA1, h-DNA2 and h-DNA3, respectively, were designed as shown in FIG. 3; the nucleic acid sequences used therein are shown in Table 1: TABLE 1
Example 1A method for detecting miRNA-21 based on target isothermal cycle amplification and nucleic acid test strip technology for non-diagnostic purposes comprises the following steps:
1) Preparing colloidal gold nanoparticles with uniform particle size by using a hydrothermal method; adding the sodium citrate solution into a reactor, heating to boiling under the magnetic stirring condition, and adding chloroauric acid solution for the first time; the molar ratio of the chloroauric acid to the sodium citrate is 1:15; stopping heating after the solution turns to light pink, cooling the solution to 90-95 ℃, adding chloroauric acid solution with the same amount as the first added amount for reaction for 20-30min, and adding chloroauric acid solution with the same amount as the first added amount again for reaction to obtain gold seed solution;
mixing a gold seed solution and a sodium citrate solution which is prepared in addition to be used as a growth solution for the next reaction, heating the growth solution to 90-95 ℃, adding chloroauric acid solution to react for 20-30min, and adding an equivalent chloroauric acid solution again to obtain a large-particle-size colloidal gold nanoparticle solution for the next reaction; wherein, gold seed solution: chloroauric acid: the molar ratio of the sodium citrate is 1:1:13.75; the particle size of the prepared colloidal gold nanoparticle solution is 13-40nm.
2) Activating sulfhydryl h-DNA1 by using TCEP, and then reacting with colloidal gold to form gold sulfide bond for preparing a colloidal gold nucleic acid probe; 20 mu L of a sulfhydryl stem-loop h-DNA probe with the concentration of 10-50 mu M, adding 20 mu L of tris (2-carboxyethyl) phosphine TCEP solution with the concentration of 20mM, activating for 1-3h, adding 2mL of nano particles with uniform particle size, and performing rotary culture reaction for 16-24 h; then adding 6 mu L of 1mg/mL of murine monoclonal primary antibody Ab1, and reacting for 1-3h; then adding 0.2mL of 3M sodium chloride solution, standing at 4 ℃ for reaction overnight, centrifuging for 15-30min at 8000-15000 rpm, removing supernatant, and re-suspending and precipitating with 0.3mL of 0.01M phosphate buffer solution PBS to obtain the colloidal gold nucleic acid probe.
3) Complementary pairing of the colloidal gold nucleic acid probe and miRNA to be detected is carried out, double-strand specific nuclease is added, and target circulation recovery and signal amplification are realized; after the reaction is finished, adding a stopping solution to inactivate enzymes to obtain a reaction solution; specifically, 5 mu L of a colloidal gold nucleic acid probe and miRNA to be detected are mixed in a phosphate buffer solution according to a proportion, 0.3U of double-strand specific nuclease and 10 x of double-strand specific nuclease buffer solution are added, and the volume ratio of the colloidal gold nucleic acid probe to the miRNA to be detected to the double-strand specific nuclease is 1:1:1, a step of; mixing the above solutions by low-speed rotation on vortex instrument, and reacting at 50deg.CAnd 50 minutes, realizing target circulation recovery and signal amplification. After the reaction, a stop solution was added and the reaction was carried out at 45℃for 5 minutes to inactivate the enzyme. Wherein, the concentrations of miRNA to be detected are respectively 0M and 0.5x10 -9 M、1x10 -9 M、2x10 -9 M、4x10 -9 M、10x10 -9 M。
4) Assembling and detecting a nucleic acid test strip: adding the reaction solution generated in the step 3) to an assembled nucleic acid test strip, and observing a detection result; the nucleic acid test strip comprises a detection pad; the nucleic acid test strip comprises a test strip clamping shell, and a sample pad, a gold pad, a detection pad and an absorption pad which are sequentially arranged in the test strip clamping shell; wherein the sample pad has a length of between 1.1 and 2 cm; the length of the gold-coated pad is 0.3-0.8cm; the length of the detection pad is 2.5-4.5cm; the length of the absorbent pad is 1.1-2cm. The detection pad comprises a detection line and a quality control line, wherein the detection line comprises deoxyribonucleotides (sd-DNA) for detection, and the concentration is 5-20 mu M; the quality control line contains a second antibody Ab2 ((Saiefeidae technology, A32723)) of Ab1 with the concentration of 0.15mg-1.0mg/mL.
The specific step of the step 4) is that the reaction liquid obtained in the step 3) is added to an assembled nucleic acid test strip, the signal collection time of the test strip is 10min, and a standard curve of the detection technology is established through the relation between the accumulation of colloidal gold signals on a raw detection line and the concentration of corresponding miRNA in the corresponding time. As shown in FIG. 4, the color development condition of the colloidal gold of the nucleic acid test strip is in direct proportion to the concentration relation of miRNA-21, and the larger the concentration is, the larger the color development intensity is, so that the invention can be applied to the detection of miRNA-21 in a sample
In the steps 2) of comparative examples 1 and 2, respectively, the colloidal gold of h-DNA2 and h-DNA3 was reacted to form gold sulfide bonds for preparing colloidal gold nucleic acid probes; the results indicate that it cannot specifically bind to the target miRNA.
The foregoing is merely exemplary of the present invention, and those skilled in the art should not be considered as limiting the invention, since modifications may be made in the specific embodiments and application scope of the invention in light of the teachings of the present invention.
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Claims (2)
1. A method for detecting miRNA-21 based on target isothermal cycle amplification and nucleic acid test strip technology for non-diagnostic purposes, which is characterized by comprising the following steps:
1) Preparing colloidal gold nanoparticles with uniform particle size by using a hydrothermal method;
2) Activating sulfhydryl h-DNA by TCEP, and then reacting with colloidal gold to form gold sulfide bond for preparing colloidal gold nucleic acid probe; wherein, the mercapto h-DNA sequence is 5'-SH-TTTTTAGCTTATCAACTACTGATCAACATCAGTCTGATAAGCTA-3';
the method specifically comprises the following steps: 10-50 mu L of mercapto stem-loop h-DNA probe with the concentration of 10-50 mu M, 10-50 mu L of tris (2-carboxyethyl) phosphine TCEP solution with the concentration of 10-50mM, 1-3h of activated tris (2-carboxyethyl) phosphine TCEP solution, 1-5mL of nano particles with uniform particle size and rotary culture reaction for 16-24 hours; then adding 4-10 mu L of 1mg/mL of murine monoclonal primary antibody Ab1, and reacting for 1-3h; adding 0.1-0.5mL of 3M sodium chloride solution, standing at 4 ℃ for reaction overnight, centrifuging for 15-30min at 8000-15000 rotating speed, removing supernatant, and re-suspending and precipitating with 0.1-0.5mL of 0.01M phosphate buffer solution PBS to obtain colloidal gold nucleic acid probe;
3) Complementary pairing is carried out on the colloidal gold nucleic acid probe and miRNA to be detected, double-strand specific nuclease is added, and target circulation recovery and signal amplification are realized; after the reaction is finished, adding a stopping solution to inactivate enzymes to obtain a reaction solution;
the method specifically comprises the following steps: mixing 3-7 mu L of colloidal gold nucleic acid probe and miRNA to be detected in a phosphate buffer according to the volume ratio of 1-3:1, adding 0.1-0.6U of double-strand specific nuclease and 10 x of double-strand specific nuclease buffer, uniformly mixing by low-speed rotation on a vortex instrument, and then placing the mixed solution in a metal bath for reaction for 20-60 minutes at 35-60 ℃ to realize target circulation recovery and signal amplification; after the reaction is finished, adding a stopping solution, and reacting for 5 minutes at 45 ℃ to inactivate enzymes;
4) Assembling and detecting a nucleic acid test strip: adding the reaction solution generated in the step 3) to an assembled nucleic acid test strip, and observing a detection result;
the method specifically comprises the following steps: adding the reaction solution obtained in the step 3) into a nucleic acid test strip, wherein the signal collection time of the nucleic acid test strip is 5-15min, and establishing a standard curve of the detection technology according to the relation between the accumulation of colloidal gold signals on a detection line and the concentration of corresponding miRNA in the corresponding time;
the nucleic acid test strip comprises a detection pad; the detection pad comprises a detection line and a quality control line, sd-DNA is protected on the detection line, and the concentration is 5-20 mu M; the base sequence number of sd-DNA is 5'-biotin-AAAACATGGTTACCGATCCAAGTTCAGTAGTTGATA-3', and the quality control line contains a second antibody Ab2 resisting Ab1, the concentration is 0.8-2mg;
the nucleic acid test strip comprises a test strip card shell, and a sample pad, a gold pad, a detection pad and an absorption pad which are sequentially arranged in the test strip card shell; wherein the sample pad has a length of between 1.1 and 2 cm; the length of the gold-coated pad is 0.3-0.8cm; the length of the detection pad is 2.5-4.5cm; the length of the absorbent pad is 1.1-2cm.
2. The method for detecting miRNA-21 based on target isothermal cycle amplification and nucleic acid test strip technology for non-diagnostic purposes according to claim 1, wherein the specific steps of the step 1) are as follows: adding the sodium citrate solution into a reactor, heating to boiling under the magnetic stirring condition, and adding the chloroauric acid solution for the first time; the mol ratio of chloroauric acid to sodium citrate is 1:10-20 parts of a base; stopping heating after the solution turns to light pink, cooling the solution to 90-95 ℃, adding chloroauric acid solution with the same amount as the first added amount for reaction for 20-30min, and adding chloroauric acid solution with the same amount as the first added amount again for reaction to obtain gold seed solution;
mixing a gold seed solution and a sodium citrate solution which is prepared in addition to be used as a growth solution for the next reaction, heating the growth solution to 90-95 ℃, adding chloroauric acid solution to react for 20-30min, and adding an equivalent chloroauric acid solution again to obtain a large-particle-size colloidal gold nanoparticle solution for the next reaction; wherein, gold seed solution: chloroauric acid: the molar ratio of the sodium citrate is 1:1:13.75; the particle size of the prepared colloidal gold nanoparticle solution is 13-40nm.
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| Title |
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| DSN结合CHA用于miRNA的可视化检测;王奎宇等;《黑龙江畜牧兽医》;20171231;13-20,285 * |
| microRNA光电化学检测及应用研究;李炳晨;《山东农业大学硕士学位论文》;20171231;全文 * |
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