阅读说明：本技术 一种核酸即时检验的方法 (Method for instant detection of nucleic acid ) 是由 汪杰 潘健 刘如明 郇伟伟 邓旭 于 2020-04-28 设计创作，主要内容包括：本发明公开了一种核酸即时检验的方法,涉及核酸检验技术领域。一种核酸即时检验的方法,所述即时检验的方法包括以下步骤待测样本加入蛋白酶K；再加入裂解液、羟基磁珠进行富集,富集后进行洗涤、干燥,使用等温扩增液重悬磁珠,静置后磁力架吸附磁珠,反应得到扩增富集磁珠；所述扩增富集磁珠与磁珠-COOH-NH2-RNA-FAM的扩增子富集液混合摇匀,孵育后吸附磁珠,去除上清液,加入的Cas13a核酸检测技术反应体系,反应观察荧光。运用不同表面修饰的磁珠,在核酸提取,扩增子富集,磁珠-RNA-荧光探针的荧光信号检测,辅助结合等温扩增和cas13a实现病原体核酸的即时检验。(The invention discloses a method for instantly testing nucleic acid, and relates to the technical field of nucleic acid testing. A method for the point-of-care testing of nucleic acid, the said method for point-of-care testing includes the following steps that protease K is added to the sample to be tested; adding lysis solution and hydroxyl magnetic beads for enrichment, washing and drying after enrichment, using isothermal amplification solution to resuspend the magnetic beads, standing, adsorbing the magnetic beads by a magnetic frame, and reacting to obtain amplified enriched magnetic beads; and mixing and shaking the amplification enrichment magnetic beads and the amplicon enrichment liquid of the magnetic beads-COOH-NH 2-RNA-FAM uniformly, adsorbing the magnetic beads after incubation, removing supernatant, adding a Cas13a nucleic acid detection technology reaction system, and reacting and observing fluorescence. Different surface modified magnetic beads are used for realizing the instant detection of pathogen nucleic acid in nucleic acid extraction, amplicon enrichment, magnetic bead-RNA-fluorescent probe fluorescent signal detection and auxiliary combination of isothermal amplification and cas13 a.)

1. A method for point-of-care testing of nucleic acids, said method comprising the steps of:

adding protease K into a sample to be detected;

adding lysis solution and hydroxyl magnetic beads for enrichment, washing and drying after enrichment, using isothermal amplification solution to resuspend the magnetic beads, standing, adsorbing the magnetic beads by a magnetic frame, and reacting to obtain amplified enriched magnetic beads;

and mixing the amplification enrichment magnetic beads and the amplicon enrichment liquid of the magnetic beads-COOH-NH 2-RNA-FAM, shaking up, adsorbing the magnetic beads after incubation, removing supernatant, adding the cas13a reaction system, and reacting to observe fluorescence.

2. The method for the point-of-care assay of nucleic acids according to claim 1, wherein the addition of proteinase K to the test sample is specifically:

adding DEPC water of a sample to be detected into an EP tube to make up the DEPC water to a preset volume, and adding proteinase K.

3. The method for immediately detecting nucleic acid according to claim 1, wherein the lysis solution and the hydroxyl magnetic beads are added and then shaken up, and then left standing at room temperature for 5-10 min.

4. The method for the real-time testing of nucleic acid according to claim 1, wherein the enriched sample is placed on a magnetic rack to adsorb magnetic beads, the supernatant is removed, a rinsing solution PW is added, the magnetic beads are resuspended, and the reaction solution is removed after standing.

5. The method for real-time nucleic acid testing according to claim 1, wherein after drying, the magnetic beads are resuspended in an isothermal amplification solution, and after standing for 5min, the magnetic beads are adsorbed by a magnetic rack and reacted for 20-30 min.

6. The method for the point-of-care testing of nucleic acids according to claim 1, wherein the incubation temperature is 37 ℃ to 40 ℃.

7. The method for point-of-care testing of nucleic acids according to claim 1, wherein the reaction time after adding cas13a is 10-30 min.

8. The method for the point-of-care assay of nucleic acid according to claim 2, wherein the reaction is carried out at 37 ℃ to 40 ℃ for 10 to 20 min.

9. The method for real-time nucleic acid assay according to claim 5, wherein after drying, the magnetic beads are resuspended in an isothermal amplification solution, and after standing for 5min, the magnetic beads are adsorbed by a magnetic rack and reacted at 37-40 ℃ for 10-20 min.

10. The method for the point-of-care assay of nucleic acids according to any one of claims 1 to 9, wherein the enrichment solution of the amplicons of the magnetic beads-COOH-NH 2-RNA-FAM comprises: magnetic bead-COOH-NH 2-RNA-FAM and a buffer system, wherein the buffer system is 20mM Tris & Cl, 1.0M NaCl, 1mM EDTA, 0.02%X-100; and, the pH was 7.8.

Technical Field

The invention relates to the technical field of high-strength fiber materials, in particular to a method for instantly testing nucleic acid.

Background

The point-of-care testing (POCT) is uniformly named by the POCT equipment technical committee of the chinese medical equipment association on the basis of multiple expert demonstrations and is defined as: the method is a detection mode which is carried out on a sampling site and can quickly obtain a detection result by utilizing a portable analytical instrument and a matched reagent. POCT meaning can be understood from two aspects: spatially, tests performed at the patient's side, i.e. "bedside tests"; temporally, a "point-of-care" test can be performed.

The detection process of nucleic acid is divided into nucleic acid extraction, targeted PCR amplification and amplicon detection, and the conventional detection methods of the processes are independently and separately completed. However, the existing on-site rapid test for nucleic acid is difficult to realize due to the complicated process and the dependence on equipment, such as QPCR and RT-PCR.

Disclosure of Invention

The present invention is directed to a method for real-time testing of nucleic acids, which solves the above-mentioned problems of the prior art.

In order to achieve the purpose, the invention provides the following technical scheme:

a method of point-of-care testing of nucleic acids, the method comprising the steps of:

adding protease K into a sample to be detected;

adding lysis solution and hydroxyl magnetic beads for enrichment, washing and drying after enrichment, using isothermal amplification solution to resuspend the magnetic beads, standing, adsorbing the magnetic beads by a magnetic frame, and reacting to obtain amplified enriched magnetic beads;

and mixing and shaking the amplification enrichment magnetic beads and the amplicon enrichment liquid of the magnetic beads-COOH-NH 2-RNA-FAM uniformly, adsorbing the magnetic beads after incubation, removing supernatant, adding the Cas13a reaction system, and reacting to observe fluorescence.

Preferably, the adding of proteinase K to the sample to be tested specifically comprises:

adding DEPC water of a sample to be detected into an EP tube to make up the DEPC water to a preset volume, and adding proteinase K.

Preferably, the lysis solution and the hydroxyl magnetic beads are added and shaken evenly, and then the mixture is kept stand for 5 to 10min at room temperature.

Preferably, the enriched sample is placed on a magnetic rack to adsorb magnetic beads, the supernatant is removed, and a rinsing liquid PW is added to weigh

Suspending the magnetic beads, standing and removing the reaction solution.

Preferably, after drying, resuspending the magnetic beads with an isothermal amplification solution, standing for 5min, adsorbing the magnetic beads with a magnetic rack, and reversing

Should be 20-30 min.

Preferably, the incubation temperature is 37 ℃ to 40 ℃.

Preferably, the reaction time after the cas13a is added into the reaction system is 10-30 min.

Preferably, the reaction condition is that the reaction is carried out for 10-20min at 37-40 ℃.

Preferably, after drying, resuspending the magnetic beads by using an isothermal amplification solution, standing for 5min, adsorbing the magnetic beads by using a magnetic rack, and reacting for 20-30min at 37-40 ℃.

Preferably, the amplicon enrichment solution of magnetic beads-COOH-NH 2-RNA-FAM comprises: magnetic bead-COOH-NH 2-RNA-FAM and a buffer system, wherein the buffer system is 20mM Tris & Cl, 1.0M NaCl, 1mM EDTA, 0.02%X-100; and, the pH was 7.8.

Compared with the prior art, the invention has the beneficial effects that:

the invention combines the application of a plurality of magnetic beads, can realize the nucleic acid extraction-amplification-signal detection in one tube, optimizes the nucleic acid extraction process to the maximum extent, combines cas13a and isothermal amplification technology, makes the instant examination of nucleic acid possible, and is the technical support for the application of isothermal amplification and cas13a technology to the instant examination. Different surface modified magnetic beads are used for realizing the instant detection of pathogen nucleic acid in nucleic acid extraction, amplicon enrichment, magnetic bead-RNA-fluorescent probe fluorescent signal detection and auxiliary combination of isothermal amplification and cas13 a. The inspection process is simple, and the biological propagation harm is reduced by the original field detection.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and those skilled in the art can also obtain other drawings according to the drawings without creative efforts.

FIG. 1 shows the results of gel electrophoresis detection;

FIG. 2 shows the fluorescence detection result of fluorescent magnetic beads.

Detailed Description

In order to enhance the understanding of the present invention, the present invention will be further described with reference to the following examples, which are only for the purpose of illustrating the present invention and are not to be construed as limiting the scope of the present invention.

The embodiment of the invention provides a method for the real-time detection of nucleic acid, which comprises the following steps:

adding protease K into a sample to be detected;

adding lysis solution and hydroxyl magnetic beads for enrichment, washing and drying after enrichment, using isothermal amplification solution to resuspend the magnetic beads, standing, adsorbing the magnetic beads by a magnetic frame, and reacting to obtain amplified enriched magnetic beads;

and mixing the amplification enrichment magnetic beads and the amplicon enrichment liquid of the magnetic beads-COOH-NH 2-RNA-FAM, shaking up, adsorbing the magnetic beads after incubation, removing supernatant, adding the cas13a reaction system, and reacting to observe fluorescence.

In the above embodiment:

magnetic bead for nucleic acid extraction:

the surface of the hydroxyl magnetic bead is modified with a large number of silicon hydroxyl groups, the silicon hydroxyl groups can be specifically combined with nucleic acid in a solution under the conditions of high salt and low pH value through hydrophobic interaction, hydrogen bond interaction, electrostatic interaction and the like, and are not combined with other impurities (such as protein), the nucleic acid can be quickly separated from a biological sample, the operation is safe and simple, the method is suitable for the instant detection of the nucleic acid, the size of the magnetic bead is optimized, the components and the reaction process of a lysate are optimized, and the isothermal amplification system is matched to directly determine whether the nucleic acid sample is transferred to the isothermal amplification reaction system after the nucleic acid is extracted, so that the reaction tube does not need to be replaced in the process.

Amplicon capture magnetic beads:

after isothermal amplification, if cas13a is used for targeted enzyme activity detection of nucleic acid, isothermal amplification reaction liquid must be removed while amplification products are retained, biotin-modified magnetic beads are used to assemble a specific buffer to complete the transfer of the liquid phase to the solid phase of the amplicon, so that the reaction system can be replaced, the reaction tube still does not need to be replaced in the process, and the addition of the magnetic beads-COOH-NH 2-RNA-FAM and the removal of FAM background signals are synchronously completed in the process.

Magnetic bead-COOH-NH 2-RNA-FAM cas13a Signal detection magnetic bead:

the instant detection of nucleic acid is realized, a simple, convenient and accurate signal detection system is needed, a magnetic bead-COOH-NH 2-RNA-FAM solid-phase probe is used for fixing a fluorescent signal on the magnetic bead, when the fluorescent signal is not cut by cas13a and is dissociated to a liquid phase, no fluorescent signal can be observed by using a visual fluorescent detection device, the background is very low, when an amplification product activates the enzyme digestion activity of cas13a, FAM is dissociated from the magnetic bead and is released, the level which is enough to be detected by a simple visual fluorescent detection device is obtained, the timely detection of nucleic acid is completed, the whole detection process utilizes hydroxyl magnetic beads to extract nucleic acid and directly release the nucleic acid to an isothermal amplification system, then an amplicon capture probe is used for capturing the amplification product, the magnetic bead-COOH-NH 2-RNA-FAM is simultaneously added in the process, the isothermal amplification reaction solution which is replaced by the adsorbed magnetic bead is directly added into the cas13a reaction system after the reaction, then the magnetic beads are adsorbed to directly detect the fluorescent signal, the sample does not leave the reaction tube in the whole process, 3 kinds of magnetic beads are utilized to realize the extremely simple and accurate real-time detection of nucleic acid,

combining the application of various magnetic beads, enriching the magnetic beads with acid, and enriching target nucleic acid; solid-phase RNA probe magnetic beads, an immobilized RNA probe and a quenching probe fluorescence signal noise reduction; and the amplicon enriches the magnetic beads, fixes the isothermal amplification DNA product and is convenient for system conversion. The method realizes the nucleic acid extraction-amplification-signal detection in one tube, optimizes the nucleic acid extraction process to the maximum extent, combines cas13a and isothermal amplification technology, makes the instant examination of nucleic acid possible, and is a technical support for the application of isothermal amplification and cas13a technology to the instant examination. Different surface modified magnetic beads are used for realizing the instant detection of pathogen nucleic acid in nucleic acid extraction, amplicon enrichment, magnetic bead-RNA-fluorescent probe fluorescent signal detection and auxiliary combination of isothermal amplification and cas13 a. Simple inspection process, and reduced biological propagation harm in original field

In one embodiment, the adding of proteinase K to the sample to be tested specifically comprises:

adding DEPC water of a sample to be detected into an EP tube to make up the DEPC water to a preset volume, and adding proteinase K.

In one embodiment, the lysate and the hydroxyl magnetic beads are added and shaken well, and then the mixture is kept standing for 5-10min at room temperature.

In one embodiment, the enriched sample is placed on a magnetic rack to adsorb magnetic beads, the supernatant is removed, a rinsing solution PW is added to resuspend the magnetic beads, and the reaction solution is removed after standing.

In one embodiment, after drying, the magnetic beads are resuspended in an isothermal amplification solution, and after standing for 5min, the magnetic beads are adsorbed by a magnetic rack and reacted for 20-30 min.

In one embodiment, the incubation temperature is 37 ℃ to 40 ℃.

In one embodiment, the reaction time after adding cas13a into the reaction system is 10-30 min.

In one embodiment, the reaction conditions are at 37 ℃ to 40 ℃ for 20 min.

In one embodiment, after drying, the magnetic beads are resuspended in isothermal amplification solution, and after standing for 5min, the magnetic beads are adsorbed by a magnetic rack and reacted at 37-40 ℃ for 20-30 min.

In one embodiment, the magnetic beads-COOH-NH 2-RNA-FAM and a buffer system of 20mM Tris. Cl, 1.0M NaCl, 1mM EDTA, 0.02%X-100; and, the pH was 7.8.

The embodiment of the invention uses different surface-modified magnetic beads to realize the instant detection of pathogen nucleic acid in nucleic acid extraction, amplicon enrichment, fluorescent signal detection of magnetic bead-RNA-fluorescent probe, and auxiliary combination of isothermal amplification and cas13 a.

The following detailed description is given with reference to specific examples:

the pathogen SRAS-CoV-2 single-stranded RNA virus of new coronary pneumonia (COVID-19), in order to simulate the nucleic acid micro-extraction environment, 50fM pseudovirus containing SRAS-CoV-2S protein sequence is added into 200ul of bovine serum to serve as a simulation sample.

The nucleic acid sequence to be detected is as follows:

GCACACGCCUAUUAAUUUAGUGCGUGAUCUCCCUCAGGGUUUUUCGGCUUUAGAACCAUUGGUAGAUUUGCCAAUAGGUAUUAACAUCACUAGGUUUCAAACUUUACUUGCUUUACAUAGAAGUUAUUUGACUCCUGGUGAUUCUUCUUCAGGUUGGACAGCUGGUGCUGCAGCUUAUUAUGUGGGUUAUCUUCAACCUAGGACUUUUCUAUUAAAAUAUAAUGAAAAUGGAACCAUUACAGAUGCUGUAGACUGUGCACUUGACCCUC；

isothermal amplification primers:

T7-CoV-2S-F：GAAATTAATACGACTCACTATAGGgcacacgcctattaatttagtgcg；

T7-CoV-2S-R：Bio-gagggtcaagtgcacagtctaca；

remarking: the sequence of the forward primer TAATACGACTCACTATAGG is a T7 promoter sequence, the amplified product is used for transfecting RNA in vitro, and the reverse primer 5' end is labeled by biotin and used for enriching an amplicon;

cas13a targets the guide crRNA sequence:

CoV-2S-CrRNA1：

GAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACGCAGCACCAGCUGUCCAACCUGAAGAAG；

CoV-2S-CrRNA2：

GAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACGCCGAAAAACCCUGAGGGAGAUCACGCA；

remarking: in order to prevent the virus mutation from off-target, 2 CrRNA guide sequences are designed:

and (3) probe primer: 5 '-/56-FAM/mARURUrGrGrCmAmArArArArrGrCmA/3 NH 2/-3';

and coupling the probe primer and a COOH modified magnetic bead to form a quenched solid phase FAM-RNA-NH 2-COOH-magnetic bead, wherein the solid phase magnetic bead can be cut off from the position of an RNA sequence by the activated Cas13a-CrRNA, and a FAM fluorescent signal is released.

The specific process is as follows:

a first part: nucleic acid extraction

Cracking

Adding DEPC water of a sample to be detected into an EP tube to be filled to 200ul, adding 50ul proteinase K, and waiting for 10min at room temperature, wherein the specific optimal condition is that the reaction is carried out for 20min at 50 ℃; the specific proteinase K is 100ug/ml proteinase K. A

Enrichment of

Adding 400ul of lysis solution, adding 50ul of Magbeads (mixing), shaking, and standing at room temperature for 5-10 min.

Specifically, the formulation of the lysis solution is 1000 ul: 50% PEG 8000 ul, 0.5M EDTA 20ul, 1% TX 100 10ul, 1M NaCl 5ul, 1M Kcl 5ul DEPC water 850 ul.

Washing machine

And (3) placing the sample in the last step on a magnetic frame to adsorb the magnetic beads, removing the supernatant, adding 600ul of PW, re-suspending the magnetic beads, standing for 1-2min, and removing the reaction solution. Specifically, the cleaning liquid PW1000ul has the formula: 1M Tris-HCl20ul, 0.5M EDTA 10ul, isopropanol 70ul, DEPC water 100ul, absolute ethanol 800 ul.

Drying

And removing residual liquid by using absorbent filter paper, wherein the filter paper can be subjected to DEPC treatment, standing at room temperature until the surfaces of the magnetic beads are completely dried, and completely drying the surfaces of the magnetic beads, or affecting subsequent reactions, wherein the process needs 10-30min approximately, and the filter paper is required to wait for patience.

Elution is carried out

Resuspending the magnetic beads by using isothermal amplification solution, standing for 5min, adsorbing the magnetic beads by using a magnetic rack, and reacting for 10-30min at 37-40 ℃.

A second part: enrichment of amplicon and addition of magnetic beads-COOH-NH 2-RNA-FAM

Adding 500ul of amplicon enrichment solution containing amplification enrichment magnetic beads (5ug) and magnetic beads-COOH-NH 2-RNA-FAM (5ug), mixing uniformly, incubating for 37 min, adsorbing the magnetic beads, removing the supernatant, adding 50ul of cas13a reaction system, reacting for 10-30min, and observing fluorescence. Specifically, 5ug of amplification enrichment magnetic beads was mixed with the amplicon enrichment solution, 5ug of magnetic beads-COOH-NH 2-RNA-FAM was mixed with the amplicon enrichment solution, and the two were mixed.

Specifically, the figure 1 in the specification is a gel electrophoresis detection result, lane 1 is a control amplification group, lanes 2-5 are negative groups, lane 6 is a positive plasmid control group, and lanes 7-10 are positive pseudovirus groups (sequentially diluted by 10 times);

the attached figure 2 of the specification is a fluorescence development detection result, after the isothermal amplification product T7 is transcribed in vitro, magnetic beads and the amplification product are added according to the steps of the experimental specification, the No. 1 sample is an Rnase control group, the No. 2 good sample is a crRNA-lacking control group, the No. 3 sample is a complete component experimental group, and the No. 4 sample is a cas 13-lacking 13a enzyme control group.

The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are merely illustrative of the principles of the invention, but that various changes and modifications may be made without departing from the spirit and scope of the invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.