CN115386658B - Single-molecule RNA quantitative detection method and system without nucleic acid amplification - Google Patents
Single-molecule RNA quantitative detection method and system without nucleic acid amplification Download PDFInfo
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Abstract
The application provides a single-molecule RNA quantitative detection method and a system without nucleic acid amplification, comprising the following steps: s1: preparing a microsphere functionally modified by a single-stranded RNA fluorescent reporter probe; s2: designing target RNA specific crRNA; s3: incubating target RNA, crRNA, cas13 protein, reaction buffer solution and S1 preparation microsphere with known concentration at proper temperature; s4: performing fluorescence imaging on the microspheres incubated in the step S3, and establishing a linear corresponding relation between the fluorescence intensity of the microspheres and the concentration of RNA; s5: and quantitatively detecting RNA in the sample by using the linear corresponding relation established by the S4. The RNA detection method can realize single-molecule level RNA quantitative analysis, does not need reverse transcription and nucleic acid pre-amplification steps, has warm detection conditions and simple operation steps, and provides a novel strategy with high sensitivity, simplicity and convenience and universality for RNA diagnosis and analysis.
Description
[ field of technology ]
The application relates to the research field of molecular diagnosis and biochemical analysis methods, in particular to a single-molecule RNA quantitative detection method and system without nucleic acid amplification.
[ background Art ]
As important genetic materials, RNA plays a vital role in various biological processes, and it is becoming increasingly important to detect RNA with high sensitivity and specificity from basic biological research to molecular biological exploration to clinical diagnostic analysis. For example, nucleic acid detection based on novel coronavirus (SRAS-CoV-2) genomic RNA, as a gold standard for novel coronavirus disease (COVID-19) diagnosis, plays a vital role in the prevention and control of COVID-19; for another example, fusion gene transcript RNA formed by chromosomal rearrangements is used as a molecular marker for various types of cancer classification, diagnosis, treatment, prognosis and monitoring of small residual foci by national integrated cancer network oncology clinical practice guidelines (National Comprehensive Cancer Network Clinical Practice in Guidelines in Oncology, NCCN guidelines) and world health organization Blue book (WHO Blue Books).
Currently, RNA detection relies mainly on nucleic acid amplification techniques, including reverse transcription-polymerase chain reaction (RT-PCR), ligase Chain Reaction (LCR), recombinase amplification techniques (RPA), loop-mediated isothermal nucleic acid amplification (LAMP), and the like. Due to the efficient exponential amplification mechanism, these methods achieve very high sensitivity, even at the single molecule level. However, these methods typically require a reverse transcription/ligation step to convert RNA to DNA, and a subsequent DNA replication step to produce detectable nucleic acid levels. However, the amplification step often introduces some troublesome problems such as loss of the target RNA molecule due to incomplete reverse transcription, amplification bias due to replication of error-prone sequences, and false positive results due to contamination. In addition, careful design of multiple target-specific probes/primers, use of multiple tool enzymes, and stringent optimization of experimental conditions are required. Therefore, there is an urgent need to develop a general RNA quantitative detection method which not only has the same sensitivity as the nucleic acid amplification technique, but also avoids the problems of the above-mentioned nucleic acid amplification.
The CRISPR/Cas13a system has target-dependent trans-cleavage activity, can specifically identify and bind target RNA through crRNA, and activate the trans-cleavage activity of the CRISPR/Cas13a, and can efficiently hydrolyze a single-stranded RNA reporter probe existing in the surrounding environment, so that amplification-free RNA detection is realized. Although the CRISPR/Cas13a system has a multiple response mechanism, it can only detect RNAs above pM, failing to meet most practical sample analysis requirements. Recently, researchers have increased the sensitivity of amplification-free RNA analysis to fM levels by tandem CRISPR/Cas systems and designing multiple crRNA CRISPR/Cas13a systems, but this undoubtedly increases the cost of analysis complicating the design; even so, quantitative detection of RNA at the aM level is still not satisfactory.
[ application ]
In view of the above, the application provides a universal, simple and convenient quantitative detection method for single-molecule RNA without nucleic acid amplification.
The RNA quantitative detection method provided by the application comprises the following steps:
s1: preparing a microsphere functionally modified by a single-stranded RNA fluorescent reporter probe;
s2: designing and synthesizing target RNA specific crRNA;
s3: incubating target RNA, crRNA, cas13 protein, reaction buffer solution and S1 preparation microsphere with known concentration at proper temperature;
s4: performing fluorescence imaging on the microsphere after the S3 incubation, and establishing a linear corresponding relation between the fluorescence intensity of the microsphere and the RNA concentration;
s5: and quantitatively detecting RNA in the sample by using the linear corresponding relation established by the S4.
In the aspect and any possible implementation manner as described above, there is further provided an implementation manner, where the S1 specifically includes:
s11: screening out single microspheres modified by streptavidin with the same size by using a microscope;
s12: designing and synthesizing a single-stranded RNA report probe marked by biotin, a fluorescent group and a quenching group;
s13: the single-stranded RNA reporter probe was immobilized on the surface of the microsphere obtained in S11 by biotin-streptavidin immune reaction.
In aspects and any one of the possible implementations described above, there is further provided an implementation, wherein the optimization of the single microsphere in S11 is selected from streptavidin-modified agarose magnetic microsphere, and the size is 1-1000 micrometers; the magnetic separation is convenient, and the streptavidin modification is convenient to fix the biotin modified single-stranded RNA reporter probe on the surface of the microsphere.
In the aspect and any possible implementation manner described above, further an implementation manner is provided, in S13, other fixing manners may be adopted, and a corresponding functional unit is required for a corresponding microsphere surface; if the surface of the microsphere can be modified with anti-FITC antibody, the corresponding RNA reporter probe does not need biotin modification.
In the aspect and any one of the possible implementation manners described above, further provided is an implementation manner, wherein in S13, the reaction of streptavidin and biotin is performed in a phosphate buffer solution (10mM,137mM NaCl,2.7mM KCl,pH 7.4), the reaction time is greater than 30 minutes, and the reaction temperature is 20-40 ℃.
In the aspect and any possible implementation manner described above, further provided is an implementation manner, in the S3, a reaction volume is 5 μl, an incubation temperature is 20-40 ℃, an optimal incubation time is 60 minutes, a final concentration of crRNA in a reaction system is 50nM, an optimal final concentration of cas13 in the reaction system is 100nM, and a reaction buffer solution pH is 7.9, including the following components: 4U RNase inhibitor, 10mM Tris-HCl,50mM NaCl,10mM MgCl 2 ，1μg/mL BSA。
In the foregoing aspect and any possible implementation manner, there is further provided an implementation manner, where the S4 specifically includes:
s41: washing the microspheres incubated with S3 once with a phosphate buffer solution (10mM,137mM NaCl,2.7mM KCl,0.05%tween-20, pH 7.4) containing a surfactant by using a magnetic separation technique, and then re-suspending the microspheres in the phosphate buffer solution (10mM,137mM NaCl,2.7mM KCl, pH 7.4);
s42: absorbing single microspheres onto a glass slide and carrying out fluorescence imaging on the single microspheres, wherein the laser wavelength is 488nm, the fluorescence collection range is 500nm-570nm, RNA with different concentrations is detected, RNA in a sample is quantitatively analyzed, and the same imaging condition is maintained;
s43: obtaining the intensity of fluorescent signals on the microsphere surface, and fitting a linear corresponding relation between the fluorescent intensity and the RNA concentration;
in the foregoing aspect and any possible implementation manner, there is further provided an implementation manner, where the S5 specifically includes:
s51: changing the RNA in the S3 into an RNA sample to be tested, and executing the same experimental steps as the S3 and the S41-S42;
s52: obtaining the intensity of fluorescent signals on the surfaces of the microspheres;
s53: and quantitatively analyzing the target RNA in the sample by using the linear corresponding relation of the S43 fitting.
In aspects and any possible implementation manner described above, there is further provided a single-molecule RNA quantitative detection system without nucleic acid amplification, the RNA quantitative detection system comprising:
the microsphere preparation unit is used for preparing microspheres functionally modified by the single-stranded RNA fluorescent reporter probe;
the crRNA design synthesis unit is used for designing and synthesizing crRNA specific to the target RNA;
a co-incubation unit for co-incubating the target RNA, crRNA, cas protein of known concentration, the reaction buffer solution and the microsphere prepared by the microsphere preparation unit at a suitable temperature;
the linear relation establishing unit is used for carrying out fluorescence imaging on the microspheres incubated by the common incubation unit and establishing a linear corresponding relation between the fluorescence intensity of the microspheres and the RNA concentration;
and the sample detection unit is used for quantitatively detecting RNA in the sample through the established linear corresponding relation.
Compared with the prior art, the application can obtain the following technical effects:
1. the application is a rapid and simple quantitative RNA detection method, which does not need reverse transcription, ligation and nucleic acid pre-amplification processes, does not need complex primer and probe design, does not need multiple tool enzymes, and does not need strict optimization of detection conditions.
2. The application relates to a universal RNA quantitative analysis method, and the prepared single microsphere modified by single-stranded RNA fluorescence report probe functionalization is universal for detecting any RNA, and only a target RNA specific crRNA is needed to be newly designed and synthesized.
3. The application is a single molecule RNA quantitative analysis method, because of adopting single microsphere as CRISPR/Cas13a trans-cleavage reaction reactor, the cleavage reaction can only be carried out in the very effective volume range of microsphere surface, thus generating local concentration increasing effect, namely RNA with the same molecular number appears in smaller volume, the concentration is larger, the concentration increasing effect can greatly increase trans-cleavage effect, and the sensitivity of RNA detection is further improved; meanwhile, all fluorescence signals are enriched on the surface of the single microsphere, and the sensitivity of RNA detection reaches a single molecular level by combining with a CRISPR/Cas13a system multiple response mechanism.
4. The application is not limited to the quantitative detection of novel coronavirus (SARS-CoV-2) RNA described in the examples, and can be generalized to quantitative analysis of any RNA molecule.
Of course, it is not necessary for any of the products embodying the application to achieve all of the technical effects described above at the same time.
[ description of the drawings ]
In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present application, and that other drawings can be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic illustration of the preparation process of a microsphere functionalized with a single-stranded RNA reporter probe according to the present application;
FIG. 2 is a schematic representation of RNA detection using functionalized single microspheres and CRISPR/Cas13a systems according to the present application;
FIG. 3 is a schematic illustration of the effect of localized concentration increase according to the present application;
FIG. 4 shows the results of the detection of IVT SARS-CoV-2-N-RNA at different concentrations according to the present application;
FIG. 5 shows the quantitative detection effect of IVT SARS-CoV-2-N-RNA at different concentrations according to the present application;
FIG. 6 is a graph showing the effect of the method on the specificity evaluation in the embodiment of the present application;
FIG. 7 is a graph showing the results of a novel coronavirus assay from a throat swab sample in accordance with embodiments of the present application.
[ detailed description ] of the application
For a better understanding of the technical solution of the present application, the following detailed description of the embodiments of the present application refers to the accompanying drawings.
The application provides a single-molecule RNA quantitative detection method without nucleic acid amplification, which comprises the following steps:
unlike classical CRISPR/Cas13a sensing systems, the present application prepares Functionalized single microspheres, i.e., functionalized-SMB, by immobilizing single-stranded RNA fluorescence reporter probes of the CRISPR/Cas13a sensing system on the surface of the single microspheres through the immune reaction of streptavidin and biotin (fig. 1). In the presence of target RNA molecules, crRNA can guide Cas13a protein to specifically recognize target RNA, after the crRNA is combined with the target RNA, the trans-cleavage reaction activity of Cas13a protein is activated, the activity can efficiently and quickly cleave a single-stranded RNA fluorescent reporter probe modified on the surface of a single microsphere, a quenching group and a fluorescent group are separated, all the fluorescent groups are fixed on the surface of the microsphere, thus, under the irradiation of laser, the fluorescence of the fluorescent group on the surface of the single microsphere is lightened, the fluorescence intensity is related to the concentration of the target RNA molecules, and thus, the quantitative detection of the target RNA molecules can be carried out (figure 2). It is worth mentioning that in the application, the Cas13a trans-cleavage reaction activated by the target RNA only generates a significant local concentration increasing effect in a very small volume of the microsphere surface, namely, the concentration increasing effect of the same molecular RNA in a small volume (fig. 3) can increase the efficiency of the Cas13a trans-cleavage reaction, so that low concentration RNA molecules can generate a significant response signal, thereby realizing the detection of single-molecule RNA.
The application provides a single-molecule RNA quantitative detection method with high sensitivity and high specificity without a nucleic acid amplification step, and the method is successfully applied to visual quantitative analysis of SARS-CoV-2 RNA.
The technical scheme for realizing the application comprises the following steps:
step 1, preparing single microspheres functionally modified by single-stranded RNA fluorescent reporter probes;
step 2, designing and synthesizing SARS-CoV-2N gene RNA specific crRNA;
step 3, incubating the IVT SARS-CoV-2N gene RNA, crRNA, cas13 protein with known concentration, the reaction buffer solution and the S1 prepared microsphere together at a proper temperature;
step 4, performing fluorescence imaging on the microspheres incubated in the step 3, and establishing a linear corresponding relation between the fluorescence intensity of the microspheres and the concentration of the RSARS-CoV-2N gene RNA;
and 5, quantitatively detecting SARS-CoV-2N gene RNA in the throat swab sample by utilizing the linear corresponding relation established in the step 4.
The application also provides a single-molecule RNA quantitative detection system without nucleic acid amplification, which comprises the quantitative detection method, and the RNA quantitative detection system comprises the following components:
the microsphere preparation unit is used for preparing microspheres functionally modified by the single-stranded RNA fluorescent reporter probe;
the crRNA design synthesis unit is used for designing and synthesizing crRNA specific to the target RNA;
a co-incubation unit for co-incubating the target RNA, crRNA, cas protein of known concentration, the reaction buffer solution and the microsphere prepared by the microsphere preparation unit at a suitable temperature;
the linear relation establishing unit is used for carrying out fluorescence imaging on the microspheres incubated by the common incubation unit and establishing a linear corresponding relation between the fluorescence intensity of the microspheres and the RNA concentration;
and the sample detection unit is used for quantitatively detecting RNA in the sample through the established linear corresponding relation.
The basic principle and specific steps of the application are as follows:
step 1: preparation of Single-stranded RNA fluorescent reporter Probe functionalized Single microspheres
First, streptavidin-modified agarose magnetic microspheres (STV-MBs) of the same size (80.+ -.3 μm) were screened using a microscope, placed in a 1.5mL centrifuge tube with low nucleic acid adsorption, washed twice with 1 XPBST (10mM,137mM NaCl,2.7mM KCl,0.05%tween-20 (water and tween-20), pH 7.4) and resuspended in 1 XPBS (10mM,137mM NaCl,2.7mM KCl, pH 7.4); subsequently, an excessive amount (2 pmol for single-sphere reaction and 2nmol for multi-sphere reaction) of single-stranded RNA reporter probe was added to the resuspended STV-MB, and the mixture was incubated with shaking at room temperature for 1 hour to immobilize the single-stranded RNA reporter Tan Zheng on the surface of the microsphere, thereby obtaining a Functionalized-SMB (FIG. 1); wherein both ends of the single-stranded RNA reporter probe are labeled with a fluorescent group and a quenching group, respectively, but the fluorescent group and biotin must be labeled on the same base.
In the embodiment, the sequence of the single-stranded RNA report probe is 5 '-UUUUC-3', the FAM fluorescent group is covalently modified on the 5 '-end U base, biotin is covalently modified, and the 3' -end quenching group BHQ1 is synthesized by Shanghai.
In order to better illustrate the RNA quantitative detection principle and demonstrate the feasibility and application value of the application, the method disclosed by the application is used for quantitatively detecting SRAS-CoV-2N gene RNA.
Example 1.
Step 2, designing and synthesizing SARS-CoV-2N gene RNA specific crRNA;
in this example, the SARS-COV-2N gene-specific crRNA targets the N gene of SARS-CoV-2, covering the reported probe sequences for RT-PCR detection of SARS-CoV-2N gene published by the American disease prevention control center, respectively. The sequence is 5'-GAUUUAGACUACCCCAAAAACGAAG GGGACUAAAACACGCUGAAGCGCUGGGGGCAAAUUGUGC-3', and is synthesized by Shanghai.
Step 3, incubating the SARS-CoV-2N gene RNA, crRNA, cas13 protein with known concentration, the reaction buffer solution and the S1 prepared microsphere together at a proper temperature;
s1: SRAS-CoV-2N gene RNA (IVT SARS-CoV-2-N-RNA) is prepared by an in vitro transcription method, and the specific steps are as follows;
(1) In vitro T7 transcribed DNA templates were obtained from DNA plasmids (pUC 57-2019-nCov-N, gold Style Co.) by PCR amplification:
mu.L of the PCR reaction mixture contained 100ng of DNA plasmid, 1 XPCR reaction buffer, 250. Mu.M dNTPs (250. Mu.M each), 500nM T7 promoter-labeled forward primer (T7-FP-SRAS-CoV-2, SEQ ID NO: 5'-TAATACGACTCACTATAGGATGTCTGATAATGGACCCCAAA ATCA-3', shanghai), 500nM reverse primer (RP-SRAS-CoV-2, SEQ ID NO: 5'-TTAGGCCTGAGTTGAGTCAGC-3', shanghai), 5U TaKaRa Taq HS DNA polymerase (Sigma) for PCR amplification. The PCR cycle included a hot start step (4 minutes at 95 ℃), a thermal cycling process (40 cycles: 20 seconds at 95 ℃, 30 seconds at 62 ℃,1 minute at 72 ℃) and a final extension (5 minutes at 72 ℃). The PCR product was confirmed by 4% agarose gel electrophoresis and purified using a SanPrep column type PCR product purification kit (Shanghai Biotechnology) to obtain a T7 transcribed DNA template.
(2) Preparation of SARS-CoV-2N Gene RNA by T7 transcription reaction: a transcription reaction mixture (50. Mu.L) containing 2. Mu. g T7 of transcribed DNA template, 1 XT 7 RNApol reaction buffer (NEB), 5mM NTP and 250U T7 RNA polymerase (NEB) was incubated isothermally at 37℃for 5 h. The transcript was then treated with DNase I at 37℃for 1h to remove the T7 transcribed DNA template. After confirming the product by 4% agarose gel electrophoresis, the transcript was purified using the Spin Column RNAclean kit (TIANGEN). Finally, the concentration of SARS-CoV-2-N-RNA was quantified using a Nanodrop One UV-Vis spectrophotometer, and the transcript length and concentration were used to further calculate the copy number.
S2: SARS-CoV-2-N-RNA detection, the specific steps are as follows:
10 μL CRISPR/Cas13a reaction mix package Functionalized-SMB (one sample added to a single microsphere), 200nM Cas13a protein, 100nM N-crRNA, 4U RRI, 1 Xreaction buffer (10 mM Tris-HCl,50mM NaCl,10mM MgCl) 2 1. Mu.g/mL BSA) and known concentrations of IVT SARS-CoV-2-N-RNA were incubated at 37℃for 60min.
Step 4, performing fluorescence imaging on the microspheres incubated in the step 3, and establishing a linear corresponding relation between the fluorescence intensity of the microspheres and the concentration of the RSARS-CoV-2N gene RNA;
fluorescence imaging of the microspheres incubated in step 3 using confocal laser fluorescence microscopy, results are shown in FIG. 4, with the concentration of IVT SARS-CoV-2-N-RNA increasing from 1aM (6 copies) to 100fM (-6.0X10) 5 Copy), the fluorescent signal of the microsphere surface is gradually enhanced, and if different colors are adopted to represent different fluorescent intensities, the change of the fluorescent intensity can be clearly distinguished by naked eyes.
The quantitative detection result of IVT SARS-CoV-2-N-RNA is shown in FIG. 5, the microsphere surface Fluorescence Intensity (FI) and the negative pair number of concentration of SARS-CoV-2-N-RNA show good linear relationship in the range of 10aM to 100fM, and the correlation equation is FI=2.56×10 5 +9.59*10 4 lgC RNA The correlation coefficient is 0.9998, which shows that the method can realize high-sensitivity RNA quantitative detection without nucleic acid amplification, meanwhile, the applicant discovers that 1aM, namely 6 molecular RNAs can generate fluorescent signals distinguishable from blank, which shows that single-molecular-level RNA detection can be realized,
to further verify the specificity of this method for RNA detection, various coronavirus (including bat-SL-COVZC45, SRAS-CoV and Human-COV-HKU 1) N gene RNAs were detected using SARS-CoV-2-N-RNA specific crRNA, and the results were shown in FIG. 6, with only SARS-CoV-2-N-RNA producing a distinct fluorescent signal, while other coronavirus RNAs producing signals were in close proximity to the blank, indicating that the method has high specificity in the detection of target RNA molecules.
And 5, quantitatively detecting SARS-CoV-2N gene RNA in the throat swab sample by utilizing the linear corresponding relation established in the step 4.
To further assess the practicality and reliability of this method, SARS-CoV-2-abMEN pseudovirus (an engineered organism) was added to NP swab sample solutions to simulate a series of SARS-CoV-2 positive NP swab samples. After viral RNA extraction using standard viral RNA extraction kit (EZ-10 Spin Column Viral Total RNA Extraction Kit, probiotics), SRAS-CoV-2 was detected in these mock-positive NP swab samples (positive P1-P3) and negative NP swab samples (negative N1-N3) using this method. As shown in FIG. 7, the sample contained 10 as compared with the negative NP sample and the blank 3 -10 5 The copy of SARS-CoV-2-abMEN pseudovirion positive samples all produced a distinct fluorescent signal.
The copy number of SARS-CoV-2-abMEN pseudovirus in the mock-positive NP swab sample was evaluated according to the correlation equation obtained in FIG. 5 and the dilution factor in the extraction step. The results are shown in Table 1, where all calculated copy numbers for these positive samples are approximately 35.6% -95% of theoretical copy numbers, indicating that some RNA molecules may be lost during the extraction process. To verify these results, the simulated SARS-CoV-2 positive NP sample was tested by the RT-PCR method reported by CDC, confirming that the method was substantially identical to the RT-PCR test results. The practicability and the reliability of the method in the aspect of clinical sample detection are fully proved.
TABLE 1 New method and RT-PCR detection of SARS-CoV-2 in analog NP samples
The application firstly provides a CRISPR/Cas13a sensing platform constructed on the surface of a single microsphere, and develops amplification-free single-molecule RNA quantitative detection. According to the application, the functionalized single microsphere coated by the single-stranded RNA fluorescent reporter probe is used as a reactor for CRISPR/Cas13a trans-cleavage reaction, and simultaneously used as a fluorescent signal enrichment and output unit, and the CRISPR/Cas13a trans-cleavage reaction is only carried out in a reaction volume with extremely small surface of the microsphere, so that a local concentration increasing effect is generated, and the cleavage reaction efficiency is increased; meanwhile, due to the enrichment effect of single microsphere signals and the multiple response mechanism of Cas13a protein, the application can detect RNA at single molecular level. This does not require reverse transcription and any nucleic acid amplification steps. Using the novel coronavirus RNA assay as a model, this method has proven to be a reliable and practical method for quantitatively determining SARS-CoV-2 in clinical samples. The application provides a new tool for molecular diagnosis and disease detection with RNA as a target.
The method and the system for quantitatively detecting the single-molecule RNA without the nucleic acid amplification provided by the embodiment of the application are described in detail. The above description of embodiments is only for aiding in the understanding of the method of the present application and its core ideas; meanwhile, as those skilled in the art will have variations in the specific embodiments and application scope in accordance with the ideas of the present application, the present description should not be construed as limiting the present application in view of the above.
Certain terms are used throughout the description and claims to refer to particular components. Those of skill in the art will appreciate that a hardware manufacturer may refer to the same component by different names. The description and claims do not take the form of an element differentiated by name, but rather by functionality. As referred to throughout the specification and claims, the terms "comprising," including, "and" includes "are intended to be interpreted as" including/comprising, but not limited to. By "substantially" is meant that within an acceptable error range, a person skilled in the art is able to solve the technical problem within a certain error range, substantially achieving the technical effect. The description hereinafter sets forth a preferred embodiment for practicing the application, but is not intended to limit the scope of the application, as the description is given for the purpose of illustrating the general principles of the application. The scope of the application is defined by the appended claims.
It should also be noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a product or system that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such product or system. Without further limitation, an element defined by the phrase "comprising one … …" does not exclude the presence of other like elements in a commodity or system comprising such elements.
It should be understood that the term "and/or" as used herein is merely one relationship describing the association of the associated objects, meaning that there may be three relationships, e.g., a and/or B, may represent: a exists alone, A and B exist together, and B exists alone. In addition, the character "/" herein generally indicates that the front and rear associated objects are an "or" relationship.
While the foregoing description illustrates and describes the preferred embodiments of the present application, it is to be understood that the application is not limited to the forms disclosed herein, but is not to be construed as limited to other embodiments, and is capable of numerous other combinations, modifications and environments and is capable of changes or modifications within the scope of the inventive concept as expressed herein, either as a result of the foregoing teachings or as a result of the knowledge or technology of the relevant art. And that modifications and variations which do not depart from the spirit and scope of the application are intended to be within the scope of the appended claims.
Claims (3)
1. A method for quantitative detection of single-molecule RNA without nucleic acid amplification, characterized in that the method for quantitative detection of RNA achieves the aim of non-diagnosis of a disease, comprising the steps of:
s1: preparing a microsphere functionally modified by a single-stranded RNA fluorescent reporter probe;
the S1 specifically comprises the following steps:
s11: adopting streptavidin to modify the single microsphere, and screening out the single microsphere modified by streptavidin with the same size by utilizing a microscope;
s12: designing a single-stranded RNA report probe marked by biotin, a fluorescent group and a quenching group, wherein the fluorescent group and the quenching group are respectively marked at two ends of the single-stranded RNA report probe, and the fluorescent group and the biotin are marked on the same base;
s13: fixing a single-stranded RNA reporter probe on the surface of the S11 single microsphere through biotin-streptavidin immune reaction;
s2: designing and synthesizing target RNA specific crRNA;
s3: incubating target RNA, crRNA, cas protein with known concentration, reaction buffer solution and S1 preparation microsphere together at a proper temperature, wherein crRNA can guide Cas13a protein to specifically recognize target RNA in the presence of target RNA molecule during incubation, and the trans-cleavage reaction activity of Cas13a protein is activated, and the activity can cleave single-stranded RNA fluorescence reporter probe modified on the surface of single microsphere to separate a quenching group from a fluorescent group;
s4: performing fluorescence imaging on the microsphere after the S3 incubation, and establishing a linear corresponding relation between the fluorescence intensity of the microsphere and the RNA concentration;
the fluorescence imaging in S4 specifically comprises: the fluorescence of the fluorescent groups on the surface of the single microsphere is lightened, and the fluorescence intensity is related to the concentration of the target RNA molecules;
s5: quantitatively detecting RNA in the sample by utilizing the linear corresponding relation established in the step S4;
the step S4 specifically comprises the following steps:
s41: washing the microspheres incubated in the step S3 with a phosphate buffer solution containing a surfactant for one time by using a magnetic separation technology, and then resuspending the microspheres in the phosphate buffer solution;
s42: sucking the single microsphere onto a glass slide, performing fluorescence imaging on the single microsphere, detecting RNA with different concentrations, quantitatively analyzing RNA in a sample, and keeping the same imaging condition;
s43: obtaining the intensity of fluorescent signals on the microsphere surface, and fitting a linear corresponding relation between the fluorescent intensity and the RNA concentration;
the step S5 specifically comprises the following steps:
s51: changing the RNA in the S3 into an RNA sample to be tested, and executing the same experimental steps as the S3 and the S41-S42;
s52: obtaining the intensity of fluorescent signals on the surfaces of the microspheres;
s53: and quantitatively analyzing the target RNA in the sample by using the linear corresponding relation of the S43 fitting.
2. The quantitative determination method according to claim 1, wherein in S13, the reaction of streptavidin and biotin is performed in a phosphate buffer solution for a reaction time of more than 30 minutes at a reaction temperature of 20-40 ℃.
3. The method according to claim 1, wherein the incubation temperature in S3 is 20-40 ℃ for 40-80 minutes.
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| CN112813143A (en) * | 2020-12-09 | 2021-05-18 | 广州市第一人民医院（广州消化疾病中心、广州医科大学附属市一人民医院、华南理工大学附属第二医院） | Amplification-free RNA quantitative detection method |
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