Optimized nucleic acid detection system based on CRISPR (clustered regularly interspaced short palindromic repeats) mediation and detection method thereof
阅读说明：本技术 一种优化的基于crispr介导的核酸检测系统及其检测方法 (Optimized nucleic acid detection system based on CRISPR (clustered regularly interspaced short palindromic repeats) mediation and detection method thereof ) 是由 费腾 黎子涵 赵文畅 马仕欣 于 2021-03-05 设计创作，主要内容包括：本发明属于核酸检测技术领域,具体涉及一种优化的基于CRISPR介导的核酸检测系统及其检测方法。其中,优化的基于CRISPR介导的核酸检测系统中,每20μL体系包括：50nM～100nM CRISPR-Cas蛋白；50nM～100nM crRNA；200～300nM单链DNA荧光信号报告分子和/或单链RNA荧光信号报告分子；0.2～1M脯氨酸或0.5～1M甜菜碱；CutSmart反应缓冲液；0.1～100ng靶标样品；以及水。本发明提出使用CutSmart反应缓冲液可以使CRISPR-Cas12a和Cas13a系统稳定、高效地进行检测。本发明发现0.2～1M L-Proline的添加可以显著提高Cas12a和Cas13a对靶标序列的检测能力,同时0.5～1M Betaine的添加也可以提升Cas13a对靶标序列的检测能力。(The invention belongs to the technical field of nucleic acid detection, and particularly relates to an optimized nucleic acid detection system based on CRISPR (clustered regularly interspaced short palindromic repeats) mediation and a detection method thereof. Wherein, in the optimized CRISPR-mediated-based nucleic acid detection system, each 20 mu L system comprises: 50nM to 100nM CRISPR-Cas protein; 50nM to 100nM crRNA; 200-300 nM single-stranded DNA fluorescent signal reporter and/or single-stranded RNA fluorescent signal reporter; 0.2-1M proline or 0.5-1M betaine; CutSmart reaction buffer; 0.1-100 ng of target sample; and water. The invention provides a method for stably and efficiently detecting CRISPR-Cas12a and Cas13a systems by using a CutSmart reaction buffer solution. The invention discovers that the addition of 0.2-1M L-Proline can obviously improve the detection capability of Cas12a and Cas13a on target sequences, and the addition of 0.5-1M Betaine can also improve the detection capability of Cas13a on the target sequences.)
1. An optimized CRISPR-based mediated nucleic acid detection system, wherein each 20 μ L system comprises:
50nM to 100nM CRISPR-Cas protein;
50nM～100nM crRNA；
200-300 nM single-stranded DNA fluorescent signal reporter and/or single-stranded RNA fluorescent signal reporter;
0.2-1M proline or 0.5-1M betaine;
CutSmart reaction buffer;
0.1-100 ng of target sample; and water.
2. The optimized CRISPR-mediated-based nucleic acid detection system according to claim 1, wherein the CRISPR-Cas protein is CRISPR-Cas12a protein or CRISPR-Cas13a protein; wherein the content of the first and second substances,
the CRISPR-Cas12a protein is selected from AsCas12a, LbCas12a, Lb4Cas12a, Lb5Cas12a, FnCas12a, HkCas12a, OsCas12a, TsCas12a, BbCas12a or BoCas12 a;
the CRISPR-Cas13a protein is selected from LwaCas13a, LbaCas13a, CamCas13a, LkuCas 13a, LshCas13a, Rca Cas13a, HheCas13a, PprCas13a, LsecCas 13a, LbmCas13a or LbnCas13 a.
3. The optimized CRISPR-based mediated nucleic acid detection system according to claim 2, wherein the CRISPR-Cas12a protein is AsCas12a and the optimized CRISPR-based mediated nucleic acid detection system is an AsCas12a detection system for detecting a DNA target nucleic acid sequence in a target sample;
in the detection system of the AsCas12a, each 20 mu L system comprises 50nM AsCas12a protein, 50nM crRNA, 250nM single-stranded DNA fluorescent signal reporter molecule, 0.2-1M proline, CutSmart reaction buffer solution, a target sample and water.
4. The optimized CRISPR-based mediated nucleic acid detection system according to claim 2, wherein the CRISPR-Cas13a protein is LwaCas13a and the optimized CRISPR-based mediated nucleic acid detection system is LwaCas13a detection system for detecting an RNA target nucleic acid sequence in a target sample;
in the LwaCas13a detection system, each 20 mu L system comprises 100nM LwaCas13a protein, 100nM crRNA, 250nM single-stranded RNA fluorescent signal reporter molecule, 0.2-1M proline or 0.5-1M betaine, CutSmart reaction buffer, a target sample and water.
5. The optimized CRISPR-based mediated nucleic acid detection system according to any of claims 1-4, wherein the molar concentration ratio of CRISPR-Cas protein to crRNA is 1: 1; the water is ultrapure water.
6. An optimized CRISPR-based mediated nucleic acid detection method based on the optimized CRISPR-based mediated nucleic acid detection system of any of claims 1 to 5, comprising the following steps:
s1: obtaining a CRISPR-Cas protein, wherein the CRISPR-Cas protein is a CRISPR-Cas12a protein for detecting DNA and/or a CRISPR-Cas13a protein for detecting RNA;
s2: determining a target nucleic acid sequence of a target sample, and obtaining a purified target nucleic acid sequence by molecular cloning and in vitro transcription technology;
s3: designing specific crRNA aiming at a target nucleic acid sequence, and obtaining purified crRNA by utilizing molecular cloning and in vitro transcription technology;
s4: synthesizing a single-stranded DNA signal reporter molecule and/or a single-stranded RNA signal reporter molecule;
s5: based on the optimized CRISPR-mediated based nucleic acid detection system of any one of claims 1-5, a reaction system is constructed, a target sample is detected, and a real-time quantitative PCR instrument is used for reading a fluorescence signal.
7. The optimized CRISPR-mediated nucleic acid detection-based method according to claim 6, wherein in step S5, a DNA target nucleic acid sequence is added into the CRISPR-Cas12a detection system and/or an RNA target nucleic acid sequence is added into the CRISPR-Cas13a detection system, and the two are respectively mixed uniformly and put into a real-time quantitative PCR instrument to react at 37 ℃ to monitor fluorescence kinetics.
8. The optimized CRISPR-based mediated nucleic acid detection method of claim 6 or 7, further comprising a step of amplifying the target nucleic acid sequence in the target sample before step S5, wherein the amplification is performed by an isothermal amplification technique, and the isothermal amplification technique comprises: RT-RPA and RT-LAMP for RNA target nucleic acid sequence amplification; and RPA and LAMP amplified against DNA target nucleic acid sequences.
9. The optimized CRISPR-based mediated nucleic acid detection method of claim 8, wherein amplifying the target nucleic acid sequence in the target sample comprises:
a) adding the target sample into an RT-RPA or RPA isothermal amplification system, and reacting at 37 ℃ for 30-60 minutes to obtain a specific product through amplification; or adding the target sample into an RT-LAMP or LAMP isothermal amplification system, and reacting for 15-30 minutes at 62 ℃ to obtain a specific product;
b) adding the specific product obtained by amplification in the step a) into a CRISPR-Cas12a detection system, and reacting for 30-60 minutes at 37 ℃.
10. The optimized CRISPR-based mediated nucleic acid detection method according to claim 9,
every 15.6 mu L of RPA or RT-RPA isothermal amplification system contains 0.5-1M betaine, 0.5-1M proline, 5% (v/v) DMSO or 5% (v/v) glycerol;
the LAMP or RT-LAMP isothermal amplification system comprises 0.5-1M proline per 10 mu L.
Technical Field
The invention belongs to the technical field of nucleic acid detection, and particularly relates to an optimized nucleic acid detection system based on CRISPR (clustered regularly interspaced short palindromic repeats) mediation and a detection method thereof.
Background
With the development of biological detection technology, nucleic acid detection technology plays an increasingly important role in the field of diagnosis because of its advantages of rapidness, specificity, accuracy, sensitivity and the like, and is widely applied to rapid detection and identification of viruses, fungi and bacteria, diagnosis of diseases and early screening of tumors. The nucleic acid detection mainly comprises a plurality of detection technologies based on PCR, isothermal amplification (mainly RPA and LAMP), gene chips and second-generation high-throughput sequencing.
At present, real-time quantitative PCR becomes the basic technology and gold standard of a molecular diagnosis method, however, the PCR technology needs repeated thermal denaturation, can not be supported by an accurate temperature control instrument, and needs professional personnel with skilled service ability to operate, thereby greatly limiting the application of the PCR technology in clinical detection. The market demand of gene molecule detection technology is continuously increased, the development trend is increasingly precise, portable and integrated, and the existing nucleic acid detection technology faces the problems of complex operation, high cost, low accuracy, limited application scene and the like. Therefore, there is a need to develop more convenient point-of-care detection systems.
In recent years, CRISPR-Cas systems are gradually applied to the field of molecular detection, wherein development of molecular detection technologies with Cas12 and Cas13 systems as cores is most representative, and the CRISPR-Cas systems become main technical branches of instant nucleic acid detection technologies. Cas12 and Cas13 systems have a similar feature: in the presence of a target nucleic acid sequence, the target nucleic acid sequence can be targeted and combined with specific DNA or RNA, and can generate a trans-cutting activity, namely non-specifically cutting nearby single-stranded DNA or RNA molecules while activating the cis-cutting activity, so that the single-stranded DNA or RNA signal reporter molecule with a fluorescent group at the 5 'end and a fluorescence quenching group at the 3' end is designed by utilizing the characteristic. The content of the target nucleic acid sequence is reflected by the fluorescence signal intensity, and the more the target sequence is in the detection system, the higher the fluorescence signal value is. In practical application, the content of a target sequence in a detection sample hardly reaches the detection limit of a CRISPR detection system, the target sequence to be detected is usually pre-amplified before detection, and in order to reduce the complexity of operation, the joining of the CRISPR detection system by using a nucleic acid isothermal amplification technology without thermal denaturation becomes a common choice. In 2017, by using a Cas13a protein combined RPA isothermal amplification technology and a reverse transcription technology, Zhang et al developed a novel method SHERLOK capable of detecting trace DNA and RNA. In 2018, Jennifer and the like develop a novel method DETECTR capable of detecting nucleic acid, and the methods combine Cas12a protein and LAMP isothermal amplification technology, so that the sensitivity can realize the detection of aM-level samples. Despite the fact that the detection method of SHERELOCK or DETECTR nucleic acid discloses the reaction buffer components of the CRISPR detection system, different laboratories or institutions use artificially prepared reaction buffers, and the detection capability of the reaction buffers is inevitably greatly different. Although isothermal amplification techniques have a constant reaction temperature and a short amplification time, they do not have high specificity for amplification of a target nucleic acid sequence. In addition, the detection sensitivity of the CRISPR detection system needs to be improved, and the long-term maintenance of the protein activity of the CRISPR detection system has a huge problem.
Disclosure of Invention
Aiming at the problems, the invention aims to screen a commercial reaction buffer solution which is easy to obtain so that a CRISPR-Cas12a/Cas13a detection system can be widely and stably applied, and provides a nucleic acid detection method for improving the specificity of an isothermal amplification technology and the detection capability of the CRISPR-Cas12a/Cas13a system by adding a compound so as to solve the problems of poor specificity of the isothermal amplification technology, instability of the CRISPR detection system, low sensitivity and the like.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the invention provides an optimized CRISPR-mediated nucleic acid detection system, wherein each 20 μ L system in the nucleic acid detection system comprises 50 nM-100 nM CRISPR-Cas protein, 50 nM-100 nM crRNA, 200-300 nM single-stranded DNA fluorescent signal reporter (ssDNA FQ reporter) and/or single-stranded RNA fluorescent signal reporter (ssRNA FQ reporter), 0.2-1M Proline (L-Proline) or 0.5-1M Betaine (Betaine), CutSmart reaction buffer, 0.1-100 ng target sample and water, wherein the molar concentration ratio of CRISPR-Cas protein to crRNA is 1: the detection effect is best when 1 is used.
In some alternative embodiments, the CRISPR-Cas protein is a CRISPR-Cas12a protein or a CRISPR-Cas13a protein.
Further, in some embodiments, the CRISPR-Cas12a protein is selected from AsCas12a, LbCas12a, Lb4Cas12a, Lb5Cas12a, FnCas12a, HkCas12a, OsCas12a, TsCas12a, BbCas12a, or BoCas12 a.
In a preferred embodiment, the CRISPR-Cas12a protein is AsCas12a (protein-encoding nucleic acid sequence reference addge plasma #123638) and the optimized CRISPR-based nucleic acid detection system is an AsCas12a detection system for detecting a DNA target nucleic acid sequence in a target sample. Preferably, each 20 mu L system in the detection system of the AsCas12a comprises 50nM AsCas12a protein, 50nM crRNA, 250nM ssDNA FQ reporter, 0.2-1M L-Proline, 10 × CutSmart reaction buffer, target sample and water.
Further, in some embodiments, the CRISPR-Cas13a protein is selected from LwaCas13a, LbaCas13a, CamCas13a, LbuCas13a, LshCas13a, RcaCas13a, HheCas13a, PprCas13a, LseCas13a, LbmCas13a, or LbnCas13 a.
In a preferred embodiment, the CRISPR-Cas13a protein is LwaCas13a (protein-encoding nucleic acid sequence reference adddge plasma #105815) and the optimized CRISPR-based mediated nucleic acid detection system is LwaCas13a detection system for detecting RNA target nucleic acid sequences in a target sample. Preferably, the LwaCas13a detection system comprises 100nM LwaCas13a protein, 100nM crRNA, 250nM ssRNA FQ reporter, 0.2-1M L-Proline or 0.5-1M Betaine, 10 × CutSmart reaction buffer, target sample and water in each 20 μ L system.
In a preferred embodiment, wherein the water is UltraPure water (Ultrapure water), the reaction system is brought to 20. mu.L by water, and the concentrations of the above components are the final concentrations of the system.
In a second aspect, the present invention provides an optimized CRISPR-based mediated nucleic acid detection method based on the above optimized CRISPR-based mediated nucleic acid detection system, specifically comprising the following steps:
s1: obtaining a CRISPR-Cas protein, wherein the CRISPR-Cas protein is a CRISPR-Cas12a protein for detecting DNA and/or a CRISPR-Cas13a protein for detecting RNA;
s2: determining a target nucleic acid sequence of a target sample, and obtaining a purified target nucleic acid sequence by molecular cloning and in vitro transcription technology;
s3: designing specific crRNA aiming at a target nucleic acid sequence, and obtaining purified crRNA by utilizing molecular cloning and in vitro transcription technology;
s4: synthesizing a single-stranded DNA signal reporter molecule and/or a single-stranded RNA signal reporter molecule;
s5: constructing a reaction system based on the optimized nucleic acid detection system based on CRISPR mediation as described in the first aspect, detecting a target sample, and reading a fluorescent signal by using a real-time quantitative PCR instrument.
Further, the preparation method of the CRISPR-Cas12a and Cas13a proteins comprises the following steps: the nucleic acid sequences of the AsCas12a and the LwaCas13a proteins are constructed into a pET-28a prokaryotic expression vector, Rosetta2(DE3) pLysS competent cells are used for transformation, IPTG is used for low-temperature induction of soluble protein expression, and affinity purification, desalting and molecular sieve purification are carried out through a His tag to obtain the target protein.
Furthermore, the target nucleic acid sequence is a partial section of S gene (coding surface Spike protein) and N gene (coding Nucleocapsid protein) of SARS-CoV-2, and the purified RNA target sequence is obtained by PCR, in vitro transcription and other technologies, and the specific sequence is shown in SEQ ID NO.1 and SEQ ID NO. 2.
Further, the preparation method of the specific crRNA comprises the following steps: aiming at the S gene and the N gene of the novel coronavirus, searching a targeting sequence containing a CRISPR-Cas12a recognition sequence (PAM) TTTV, and designing crRNA with the length of 20 nt; cas13a has no explicit recognition sequence (PFS) restriction and a crRNA of 28nt length was designed. When synthesizing the DNA oligo, adding a reverse complementary sequence of a T7 promoter at the 3' end, then carrying out annealing complementary pairing on the DNA oligo with a T7 promoter (T7-F) to form incomplete double-stranded DNA, carrying out in vitro transcription under the action of T7 RNA polymerase, and obtaining the target crRNA through RNA purification, wherein the specific sequence of the crRNA is shown in SEQ ID NO. 4-7.
Further, the single-stranded dna (ssDNA) and rna (ssRNA) fluorescent signal reporter (FQ reporter) are applied to CRISPR-Cas12a and Cas13a detection systems, respectively, and 6-carboxyfluorescein (6-FAM) is used at the 5 'end of the ssDNA/ssRNA FQ reporter, and the fluorescence quencher (BHQ1) is used at the 3' end thereof for labeling, wherein the labeling products are as follows:
ssDNA FQ reporter：5’6-FAM-TTATT-BHQ1 3’(SEQ ID NO.8)
ssRNA FQ reporter：5’6-FAM-UUUUUU-BHQ1 3’(SEQ ID NO.9)。
further, a DNA target nucleic acid sequence is added into the CRISPR-Cas12a detection system, an RNA target nucleic acid sequence is added into the CRISPR-Cas13a detection system, the DNA target nucleic acid sequence and the RNA target nucleic acid sequence are uniformly mixed and put into a real-time quantitative PCR instrument (Applied Biosystems) for reaction at 37 ℃, and the fluorescence kinetics is monitored.
Further, when detecting the trace target nucleic acid sequence, a step of amplifying the target nucleic acid sequence in the target sample is further included before step S5. The amplification adopts an isothermal amplification technology, and the isothermal amplification technology comprises RT-RPA and RT-LAMP for RNA target nucleic acid sequence amplification; and RPA and LAMP amplified against DNA target nucleic acid sequences.
Specifically, adding a target sample into an RT-RPA or RPA isothermal amplification system, and reacting at 37 ℃ for 30-60 minutes to obtain a specific product through amplification; or adding the target sample into an RT-LAMP or LAMP isothermal amplification system, carrying out amplification at 62 ℃ for 15-30 minutes to obtain a specific product, adding the specific product obtained by amplification into a CRISPR-Cas12a detection system, and carrying out reaction at 37 ℃ for 30-60 minutes.
Furthermore, in the process of combining the isothermal amplification technology with the CRISPR detection system, 0.5-1M beta, 0.5-1M 1M L-Proline, 5% (v/v) DMSO or 5% (v/v) Glycerol (Glycerol) is added into each 15.6 mu L of RPA or RT-RPA isothermal amplification system, so that the detection signal of the CRISPR system can be improved; 0.5-1M L-Proline is added into each 10 mu L LAMP or RT-LAMP isothermal amplification system, and the detection signal of the CRISPR system can also be obviously improved.
The invention has the advantages that:
(1) the invention provides a method for stably and efficiently detecting CRISPR-AsCas12a and LwaCas13a by using a CutSmart reaction buffer solution.
(2) The invention discovers that the addition of 0.2-1M L-Proline can obviously improve the detection capability of AsCas12a and LwaCas13a on target sequences, and the addition of 0.5-1M Betaine can also improve the detection capability of LwaCas13a on target sequences.
(3) The invention discovers that the amplification of a specific product can be obviously improved by adding 0.5-1M L-Proline into an RT-LAMP amplification system; the amplification of specific products can be improved by adding 0.5-1M Betaine, 0.5-1M L-Proline, 5% DMSO or 5% Glycerol to an RT-RPA amplification system.
(4) The invention discloses an optimized nucleic acid detection system based on CRISPR mediation and a detection method thereof, solves the problems of poor specificity of isothermal amplification technology, instability and low sensitivity of a CRISPR detection system, and provides an efficient, accurate and stable detection method for clinical diagnosis and laboratory research.
Drawings
FIG. 1 is a schematic diagram of the principle of CRISPR-Cas12a/Cas13a nucleic acid detection;
FIG. 2 is a schematic diagram of the genomic location of SARS-CoV-2 target sequence and crRNA;
FIG. 3 is a comparison of the effect of different reaction buffers on the detection ability of AsCas12 a;
FIG. 4 is a comparison of the effect of different reaction buffers on the detectability of LwaCas13 a;
FIG. 5 is a schematic diagram of the effect of added compounds on the CRISPR-Cas12a/Cas13a detection system;
FIG. 6 shows the fluorescence detection result of detecting SARS-CoV-2S gene by AsCas12a after adding different compounds;
FIG. 7 shows the result of dynamic monitoring fluorescence signal of detecting SARS-CoV-2S gene by the L-Proline added AsCas12a detection system;
FIG. 8 shows the fluorescence detection result of the gene SARS-CoV-2N detected by LwaCas13a after adding different compounds;
FIG. 9 shows the result of dynamic monitoring fluorescence signal of the detection system of LwaCas13a with the addition of L-Proline for detecting SARS-CoV-2N gene;
FIG. 10 is a schematic diagram of the detection of specific products using the AsCas12a system with the addition of compounds during RPA and LAMP amplification;
FIG. 11 shows the fluorescence detection result (N gene) of RT-LAMP amplification products after addition of different compounds based on the AsCas12a system;
fig. 12 shows that the addition of 0.5M L-Proline significantly improved the ability to amplify specific products of RT-LAMP (N gene, N ═ 3);
FIG. 13 shows the results of fluorescence detection of RT-RPA amplification products (S gene) after addition of different compounds based on the AsCas12a system;
FIG. 14 shows that the addition of 1M Betaine, 1M L-Proline and 5% DMSO significantly improves the ability of RT-RPA to amplify specific products (S gene).
Detailed Description
The invention is further illustrated by the following examples, but not by way of limitation, in connection with the accompanying drawings. The following provides specific materials and sources thereof used in embodiments of the present invention. However, it should be understood that these are exemplary only and not intended to limit the invention, and that materials of the same or similar type, quality, nature or function as the following reagents and instruments may be used in the practice of the invention. The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
In the following examples: twist AmpTMBasic Kit was purchased from TwistDx corporation; WarmStart RTx Reverse Transcriptase (# M0380L), ProtoScript II Reverse Transcriptase (# M0368L), Bst 2.0WarmStart DNA Polymerase (# M0538S), NEB2 Buffer (# B7002S), NEB2.1Buffer (# B7202S), NEB3 Buffer (# B7003S), NEB3.1 Buffer (# B7203S), Isothermal Amplification Buffer (# B0537S), and CutSmart Buffer (# B7204S) were all available from NEB corporation; takara T Buffer (# SD6092) and Takara K Buffer (# SD6076) were purchased from Takara; t7 RNA Polymerase (#30223-1) was purchased from NxGen; conventional reagents such as Tris-HCl, MgCl2DMSO and glycerol, etc. were purchased from Thermo Fisher; FQ reporter nucleic acid sequence synthesis was accomplished by Shanghai Huajin Biotechnology, Inc.; the conventional primers were synthesized by Suzhou Hongxn Biotechnology Ltd.
The target sequences of the S and N genes of SARS-CoV-2, and the nucleic acid sequence information of crrnas of CRISPR-Cas12a and Cas13a in the examples of the present invention are shown in tables 1 and 2.
TABLE 1 target sequences of the S and N genes of SARS-CoV-2
TABLE 2 CRISPR-Cas12a/Cas13a CRRNA targeting S and N genes
Example 1: detection of SARS-CoV-2 Using the present invention
The principle schematic diagram of CRISPR-Cas12a/Cas13a nucleic acid detection is shown in fig. 1, Cas12a and Cas13a bind to their corresponding crrnas to form complexes, target DNA and RNA target sequences under the guidance of specific crRNA, respectively, and generate a trans-cleavage activity, that is, non-specifically cleave nearby single-stranded DNA or RNA FQ reporter while activating cis-cleavage activity, thereby generating a fluorescence signal that can be read, and by using this correlation, the content of the target sequence can be determined by the strength of the fluorescence signal.
The genome mapping scheme of SARS-CoV-2 target sequence and crRNA is shown in FIG. 2, the positions of the S gene and N gene target sequence on SARS-CoV-2 genome are shown in the figure as most parts, and the designed positions of specific crRNA are marked on the target sequences of S gene and N gene, and are indicated by gray bars.
In the embodiment, the DNA target sequence of the S gene is annealed, complemented and paired by two DNA oligos to form double-stranded DNA, the sequences at two ends of the DNA are extended by using a PCR technology, and the DNA target sequence of the S gene is obtained by purification and named as SARS-CoV-2-S-DNA; when the S gene DNA is elongated, a T7 promoter sequence is introduced at the 5' end, and the DNA fragment is transcribed into RNA by T7 RNA polymerase (NxGen), which is named SARS-CoV-2-S-RNA.
In this example, a 20 μ L system is adopted for detection, as shown in table 3, but not limited thereto, including the adjustment of the ratio of the corresponding components:
TABLE 3 CRISPR-Cas12a/Cas13a detection system for SARS-CoV-2 virus
Wherein 10 × Buffer is prepared from NEB2, NEB2.1, NEB3, NEB3.1, Takara T, Takara K, Isothermal Amplification Buffer and CutSmart reaction Buffer by using H2The test group with O as reaction buffer served as a negative control.
After the preparation of the reaction system is completed, the reaction system is uniformly mixed and put into a real-time quantitative PCR instrument (Applied Biosystems) for reaction at 37 ℃, the fluorescence kinetics is monitored, the detection is carried out once every 1 minute, and the detection lasts for 90 minutes. And subtracting the background ROX value from the fluorescence FAM value collected at each time point, and analyzing and comparing by using the processed fluorescence signal value as raw data.
As a result: as shown in FIGS. 3 and 4, both AsCas12a and LwaCas13a can stably and efficiently detect target sequences under the conditions of a CutSmart reaction buffer, and the detection activity of the kit is remarkably superior to that of the kit in other reaction buffers.
Example 2: addition of compound improves detection capability of CRISPR-Cas12a/Cas13a on SARS-CoV-2
A schematic diagram of adding compounds in a CRISPR-Cas12a/Cas13a detection system is shown in FIG. 5, SARS-CoV-2-S-DNA is used as a target sequence of an AsCas12a detection system, SARS-CoV-2-N-RNA is used as a target sequence of an LwaCas13a detection system, and different compounds are respectively added in the two detection systems to screen compounds capable of remarkably improving CRISPR detection signals.
In this embodiment, a 20 μ L system is adopted as shown in table 4, but not limited thereto, and includes the following components in proportion:
TABLE 4 CRISPR-Cas12a/Cas13a detection system for adding different compounds to target SARS-CoV-2 virus
Wherein the screened compounds include 0.2M Betaine, 0.5M Betaine, 1M Betaine, 0.2M L-Proline, 0.5M L-Proline, 1M L-Proline, 0.2M Urea, 0.5M Urea, 1M Urea, 1% Formamide, 5% Formamide, 10% Formamide, 1% DMSO, 5% DMSO, 10% DMSO, 1% Glycerol, 5% Glycerol, and 10% Glycerol (percentages are volume percentages); the detection group without the compound is used as a control group and is named as no chemical; the test group with neither compound nor target sequence added served as a negative control and was designated as no template.
In this example, the detection ability of CRISPR-Cas12a/Cas13a was determined using fluorescence signal intensity. And (3) measuring the fluorescence of the detection reaction by using a real-time quantitative PCR instrument, carrying out the reaction at 37 ℃, and monitoring the fluorescence kinetics, wherein a Reporter channel selects FAM, a Quencher channel selects NFQ-MGB, the detection is carried out once every 1 minute, and the detection lasts for 1 hour. And subtracting the background ROX value from the fluorescence FAM value collected at each time point, and performing data analysis by using the processed fluorescence signal value. The effect of adding different compounds on the detection ability of CRISPR-Cas12a/Cas13a was compared using endpoint signal values after 1 hour of detection.
As shown in FIG. 6, compared with a no chemical control group, the fluorescence detection result of SARS-CoV-2S gene detected by AsCas12a added with different compounds is that the detection capability of the AsCas12a system can be improved by 5-7 times by adding 0.2M L-Proline, 0.5M L-Proline and 1M L-Proline, and the detection capability of the AsCas12a system is improved by about 2 times by adding 10% of Glycerol; the result of dynamic monitoring of fluorescence signals of SARS-CoV-2S gene detected by the L-Proline added AsCas12a detection system is shown in FIG. 7, and the increase rate of fluorescence signals is significantly improved by the addition of L-Proline.
The fluorescence detection result of the SARS-CoV-2N gene detected by the LwaCas13a after different compounds are added is shown in FIG. 8, compared with a no chemical control group, the addition of 0.2M L-Proline, 0.5M L-Proline and 1M L-Proline can improve the detection capability of the LwaCas13a system by more than 2 times, and the addition of 1M Betaine also improves the detection capability of the LwaCas13a system by about 2 times; the results of dynamic monitoring of fluorescence signals of SARS-CoV-2N gene detected by the L-Proline added LwaCas13a detection system are shown in FIG. 9, and the increase rate of fluorescence signals is significantly improved by the addition of L-Proline.
In the embodiment, the addition of L-Proline obviously improves the detection activities of CRISPR-Cas12a and Cas13 a.
Example 3: detection of trace amount of SARS-CoV-2 by combination of AsCas12a system and isothermal amplification technology
Adding compounds in the RPA and LAMP amplification process, using an AsCas12a system to detect specific products as a schematic diagram shown in FIG. 10, adding different compounds in the amplification process to amplify target sequences by using SARS-CoV-2-S-RNA and SARS-CoV-2-N-RNA as templates of an RT-RPA and RT-LAMP amplification system respectively, then carrying out PCR purification on the amplification products to remove the added compounds, interfering substances such as DNA polymerase and reverse transcriptase and the like, and using the purified DNA as the input of an AsCas12a detection system for detection.
In this example, RT-LAMP amplification was performed using a 10. mu.L system as shown in Table 5, but the method is not limited thereto, and includes the following steps:
TABLE 5 target SARS-CoV-2-N-RNA RT-LAMP amplification System with addition of different Compounds
Wherein the added compounds comprise 0.5M Betaine, 1M Betaine, 0.5M L-Proline, 1M L-Proline, 0.5M Urea, 1M Urea, 1% Formamide, 5% DMSO, 10% DMSO, 5% Glycerol and 10% Glycerol (the percentage is volume percentage), and SARS-CoV-2-N-RNA templates of all the amplification systems of the added compounds are 103Copying; the N-RNA template is 103Copy and no compound amplification system as control, designated 103copies; the N-RNA template is 106The amplification system with copy and no compound as positive control group was named 106copies; the amplification system with neither compound nor target sequence added was designated as a negative control, 0 copy.
Specific primer sequences used in the RT-LAMP amplification system are shown in Table 6.
TABLE 6 RT-LAMP amplification primer sequences targeting SARS-CoV-2-N-RNA
In this example, RT-RPA amplification was performed using a 15.6. mu.L system as shown in Table 7, but not limited thereto, including the adjustment of the ratio of the corresponding components:
TABLE 7 RT-RPA amplification System for the target SARS-CoV-2-S-RNA addition of different Compounds
Wherein the added compounds comprise 0.5M Betaine, 1M Betaine, 0.5M L-Proline, 1M L-Proline, 0.5M Urea, 1M Urea, 1% Formamide, 5% DMSO, 10% DMSO, 5% Glycerol and 10% Glycerol (the percentage is volume percentage), and SARS-CoV-2-S-RNA templates of all the amplification systems of the added compounds are 103Copying; S-RNA template is 103Copy and no compound amplification system as control, designated 103copies; S-RNA template is 106The amplification system with copy and no compound as positive control group was named 106copies; the amplification system with neither compound nor target sequence added was designated as a negative control, 0 copy.
Specific primer sequences used in the RT-RPA amplification system are shown in Table 8.
TABLE 8 RT-RPA amplification primer sequences targeting SARS-CoV-2-S-RNA
After the amplification products of RT-LAMP and RT-RPA are purified by PCR, the amplification products are respectively detected by using an AsCas12a detection system, the detection system refers to Table 3, wherein a detection group without any nucleic acid sequence in the detection system is set as a control group and is named as CRISPR Ctrl.
CRISPR detection is a specific process that activates its nuclease activity only in the presence of sequences that crRNA can target. Based on the specific detection capability, because the amplification specificity of the isothermal amplification technology is poor, the content of the target sequence in the amplification product input into the detection system is reflected by the fluorescence signal value of the detection system, so that the stronger the fluorescence signal, the more the specific target sequence input into the CRISPR detection system is, and the more the added compound improves the specificity of the amplification system.
As shown in FIG. 11, the fluorescence detection result (N gene) of RT-LAMP amplification products after detection of different compounds based on the AsCas12a system is that the fluorescence signal input by the sample of the AsCas12a detection system, which is obtained by adding amplification products of 0.5M L-Proline and 1M L-Proline to the RT-LAMP amplification system, is significantly higher than 103The copies group and the other compound addition group showed that the addition of 0.5M L-Proline and 1M L-Proline amplified more specific products by RT-LAMP. Furthermore, FIG. 12 shows the effect of the addition of 0.5M L-Proline on the amplification specificity of RT-LAMP, and the addition of 0.5M L-Proline at 103copies and 105The specificity of the RT-LAMP amplification system is obviously improved under the input level of the copies template.
The fluorescence detection result (S gene) of RT-RPA amplification products after detection of different compounds based on the AsCas12a system is shown in FIG. 13, wherein 0.5M Betaine, 1M Betaine, 0.5M L-Proline, 1M L-Proline, 5% DMSO and 5% Glycerol are added in the RT-RPA amplification system, and the fluorescence signal of the AsCas12a detection system is obviously higher than 103The copies group and the other compound addition group show that the addition of the compounds can make RT-RPA amplify more specific products. In addition, the results of detecting the dynamic fluorescence signals of RT-RPA amplification products with 1M Betaine, 1M L-Proline and 5% DMSO added based on the AsCas12a system are shown in FIG. 14, which is 103The copy S gene RNA is used as a template of an RT-RPA amplification system, and the content of an amplification product of the copy S gene RNA cannot reach the detection limit of an AsCas12a detection system; after the three compounds are added into an RT-RPA amplification system, the amplification product can enable the fluorescence signal detected by AsCas12a to be rapidly increased.
The above description of exemplary embodiments has been presented only to illustrate the technical solution of the invention and is not intended to be exhaustive or to limit the invention to the precise form described. Obviously, many modifications and variations are possible in light of the above teaching to those skilled in the art. The exemplary embodiments were chosen and described in order to explain certain principles of the invention and its practical application to thereby enable others skilled in the art to understand, implement and utilize the invention in various exemplary embodiments and with various alternatives and modifications. It is intended that the scope of the invention be defined by the following claims and their equivalents.
Sequence listing
<110> university of northeast
<120> an optimized nucleic acid detection system based on CRISPR mediation and detection method thereof
<130> 2020S1099
<160> 17
<170> PatentIn version 3.5
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