CN117778386A

CN117778386A - CrRNA and CRISPR diagnostic kit for single base mutant RNA detection

Info

Publication number: CN117778386A
Application number: CN202311668466.4A
Authority: CN
Inventors: 张开翔; 李亚楠; 徐汝; 全丰蕾; 高华; 张振中
Original assignee: Zhengzhou University
Current assignee: Zhengzhou University
Priority date: 2023-12-06
Filing date: 2023-12-06
Publication date: 2024-03-29

Abstract

The invention belongs to the research field of molecular diagnosis and biochemical analysis methods, and particularly relates to a crRNA and CRISPR diagnostic kit for single-base mutant RNA detection. In the crRNA of the present invention, the site for recognizing the single base mutation is located at the 3 rd position of the 5' end of the crRNA spacer region, and the crRNA further has any one of the following designs: the 2, 4 or 5 position of the 5' end of the crRNA spacer comprises 1-3 mismatches; or adding n bases at the 3 'end of the crRNA, and complementarily pairing with the first n bases at the 5' end of the crRNA spacer to form a hairpin structure. The invention also constructs a CRISPR diagnostic kit by using the crRNA. The invention can overcome the problem of insufficient single base mutation detection capability of CRISPR diagnosis, realizes the specificity and high-sensitivity detection of RNA, and has good application prospect.

Description

CrRNA and CRISPR diagnostic kit for single base mutant RNA detection

Technical Field

The invention belongs to the research field of molecular diagnosis and biochemical analysis methods, and particularly relates to a crRNA and CRISPR diagnostic kit for single-base mutant RNA detection.

Background

An RNA virus is a single stranded RNA virus, the genetic material of which is RNA. Compared to DNA viruses, RNA viruses are more prone to mutation due to the lack of a proofreading mechanism during replication of RNA, resulting in a higher error rate. The mutation rate of RNA viruses is high, but the adaptation of each mutant may be different. This property makes RNA viruses highly diverse and adaptable, but also makes them difficult to prevent and treat. Common RNA viruses include influenza virus, aids virus, ebola virus, coronavirus, and the like.

Coronaviruses are a type of enveloped RNA viruses widely existing in nature, can cause diseases such as influenza, middle East Respiratory Syndrome (MERS), severe Acute Respiratory Syndrome (SARS) and the like, and bring great pressure to human health, social stability and global economy. Rapid variation of coronaviruses has an important impact on viral transmission, pathogenicity and efficacy of therapeutic interventions. Because coronaviruses have the characteristics of high transmission speed, serious clinical symptoms and the like, an economic and efficient rapid detection strategy needs to be developed to analyze and monitor variant strains of the coronaviruses in real time. In addition, many variants of coronaviruses contain dominant genotypes that occur independently, suggesting that specific mutation site assays may be an effective strategy for predicting the direction of evolution of a dangerous virus strain. However, the lack of rapid and immediate monitoring of coronavirus variants is currently severely hampering related epidemiological and clinical studies. Therefore, the development of a novel rapid coronavirus variant analysis and detection method is helpful for tracking the evolution trend of specific variants in the crowd and improving the prediction and prevention and control capability of related diseases.

The current gold standard method for detecting variants of RNA viruses such as coronaviruses is whole genome sequencing, and can obtain the whole sequence information of the virus strains for the accurate typing of different types of viruses. However, the high cost of sequencing-related experiments limits the application of sequencing methods to crowd-wide virus real-time monitoring. Nucleic acid detection based on polymerase chain reaction (Polymerase Chain Reaction, PCR) has also been widely used for diagnosing various mutant subtypes of coronavirus, but is limited by the need for precise temperature control of instruments and specialized experimenters, relatively complex probe designs, and nucleic acid amplification bias of the PCR reaction itself, which are currently mainly used for detection applications in central laboratories, and are relatively difficult to use for simple and rapid point-of-care test (POCT) molecular diagnosis. Therefore, the development of a rapid, sensitive, low-cost, portable and easy-to-operate detection system with single base specificity is of great importance for detection studies of coronavirus variants.

CRISPR diagnostic (CRISPR-Dx) technology can specifically recognize target sequences through CRISPR RNA (crRNA) and output detection signals by activating CRISPR-associated protein (Cas) to cleave DNA or RNA reports. Commercial detection platforms based on CRISPR-Dx, such as specific high sensitivity enzyme reporter Solution (SHERLOCK) and DNA endonuclease targeted CRISPR trans-report (detect), have been successfully developed for detection of RNA viruses such as SARS-CoV-2. Advances in amplification-free CRISPR-Dx technology, including superactive lwaca 13a effectors, CRISPR-cell phone microscopy, gwaca 13a systems based on gfets, and ECRISPR, effectively avoid cumbersome laboratory settings, shorten detection times, and thereby promote the application of CRISPR-Dx in pathogen screening and infectious disease diagnosis.

However, the use of CRISPR-Dx in the identification of RNA viral variants remains a challenge. In general, cas proteins, particularly Cas9 and Cas13a, are tolerant of single base mismatches between the crRNA and the target sequence. Thus, CRISPR-Dx has limited discrimination in recognizing single base mutations in the genome of RNA viral variants. It is also a great challenge to explore highly sensitive, target-free pre-amplified CRISPR-Dx technology to rapidly detect and identify RNA viruses and variants thereof with single nucleotide resolution.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides a crRNA and CRISPR diagnostic kit for single-base mutant RNA virus detection.

A crRNA for single base mutant RNA detection, the site for identifying the single base mutation being located at the 3 rd position of the 5' end of the crRNA spacer;

the crRNA also has a design of any one of the following:

1) The 2, 4 or 5 position of the 5' end of the crRNA spacer comprises 1-3 mismatches;

2) Adding n bases at the 3 'end of the crRNA, and complementarily pairing with the first n bases at the 5' end of the crRNA spacer to form a hairpin structure; n has a value of 8-10.

Preferably, the crRNA has a design of any one of:

the 2 nd and 4 th positions of the 5' end of the crRNA spacer region are mismatched, and the nucleotide sequence of the crRNA is shown as SEQ ID NO. 1;

or, the 5 th position of the 5' end of the crRNA spacer region is mismatched, and the nucleotide sequence of the crRNA is shown as SEQ ID NO. 6;

or, 2 nd, 4 th and 5 th positions of the 5' end of the crRNA spacer region are mismatched, and the nucleotide sequence of the crRNA is shown as SEQ ID NO. 4;

or, the 3 '-end of the crRNA is added with 8 bases and complementarily paired with the first 8 bases at the 5' -end of the crRNA spacer region to form a hairpin structure; the nucleotide sequence of the crRNA is shown in any one of SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO. 5.

The invention also provides a design method of the crRNA for single-base mutant RNA detection, which comprises the following steps:

step 1, setting a site for identifying single base mutation at the 3 rd position of the 5' end of the crRNA spacer according to the sequence of single base mutant RNA to be identified;

step 2, designing the crRNA according to any one of the following modes:

2) Adding n bases at the 3 'end of the crRNA, and complementarily pairing with the first n bases at the 5' end of the crRNA spacer to form a hairpin structure; n is 8-10;

and 3, designing the sequence of the rest part of the crRNA according to the sequence of the single-base mutant RNA to be identified.

The invention also provides application of the crRNA for single-base mutant RNA detection in preparing a CRISPR diagnostic kit for single-base mutant RNA detection.

The invention also provides a CRISPR diagnostic kit for single-base mutant RNA detection, which comprises Cas13a and the crRNA for single-base mutant RNA detection.

Preferably, the kit further comprises: topology DNA/RNA nanocollar, T4PNK, phi29DNA polymerase, nucleic acid dye, pyrophosphatase and reaction buffer.

Preferably, the topological DNA/RNA nanoloop is used for responding to trans-cleavage activity and signal amplification of Cas13a, and is formed by topological hybridization of two circular DNA strands, wherein one DNA loop contains 2-4 continuous RNA bases U.

Preferably, the nucleic acid dye is selected from 1 XSYBRGreenI.

Preferably, the composition of the reaction buffer is:

0.5～1×phi29 buffer、

5～8mM Mg(COOH) ₂ 、

30～35mM KCOOH、

0.01～0.1％v/v Tween 20、

0.4～1mM DTT、

0.5～1×HOLMES Buffer 2、

0.5～1mMdTNP mix。

preferably, the kit further comprises a gene synthesis sample to be tested.

In the present invention, the RNA to be detected is an RNA molecule which is highly correlated with the occurrence and progression of a disease, and dynamically reflects the cell state and regulatory processes, for example: mRNA, non-coding RNA, circular RNA, RNA virus, and the like.

The "crRNA" is an RNA molecule in the CRISPR/Cas13a system for guiding Cas13a protein to locate and cleave a target RNA, the sequence of which comprises two parts: framework sequences and spacer (spacer) sequences. The crrnas of the present invention design spacer regions.

Aiming at the problem that single mismatch of a target can be tolerated by Cas13a/crRNA, so that the specificity of CRISPR-Dx detection of single-base mutant RNA (such as RNA virus) is insufficient, the invention provides a novel crRNA design concept. Specifically, the site to be identified is placed at the 3 rd position of the 5 'end of the crRNA spacer, then single mismatch is added at the 2 nd position of the 5' end of the crRNA spacer, so that the crRNA identification mutant type is 1 mismatch, the wild type is 2 mismatches, and the activation effects of the two on Cas13a with different degrees are distinguished. If mutant and wild type cannot be distinguished, a different number or position of mismatches are added, or crRNA is engineered into hairpin structures. When the single base mutation to be detected is not present, the crrnas with different numbers of synthetic mismatches generate protrusions in the targeted recognition process, so that stricter distinction is generated at the target position, and the capability of the Cas13a/crRNA probe to recognize the single base mutation is enhanced. The specific principle is as follows: recognition of wild-type and mutant targets by Cas13a/crRNA probes is achieved by complementary pairing of the spacer region of the crRNA with the target base. The smaller the gibbs free energy of both, the more stable its binding, the stronger the trans-cleavage activity of Cas13 a. However, in the presence of single base mutations, crrnas bind similarly to wild-type and mutant targets and are therefore indistinguishable. By increasing the number or position of mismatches, or modifying the crRNA to a hairpin structure, the gibbs free energy between the wild-type target and the crRNA can be increased, making the binding of the two more unstable, thereby achieving a significant distinction between wild-type and mutant.

Based on the principle, the following beneficial effects are realized through the technical scheme of the invention:

1. the crRNA designed according to the method can specifically identify a plurality of RNA single base mutation sites, and can effectively improve the sensitivity of the CRISPR-Dx technology to single base mutation RNA detection.

2. When the crRNA provided by the invention is used for CRISPR-Dx detection, a target pre-amplification step and one-pot analysis of whole-process closed tube are not needed, so that the problems of biosafety and false positive caused by aerosol pollution are avoided.

3. The design principle of the Cas13a/crRNA probe provided by the invention can be applied to crRNA engineering design of a plurality of RNA single base mutation sites.

4. In a preferred scheme, the CRISPR-Dx detection kit provided by the invention can be used for detection by a one-pot method, the detection process is quite simple, and the separation and temperature control processes are omitted.

5. In a preferred scheme, the invention constructs a report and signal amplification system of the topological DNA/RNA nano-collar as Cas13a, and the ultra-sensitive detection of the low-abundance mutant sites can be realized by using the rolling ring amplification strategy mediated by the topological DNA/RNA nano-collar as a high-efficiency signal amplification module.

6. The method provided by the invention can design crRNA of various mutation sites, realize detection of various mutation sites, and can be suitable for accurate identification of various pathogen subtypes by a combined coding mode.

It should be apparent that, in light of the foregoing, various modifications, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.

The above-described aspects of the present invention will be described in further detail below with reference to specific embodiments in the form of examples. It should not be understood that the scope of the above subject matter of the present invention is limited to the following examples only. All techniques implemented based on the above description of the invention are within the scope of the invention.

Drawings

Fig. 1: the Cas13a/crRNA probe design principle proposed by the present invention and the ability of Cas13a to recognize SNPs in example 1 using the high-frequency mutation site of the novel coronavirus as a target are explored. Wherein fig. 1A is a engineered flow for Cas13a to recognize single base mutations. FIG. 1B is a schematic representation of crRNA engineering classification. FIGS. 1C-H show the modification and recognition of 6 crRNAs.

Fig. 2: schematic design and preparation of topologically DNA/RNA nanocollars in example 2 of the present invention.

Fig. 3: synthesis of topologically DNA/RNA nanocollars in example 2 of the invention. Wherein FIG. 3A shows the steps of preparing and purifying the topological DNA/RNA nanocollar, and removing the isomer by polyacrylamide gel purification to obtain the topological double ring with strong interlocking capability. FIG. 3B is a topological DNA/RNA nanocollar synthesized by 12% polyacrylamide gel electrophoresis analysis. Fig. 3C is a study of the topological interlocking structure of topological DNA/RNA nanocollars.

Fig. 4: the one-pot method of example 2 of the present invention was limited. FIG. 4A shows the detection limit of the synthesized sample (N gene). FIGS. 4B-C show pseudovirus (N gene) detection limits. Fig. 4D is the result of 20 replicates of the low copy sample. Statistical results are shown as mean ± SD (n=3).

Fig. 5: in the embodiment 3 of the invention, the feasibility of different variant strains is judged by a one-pot method. FIG. 5A is a schematic representation of mutation site combination coding. FIGS. 5B-H are combinations of 9 crRNAs recognizing different variants.

Fig. 6: the one-pot method of example 4 of the present invention was used for analysis of clinical samples. FIG. 6A combinatorial coding analysis of three clinical variant sequencing samples. Fig. 6B is a schematic diagram of a portable small instrument. Fig. 6C is a visual analysis of a clinical variant of the portable small instrument and fig. 6D-E are results of the portable small instrument analysis.

Detailed Description

Except for the specific descriptions, the experimental materials, reagents and equipment in the examples can be obtained through conventional purchase channels; the experimental methods used in the examples are conventional methods unless otherwise specified.

EXAMPLE 1 crRNA for Single base mutant RNA Virus detection

The present example provides crRNA for single base mutant RNA virus detection, which is designed as shown in FIG. 1A, comprising the following specific steps:

step 1, setting a site for identifying single base mutation at the 3 rd position of the 3' end of the crRNA according to the sequence of the single base mutant RNA virus to be identified;

step 2, designing the crRNA according to any one of the following modes:

step 3, designing the sequence of the rest part of the crRNA according to the sequence of the single base mutant RNA virus to be identified.

According to the above method, in this example, taking the characteristic mutation (shown in FIG. 1B, Q493R, K444T, W152R, V83A, F486S, Δ69/70, N501Y) of different Omeganone variants of COVID-19 as an example, crRNA probes capable of specifically recognizing mutant strains were designed, and 6 crRNA sequences were designed as follows in the manner shown in FIG. 1C (SM 1: mismatch at 5 'end of crRNA spacer, mismatch at 5' end of SM2: crRNA spacer, mismatch at 2 nd and 4 th end of crRNA spacer, mismatch at 2 nd, 4 th end of crRNA spacer, 5 th end of crRNA spacer, 8 bases added at 3 'end of Hairpin-spacer, complementary pairing with the first 8 bases at 5' end of crRNA spacer, to form Hairpin structure):

in addition, the present example also examined the sequences of conserved regions of the N gene and O gene as a verification of new coronaviruses. 2 crRNA sequences were as follows:

the sequences of the above 9 genes and mutants thereof (wherein MT is mutant and WT is wild-type) are shown in the following table:

the Cas13a/crRNA complex composed of the crrnas described above was validated for its ability to recognize SNPs by the following method:

(1) Preparation of crRNA: corresponding crRNA (SEQ ID NO.1-SEQ ID NO. 9) is designed according to the different mutation target RNA sequences screened, and then a transcription template containing a T7 promoter, a T7 forward primer and a reverse primer are ordered for PCR reaction to serve as a double-stranded DNA template for transcribing the crRNA. In vitro transcription synthesis was performed using a Hiscibe T7 rapid and efficient RNA in vitro synthesis kit (Hiscibe T7 Quick High Yield RNA Synthesis) incubated overnight (8 h above) at 37 ℃. Finally, purifying crRNA by using a TRlzon method, and determining the concentration of the obtained crRNA solution by using a Nanodrop nucleic acid quantitative instrument, and storing the crRNA solution in a refrigerator at the temperature of-80 ℃ to prevent degradation.

(2) Recognition of single base mutation sites by Cas13 a/crRNA: mu.L of 100nM Cas13a, 1. Mu.L of 100nM crRNA (SEQ ID NO.1-SEQ ID NO. 9), 2. Mu.L of 100nM Target RNA, 2. Mu.L of 1. Mu.M ssRNA reporter, 2. Mu.L of 10 XHOLMES Buffer 2 and 11. Mu.L of DEPC treated water were mixed on ice. The reaction system was incubated at 37℃for 30 minutes, and then fluorescence signals (excitation wavelength: 488nm, emission wavelength: 520 nm) were detected using an enzyme-labeled instrument. Wherein, the nucleotide sequence of ssRNA reporter is: u (FAM) UUUU (BHQ-1).

FIGS. 1D-H are crRNA screens of different mutation sites, and the horizontal scale red represents that the mutant and wild type of the sites can be effectively distinguished by the modification mode of the crRNA.

The experimental results show that aiming at different mutation targets of the novel coronavirus, effective distinction of single-base mutation of mutation sites and wild sites can be realized by modifying the structure of crRNA, including adding mismatch, modifying into hairpin shape and the like.

Example 2 CRISPR diagnostic kit and CRISPR diagnostic method for single base mutant RNA Virus detection

The present example provides a kit for single-base mutant RNA virus detection and a CRISPR diagnostic method for single-base mutant RNA virus detection using the same. The crRNA used in this example was designed according to the method of example 1. For example, using the characteristic mutations of the different Omikovia variants of COVID-19 (Q493R, K444T, W152R, V83A, F486S, Δ69/70, N501Y) as an example, the crRNA sequences were designed as shown in SEQ ID NO.1-SEQ ID NO. 7.

1. Composition of the kit (10 parts)

Component (A)	Specification of specification
		crRNA	10 uL/pipe (10 uM)
Cas13a	10 uL/tube (10 pmol/uL)
		Topological DNA/RNA nanocollars	50 uL/pipe (300 pM)
T4PNK	20 uL/pipe (10U/uL)
		phi29DNA polymerase	10 uL/pipe (10U/uL)
10000 XSYBRGreenI nucleic acid dye	10 uL/tube
		Pyrophosphatase enzyme	10 uL/pipe (0.1U/uL)
10×phi29buffer	50 uL/tube
		10×HOLMES Buffer 2	50 uL/tube
10mMdTNP mix	50 uL/tube
		DEPC treated water	1 mL/tube

A topological DNA/RNA nanoloop is used to respond to trans-cleavage activity and signal amplification of Cas13a, which is formed by topological hybridization of two circular DNA strands. One of the topological DNA/RNA nano-collars is used as an identification ring and contains 2-4 continuous RNA bases U for trans-shearing of Cas13 a; one acts as a reporting ring, functioning as a template for RCA. After cleavage of the recognition loop by Cas13a, the template becomes stranded as a primer and the RCA reaction takes place with the reporter loop as RCA.

In this example, the sequences of the two circular DNA strands are as follows:

1. ^C DNA _R (SEQ ID NO.26):

ATACAATAGGACTCAATTCTGCTTGACAACTACTGCGTCTATTTTCA CCTCGCATACAGTCCGGGG

2. ^C DNA _u (SEQ ID NO.27):

CTCCCACTTGAGTTCCTGACTTTCCTATTGTATCCCCGGACTGTTTT AUUUUTCATCATTTTCATCACGCG

the synthesis process of the topological DNA/RNA nano-collar is schematically shown in FIG. 2A, and comprises the following steps:

(1) Preparation of topologic DNA/RNA nanocollars: first, 2. Mu.L of 10. Mu.M ^C DNA _u Loop (containing 4 bases "U", cleavable by Cas13a/crRNA in trans), 3. Mu.L of 10. Mu.M 5' phosphorylated ^C DNA _R In 1×T4DNA ligationThe mixture was homogenized in enzyme-containing buffer, and the final volume was 19.5. Mu.L. Heating at 95 ℃ for 5 minutes, then reducing to 16 ℃ at the speed of 0.1 ℃/s, then adding 0.5 mu L T DNA ligase, and standing at room temperature for reaction for 8 hours to obtain the topology DNA/RNA nano collar. 1×t4 DNA ligase buffer: 40mM Tris-HCl,10mM MgCl ₂ ，10mM DTT，0.5mM ATP，pH 7.8，25℃。

(2) Purification of topologic DNA/RNA nanocollars: and (3) treating the connection reaction product by using Exonuclease I (Exonecut I, exo I) and Exonuclease III (Exonecut III, exo III), reacting at 37 ℃ for 8 hours, incubating at 70 ℃ for 5 minutes after the reaction is finished, and obtaining the topological DNA/RNA nano-collar, and preserving at-20 ℃ for later use.

Characterization of the synthesized topological DNA/RNA nanocollar, the results of polyacrylamide gel electrophoresis of the preparation and purification of the topological DNA/RNA nanocollar are shown in fig. 3A, and two bands of the last lane (double ring after exonuclease treatment) of the left graph are subjected to gel cutting recovery and verification, wherein the upper band (red arrow) does not generate RCA under the action of Phi29 enzyme, and RCA only occurs after RNaseA treatment, which indicates that RCA is the target double ring of the study.

The result of the topological DNA/RNA nanocollar electrophoresis is shown in fig. 3B, lane 5: and cutting the gel after exonuclease treatment to recover the upper layer strip to obtain the double-ring product of the experiment. The topology verification of the topological DNA/RNA nanocollar is shown in fig. 3C, where when two single-ring specific primers are administered, RCA reaction can occur in single-ring, but RCA does not occur in double-ring pair of two primers, demonstrating the stability of double-ring structure. The experimental results show that the topological DNA/RNA nano-collar is successfully synthesized and has structural stability.

2. CRISPR diagnostic method

The one-pot detection solution system was prepared according to the following solution: 9.1. Mu.L of DEPC treated water, 1. Mu.L of 10 XHOLMES Buffer 2, 1. Mu.L of 10 XPhi 29 Buffer and 1. Mu.L of 10mM dNTP mix, then 2. Mu.L of 300pM DNA/RNA nanocollar, 2. Mu.L of 100nM Cas13a, 1. Mu.L of 100nmcrRNA, 0.5. Mu. L T4PNK, 0.2. Mu.L of Phi29DNA polymerase, 0.2. Mu.L of pyrophosphatase and 2. Mu.L of 10 XSYBR Green I were sequentially added, and the reaction system was prepared on ice and stirred uniformly to prepare a detection solution. The 10×phi29 buffer formulation was: 330mM Tris-HCl,100mM MgCl2,660mMKCl,1% (v/v) Tween 20,10mM DTT,pH 7.9, 25 DEG C

And adding a sample to be detected into the detection liquid, placing the reaction system at 37 ℃ for incubation for 1 hour, and then detecting a fluorescent signal (excitation wavelength: 488nm and emission wavelength: 520 nm) by using an enzyme-labeled instrument, so that the sample to be detected can be detected.

Serial concentration gradient diluted gene synthesis samples to be detected (the final detection concentrations are respectively 10nM,1nM,100pM,10pM,1pM,100fM,10fM,1fM,100aM,10aM,1aM and 0.1 aM) are added into detection liquid, the reaction system is incubated at 37 ℃ for 1 hour, and then a standard curve can be drawn by using an enzyme-labeled instrument to detect fluorescence signals (excitation wavelength: 488nm and emission wavelength: 520 nm).

Example 3 detection Limit investigation of CRISPR diagnostic methods

In this embodiment, the detection limit of the one-pot detection system of embodiment 2 is examined by using a pseudo virus, and the specific steps are as follows:

1. preparation of pseudoviral RNA: pseudoviruses (ordered in biological medicine technology Co., ltd. In Fubai, suzhou, product number: FNRV2593, pseudoviruses are complete sequences of ORF1a/b gene sequences, S gene 1.3kb sequences, E genes, M genes and N genes of retrovirus outer membrane wrapping parts, and no mutation sites) are taken out from a refrigerator at-20 ℃, placed on ice for melting or naturally melted at 4 ℃, and can be subjected to relevant experimental operation after being completely melted; placing the pseudovirus with the amount required by the current experiment in an EP tube, and inactivating the pseudovirus for 30min at 56 ℃; viral RNA was then rapidly prepared from the pseudoviral sample using a viral RNA rapid extraction kit.

2. Drawing a standard curve: and ordering the pseudovirus standard plasmid and carrying out quantitative detection experiments by using RT-qPCR to obtain a standard curve of Ct value and copy number of the pseudovirus standard plasmid. Then, RT-qPCR is carried out on the prepared pseudovirus RNA to obtain a Ct value of the pseudovirus RNA, and the Ct value is substituted into a standard curve to obtain the copy number of the pseudovirus.

The nucleotide sequence of the standard plasmid (SEQ ID NO. 28) is:

ATGTCTGATAATGGACCCCAAAATCAGCGAAATGCACCCCGCATTACGTTTGGTGGACCCTCAGATTCAACTGGCAGTAACCAGAATGGAGAACGCAGTGGGGCGCGATCAAAACAACGTCGGCCCCAAGGTTTACCCAATAATACTGCGTCTTGGTTCACCGCTCTCACTCAACATGGCAAGGAAGACCTTAAATTCCCTCGAGGACAAGGCGTTCCAA。

3. pseudoviral RNA detection limit: pseudoviral RNA of known copy number was subjected to serial concentration gradient dilutions (final concentrations of detection set at 5.3,4.3,3.3,2.3,1.3,0.3 in lg (copies/μl), respectively). Then, referring to the method of the "second part", a detection liquid is prepared. Wherein, an N-gene (SEQ ID NO. 24) synthetic sample is used as a synthetic sample of the gene to be detected, and the crRNA sequence is shown as SEQ ID NO. 8. Pseudoviral RNA was added at various concentrations, the reaction system was incubated at 37℃for 1 hour, and then fluorescent signals (excitation wavelength: 488nm, emission wavelength: 520 nm) were detected using an enzyme-labeled instrument.

The detection limit of the N gene of the synthetic sample by the one-pot method is shown in FIG. 4A, and the detection limit is 0.1aM; the extraction and detection limit of one-pot method on pseudoviruses is shown in FIG. 4B, and the detection limit is 10 ^{^0.3} The copies/. Mu.L, i.e., 1.99 copies/. Mu.L; stability of detection of low copy samples by one-pot method see fig. 4C, and 20 repeated detection of low copy samples was performed to investigate the stability and reproducibility of the method, and 20 repeated experiments showed that excellent detection analysis was also maintained for low copy samples.

The experimental result shows that the one-pot detection system formula provided by the method has the advantages of high sensitivity and robustness.

Example 4 feasibility study of identifying different variants based on Cas13a probe and one-pot detection system of topological DNA/RNA nanocollars

This example examines the feasibility of the one-pot assay system of example 2 to identify RNA virus variants, comprising the steps of:

designing the sequences of different variant plasmids: and (3) carrying out sequence design of plasmids of different variants according to mutation conditions of the variants in 9 sites in the A diagram (SEQ ID NO.29-SEQ ID NO. 37), inserting the 9 sites into a PUC57 plasmid, adding 10nt sequences before and after each site, adding a T7 promoter sequence at the forefront end of the inserted sequence to construct different variants, and then carrying out transcription and purification by using a T7 in vitro transcription kit to obtain RNA targets of the different variants. The purified variant RNA is quantified by an ultra-micro spectrophotometer and stored at-20 ℃ for later use.

The sequences of the 7 different variant plasmids were as follows:

wild(SEQ ID NO.29)：

TAATACGACTCACTATAGGGAGCCTAAAAAGGACAAAAAGAAGAAGGC

TGATGAAACTCAAGCCTTACTTGGTGCCACTTCTGCTGCTCTTCAACCT

GAAGAAGAGCAAGAAGAAGACTTTCCTTTACAATCATATGGTTTCCAA

CCCACTAATGGTGTTGGTTTGGTGTTGAAGGTTTTAATTGTTACTTTCCT

TTACAATCATATGGTTTCTTGGTTCCATGCTATACATGTCTCTGGGACCA

ATGGTACTAAGAGGTCGTTATAGCTTGGAATTCTAACAATCTTGATTCTA

AGGTTGGTGGTAATTTTGGGTGTTTATTACCACAAAAACAACAAAAGTT

GGATGGAAAGTGTGGGACCAATGGTACTAAGAGGTTTGATAACCCTGT

CCTACCATTTAAGGCCGGTAGCACACCTTGTAATGGTGTTGAAGGTTTTAATTGTTACTT；

BA.2(SEQ ID NO.30)：

TAATACGACTCACTATAGGGAGCCTAAAAAGGACAAAAAGAAGAAGGC

TGATGAAACTCAAGCCTTACTTGGTGCCACTTCTGCTGCTCTTCAACCT

GAAGAAGAGCAAGAAGAAGACTTTCCTTTACAATCATATGGTTTCCAA

CCCACTTATGGTGTTGGTTTGGTGTTGAAGGTTTTAATTGTTACTTTCCT

TTACGATCATATGGTTTCTTGGTTCCATGCTATACATGTCTCTGGGACCA

ATGGTACTAAGAGGTCGTTATAGCTTGGAATTCTAACAATCTTGATTCTA

AGGTTGGTGGTAATTTTGGGTGTTTATTACCACAAAAACAACAAAAGTT

GGATGGAAAGTGTGGGACCAATGGTACTAAGAGGTTTGATAACCCTGT

CCTACCATTTAAGGCCGGTAGCACACCTTGTAATGGTGTTGAAGGTTTTAATTGTTACTT；

BA.5(SEQ ID NO.31)：

TAATACGACTCACTATAGGGAGCCTAAAAAGGACAAAAAGAAGAAGGC

TGATGAAACTCAAGCCTTACTTGGTGCCACTTCTGCTGCTCTTCAACCT

GAAGAAGAGCAAGAAGAAGACTTTCCTTTACAATCATATGGTTTCCAA

CCCACTTATGGTGTTGGTTTGGTGTTGAAGGTTTTAATTGTTACTTTCCT

TTACAATCATATGGTTTCTTTTCCAATGTTACTTGGTTCCATGCTATATCT

GGGACCAATGGTACCGTTATAGCTTGGAATTCTAACAATCTTGATTCTAA

GGTTGGTGGTAATTTTGGGTGTTTATTACCACAAAAACAACAAAAGTTG

GATGGAAAGTGTGGGACCAATGGTACTAAGAGGTTTGATAACCCTGTC

CTACCATTTAAGGCCGGTAGCACACCTTGTAATGGTGTTGAAGGTTTTAATTGTTACTT；

BQ.1(SEQ ID NO.32)：

TAATACGACTCACTATAGGGAGCCTAAAAAGGACAAAAAGAAGAAGGC

TGATGAAACTCAAGCCTTACTTGGTGCCACTTCTGCTGCTCTTCAACCT

GAAGAAGAGCAAGAAGAAGACTTTCCTTTACAATCATATGGTTTCCAA

CCCACTTATGGTGTTGGTTTGGTGTTGAAGGTTTTAATTGTTACTTTCCT

TTACAATCATATGGTTTCTTTTCCAATGTTACTTGGTTCCATGCTATATCT

GGGACCAATGGTACCGTTATAGCTTGGAATTCTAACAATCTTGATTCTAC

GGTTGGTGGTAATTTTGGGTGTTTATTACCACAAAAACAACAAAAGTTG

GATGGAAAGTGTGGGACCAATGGTACTAAGAGGTTTGATAACCCTGTC

CTACCATTTAAGGCCGGTAGCACACCTTGTAATGGTGTTGAAGGTTTTAATTGTTACTT；

CH1.1(SEQ ID NO.33)：

TAATACGACTCACTATAGGGAGCCTAAAAAGGACAAAAAGAAGAAGGC

TGATGAAACTCAAGCCTTACTTGGTGCCACTTCTGCTGCTCTTCAACCT

GAAGAAGAGCAAGAAGAAGACTTTCCTTTACAATCATATGGTTTCCAA

CCCACTTATGGTGTTGGTTTGGTGTTGAAGGTTTTAATTGTTACTTTCCT

TTACAATCATATGGTTTCTTGGTTCCATGCTATACATGTCTCTGGGACCA

ATGGTACTAAGAGGTCGTTATAGCTTGGAATTCTAACAATCTTGATTCTA

AGGTTGGTGGTAATTTTGGGTGTTTATTACCACAAAAACAACAAAAGTC

GGATGGAAAGTGTGGGACCAATGGTACTAAGAGGTTTGATAACCCTGT

CCTACCATTTAAGGCCGGTAGCACACCTTGTAATGGTGTTGAAGGTTTTAATTGTTACTT；

XBB.1(SEQ ID NO.34)：

TAATACGACTCACTATAGGGAGCCTAAAAAGGACAAAAAGAAGAAGGC

TGATGAAACTCAAGCCTTACTTGGTGCCACTTCTGCTGCTCTTCAACCT

GAAGAAGAGCAAGAAGAAGACTTTCCTTTACAATCATATGGTTTCCAA

CCCACTTATGGTGTTGGTTTGGTGTTGAAGGTTTTAATTGTTACTTTCCT

TTACAATCATATGGTTTCTTGGTTCCATGCTATACATGTCTCTGGGACCA

ATGGTACTAAGAGGTCGTTATAGCTTGGAATTCTAACAATCTTGATTCTA

AGGTTGGTGGTAATTTTGGGTGTTTATTACCACAAAAACAACAAAAGTT

GGATGGAAAGTGTGGGACCAATGGTACTAAGAGGTTTGATAACCCTGC

CCTACCATTTAAGGCCGGTAGCACACCTTGTAATGGTGTTGAAGGTTCTAATTGTTACTT；

XBB1.5(SEQ ID NO.35)：

TAATACGACTCACTATAGGGAGCCTAAAAAGGACAAAAAGAAGAAGGC

TGATGAAACTCAAGCCTTACTTGGTGCCACTTCTGCTGCTCTTCAACCT

GAAGAAGAGCAAGAAGAAGACTTTCCTTTACAATCATATGGTTTCCAA

CCCACTTATGGTGTTGGTTTGGTGTTGAAGGTTTTAATTGTTACTTTCCT

TTACAATCATATGGTTTCTTGGTTCCATGCTATACATGTCTCTGGGACCA

ATGGTACTAAGAGGTCGTTATAGCTTGGAATTCTAACAATCTTGATTCTA

AGGTTGGTGGTAATTTTGGGTGTTTATTACCACAAAAACAACAAAAGTT

GGATGGAAAGTGTGGGACCAATGGTACTAAGAGGTTTGATAACCCTGC

CCTACCATTTAAGGCCGGTAGCACACCTTGTAATGGTGTTGAAGGTTTTAATTGTTACTT。

detection of different variants: the 9 crrnas (SEQ ID nos. 1-9) recognize respectively transcribed different variant RNA targets, and examine whether different sites in the variants can be distinguished, as well as the wild-type and mutant sites between the different variants. The detection system is described in example 2 in the second section in CRISPR diagnostic method.

Schematic representation of combinatorial coding for identification of different variants is shown in FIG. 5A; results of identifying different variants based on Cas13a probes and a one-pot detection system of topological DNA/RNA nanocollars are shown in fig. 5b-h, where each column color represents one crRNA (see right panel of panel a). The results are explained by taking BA.2 as an example, and firstly N gene and O gene are taken as conserved region sequences and can be identified and detected by a one-pot method; for the other 7 sites, ba.2 was single base mutated at two sites of N501Y, Q493R, the other 5 sites were wild type, and the detection results also indicated that signals above the threshold were generated at two sites of N501Y, Q493R, the other 5 wild type sites were background signals.

The above experimental results show that the method has the ability to identify different variants.

Example 5 identification of different variants in clinical samples using a one-pot detection system based on Cas13a probe and topological DNA/RNA nanocollars

This example examined the feasibility of the one-pot assay system of example 2 to identify RNA virus variants, and 3 novel coronavirus samples of clinical origin were assayed in one pot using a microplate reader and a portable small instrument (Major Science co., ltd, bluViewTransilluminator, MBE-300), respectively, for analysis of the results. Using 9 crRNAs (SEQ ID NO. 1-9) to respectively perform one-pot identification verification on clinical samples, and judging which type of variant the samples belong to by referring to a combined coding schematic diagram according to experimental results; meanwhile, 3 clinical samples were subjected to whole genome sequencing of the novel coronavirus RNA, and the variant type was further verified.

The results of the analysis of 3 samples by using the enzyme-labeled instrument are shown in fig. 6A-C, and the variant types corresponding to the upper right corners S1, S2 and S3 are the results identified by whole genome sequencing, and the one-pot method judging result is consistent with the results. The results of the analysis of 3 samples using a portable small instrument are shown in fig. 6D-E, the small instrument directly reads the results visually through blue light irradiation, and the instrument size is similar to the size of a mobile phone; the mutation site identified by the one-pot method was detected in whole genome sequencing both as a result of blue light irradiation (E left) and as a result of ultraviolet photographing by a gel imager (right), wherein the 3*3 position is consistent with the right panel of fig. 5A.

The experimental results show that the one-pot detection system based on the Cas13a probe and the topological DNA/RNA nanocollar can identify different variants in clinical samples.

According to the embodiment, the invention provides a novel crRNA and CRISPR diagnostic kit, which can overcome the problem of insufficient single-base mutation detection capability of CRISPR diagnosis, realize specific and high-sensitivity detection of single-base mutant RNA viruses such as COVID-19 and the like, and has good application prospects.

Claims

1. A crRNA for single base mutant RNA detection, characterized in that: the site for identifying the single base mutation is positioned at the 3 rd position of the 5' end of the crRNA spacer;

the crRNA also has a design of any one of the following:

2. The crRNA for single base mutant RNA detection according to claim 1, wherein: the crRNA has a design of any one of the following:

3. A method of designing crRNA for single base mutant RNA detection according to claim 1 or 2, comprising the steps of:

step 2, designing the crRNA according to any one of the following modes:

4. Use of crRNA for single base mutant RNA detection according to claim 1 or 2 for the preparation of a CRISPR diagnostic kit for single base mutant RNA detection.

5. A CRISPR diagnostic kit for single base mutant RNA detection, comprising Cas13a and the crRNA for single base mutant RNA detection of claim 1 or 2.

6. The CRISPR diagnostic kit of claim 5, further comprising: topology DNA/RNA nanocollar, T4PNK, phi29DNA polymerase, nucleic acid dye, pyrophosphatase and reaction buffer.

7. The CRISPR diagnostic kit according to claim 6, wherein: the topological DNA/RNA nano-collar is used for responding to the trans-cleavage activity and signal amplification of Cas13a, and is formed by topological hybridization of two circular DNA chains, wherein one DNA ring contains 2-4 continuous RNA bases U.

8. The CRISPR diagnostic kit according to claim 6, wherein: the nucleic acid dye is selected from 1 XSYBRGreenI.

9. The CRISPR diagnostic kit according to claim 6, wherein: the reaction buffer comprises the following components:

0.5～1×phi29 buffer、

5～8mM Mg(COOH) ₂ 、

30～35mM KCOOH、

0.01～0.1％v/v Tween 20、

0.4～1mM DTT、

0.5～1×HOLMES Buffer 2、

0.5～1mMdTNP mix。

10. the CRISPR diagnostic kit according to claim 5, wherein: the kit also comprises a gene synthesis sample to be detected.