CN117778386A - A crRNA and CRISPR diagnostic kit for single-base mutation RNA detection - Google Patents

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

A crRNA and CRISPR diagnostic kit for single-base mutation RNA detection

Technical field

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

Background technique

RNA virus is a single-stranded RNA virus whose genetic material is RNA. Compared with DNA viruses, RNA viruses are more prone to mutation. This is because the RNA replication process lacks a proofreading mechanism, resulting in a higher error rate. RNA viruses have a higher mutation rate, but the adaptability of each mutant strain may be different. This characteristic makes RNA viruses highly diverse and adaptable, but also makes them difficult to prevent and treat. Common RNA viruses include influenza viruses, HIV, Ebola viruses, and coronaviruses.

Coronavirus is a type of enveloped RNA virus widely found in nature. It can cause diseases such as influenza, Middle East Respiratory Syndrome (MERS) and Severe Acute Respiratory Syndrome (SARS), bringing serious consequences to human health, social stability and the global economy. huge stress. The rapid mutation of coronavirus has an important impact on the transmissibility and pathogenicity of the virus and the efficacy of therapeutic interventions. Because coronavirus has the characteristics of rapid transmission and severe clinical symptoms, it is necessary to develop a cost-effective and rapid detection strategy to conduct real-time analysis and monitoring of coronavirus mutant strains. In addition, many coronavirus mutant strains contain independently emerging dominant genotypes, suggesting that specific mutation site determination may be an effective strategy for predicting the evolutionary direction of dangerous virus strains. However, there is currently a lack of rapid and immediate methods for monitoring coronavirus variants, which seriously hinders related epidemiological and clinical research. Therefore, the development of new rapid coronavirus mutant strain analysis and detection methods can help track the evolution trend of specific mutant strains in the population and improve the prediction and prevention and control capabilities of related diseases.

The current gold standard method for detecting mutant strains of RNA viruses such as coronavirus is whole-genome sequencing, which can obtain the full sequence information of virus strains and be used for accurate typing of different types of viruses. However, the high cost of sequencing-related experiments limits the application of sequencing methods for real-time surveillance of viruses at a population scale. Nucleic acid detection based on polymerase chain reaction (Polymerase Chain Reaction, PCR) has also been widely used to diagnose multiple mutant subtypes of coronavirus, but it is limited by the need for precise temperature control instruments, professional laboratory personnel, and more complex Problems such as probe design and nucleic acid amplification bias in the PCR reaction itself are currently mainly used for detection applications in central laboratories, and are 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 crucial for the detection of coronavirus mutant strains.

CRISPR diagnostic (CRISPR-Dx) technology can specifically recognize target sequences through CRISPR RNA (crRNA) and output detection signals by activating CRISPR-associated proteins (Cas) to cleave DNA or RNA reporters. Commercial detection platforms based on CRISPR-Dx, such as specific high-sensitivity enzyme reporter solution (SHERLOCK) and DNA endonuclease-targeted CRISPR trans reporter (DETECTR), have been successfully developed for the detection of RNA viruses such as SARS-CoV-2 . Advances in amplification-free CRISPR-Dx technology, including superactive LwaCas13a effectors, CRISPR-mobile microscopy, gFET-based LwaCas13a systems, and ECRISPR, have effectively avoided cumbersome laboratory setups and shortened detection times, thus promoting CRISPR- Application of Dx in pathogen screening and infectious disease diagnosis.

However, the application of CRISPR-Dx in the identification of RNA virus variants still faces challenges. In general, Cas proteins, especially Cas9 and Cas13a, can tolerate single-base mismatches between crRNA and target sequences. Therefore, CRISPR-Dx has limited discriminatory power in identifying single-base mutations in the genomes of RNA virus variants. Exploring highly sensitive, target-free pre-amplification CRISPR-Dx technology to rapidly detect and identify RNA viruses and their variants with single-nucleotide resolution is very necessary and also a huge challenge.

Contents of the invention

In view of the problems of the prior art, the present invention provides a crRNA and CRISPR diagnostic kit for detecting single-base mutation RNA viruses.

A crRNA for single base mutation RNA detection, the site used to identify the single base mutation is located at the 3rd position of the 5' end of the crRNA spacer region;

The crRNA also has any of the following designs:

1) Positions 2, 4 or 5 at the 5’ end of the crRNA spacer region include 1-3 mismatches;

2) Add n bases to the 3' end of crRNA, which complementarily pair with the first n bases at the 5' end of the crRNA spacer to form a hairpin structure; the value of n is 8-10.

Preferably, the crRNA has any of the following designs:

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

Or, the 5th position at the 5' end of the crRNA spacer is mismatched, and the nucleotide sequence of the crRNA is shown in SEQ ID NO.6;

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

Alternatively, 8 bases are added to the 3' end of the crRNA, which are complementary to the first 8 bases at the 5' end of the crRNA spacer 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 present invention also provides the above-mentioned design method of crRNA for single-base mutation RNA detection, which includes the following steps:

Step 1, according to the sequence of the single-base mutation RNA to be identified, setting the site for identifying the single-base mutation at the third position of the 5' end of the crRNA spacer region;

Step 2, design the crRNA in any of the following ways:

1) Positions 2, 4 or 5 at the 5’ end of the crRNA spacer region include 1-3 mismatches;

2) Add n bases to the 3’ end of the crRNA and complementary pair with the first n bases at the 5’ end of the crRNA spacer to form a hairpin structure; the value of n is 8-10;

Step 3: Design the sequence of the remaining part of the crRNA based on the sequence of the single-base mutant RNA to be identified.

The present invention also provides the use of the above crRNA for single base mutation RNA detection in preparing a CRISPR diagnostic kit for single base mutation RNA detection.

The present invention also provides a CRISPR diagnostic kit for detecting single-base mutation RNA, wherein the kit comprises Cas13a and the above-mentioned crRNA for detecting single-base mutation RNA.

Preferably, the kit also includes: topological DNA/RNA nanothread, T4PNK, phi29 DNA polymerase, nucleic acid dye, pyrophosphatase and reaction buffer.

Preferably, the topological DNA/RNA nanoloop is used to respond to the trans-cutting activity and signal amplification of Cas13a, and the topological DNA/RNA nanoloop is formed by topological hybridization of two circular DNA chains, wherein one DNA loop contains 2-4 consecutive RNA bases U.

Preferably, the nucleic acid dye is selected from 1×SYBRGreenI.

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 also includes a synthetic sample of the gene to be tested.

In the present invention, the RNA detected is an RNA molecule that is highly related to the occurrence and development of diseases and can dynamically reflect cell status and regulatory processes, such as: mRNA, non-coding RNA, circular RNA, and RNA viruses.

The "crRNA" is an RNA molecule used in the CRISPR/Cas13a system to guide the Cas13a protein to locate and cleave target RNA. Its sequence includes two parts: a backbone sequence and a spacer sequence. The crRNA of the present invention has a spacer region designed.

Aiming at the problem that Cas13a/crRNA can often tolerate a single mismatch of the target, resulting in insufficient specificity of CRISPR-Dx in detecting single-base mutant RNA (such as RNA viruses), the present invention provides a new crRNA design idea. Specifically, the site to be recognized is placed at the 3rd position at the 5' end of the crRNA spacer, and then a single mismatch is added at the 2nd position at the 5' end of the crRNA spacer, so that the crRNA recognizes the mutant type as 1 mismatch and recognizes the wild type. There are two mismatches, and they are distinguished by their different degrees of activation effects on Cas13a. If the mutant cannot be distinguished from the wild type, then add different numbers or positions of mismatches, or modify the crRNA into a hairpin-like structure. When the single-base mutation to be detected is not present, this different number of synthetic mismatched crRNAs will create a bulge during the target recognition process, resulting in tighter discrimination at the target position, thereby enhancing Cas13a/crRNA The ability of a probe to identify single base mutations. The specific principle is: the identification of wild-type and mutant targets by the Cas13a/crRNA probe is achieved through complementary pairing of the spacer region of crRNA with the target base. When the Gibbs free energy of the two is smaller, their combination is more stable, and the trans-cleavage activity of Cas13a is stronger. However, in the presence of single-base mutations, crRNA binds to wild-type and mutant targets similarly and therefore cannot be distinguished. By adding different numbers or positions of mismatches, or transforming crRNA into a hairpin-like structure, the Gibbs free energy between the wild-type target and crRNA can be increased, making the combination between the two more unstable, thereby achieving wild-type and crRNA Significant differentiation of mutant forms.

Based on the above principles, the following beneficial effects are achieved through the technical solution of the present invention:

1. The crRNA designed according to the method of the present invention can specifically identify a variety of RNA single-base mutation sites, and can effectively improve the sensitivity of CRISPR-Dx technology for detecting single-base mutation RNA.

2. When using the crRNA of the present invention for CRISPR-Dx detection, the target pre-amplification step is not required, and the one-pot analysis with closed tubes throughout the entire process avoids biosafety and false positive problems caused by aerosol contamination.

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

4. In a preferred embodiment, the present invention provides a CRISPR-Dx detection kit that can be used for detection in a one-pot method. The detection process is quite simple and the separation and temperature control processes are omitted.

5. In the preferred solution, the present invention constructs a topological DNA/RNA nano-loop as a reporting and signal amplification system for Cas13a, and uses a rolling circle amplification strategy mediated by a topological DNA/RNA nano-loop as an efficient signal amplification system. module, capable of ultra-sensitive detection of low-abundance mutant sites.

6. The method provided by the present invention can design a variety of crRNAs with different mutation sites, realize the detection of a variety of different mutation sites, and can be applied to the accurate identification of multiple pathogen subtypes through combined encoding.

Obviously, according to the above content of the present invention, according to the common technical knowledge and common means in the field, without departing from the above basic technical idea of the present invention, various other forms of modifications, replacements or changes can also be made.

The above contents of the present invention will be further described in detail below through specific implementation methods in the form of examples. However, this should not be understood to mean that the scope of the above subject matter of the present invention is limited to the following examples. All technologies implemented based on the above contents of the present invention belong to the scope of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: Cas13a/crRNA probe design principles proposed by the present invention and Example 1 uses the high-frequency mutation site of the new coronavirus as a target to explore the ability of Cas13a to recognize SNP. Figure 1A shows the transformation process for Cas13a to recognize single base mutations. Figure 1B is a schematic diagram of crRNA modification categories. Figure 1C-H shows the transformation and recognition of 6 crRNAs.

Figure 2: Schematic diagram of the design and preparation of topological DNA/RNA nano-rings in Example 2 of the present invention.

Figure 3: Synthesis of topological DNA/RNA nanothrust in Example 2 of the present invention. Figure 3A shows the preparation steps and purification process of topological DNA/RNA nano-rings. It is necessary to remove isomers through polyacrylamide gel purification to obtain topological double rings with strong interlocking ability. Figure 3B shows the synthesized topological DNA/RNA nanothrust analyzed by 12% polyacrylamide gel electrophoresis. Figure 3C shows the investigation of the topological interlocking structure of topological DNA/RNA nano-rings.

Figure 4: Detection limit of one-pot method in Example 2 of the present invention. Figure 4A shows the detection limit of the synthetic sample (N gene). Figure 4B-C shows the detection limit of pseudovirus (N gene). Figure 4D shows the results of 20 repeated experiments on low-copy samples. Statistical results are shown as mean±SD (n=3).

Figure 5: The feasibility of one-pot method to determine different mutant strains in Example 3 of the present invention. Figure 5A is a schematic diagram of the combination coding of mutation sites. Figure 5B-H shows the combination of nine crRNA recognition variants.

Figure 6: The one-pot method used in the analysis of clinical samples in Example 4 of the present invention. Figure 6A Combinatorial coding analysis of three clinical variant sequencing samples. Figure 6B is a schematic diagram of a portable small instrument. Figure 6C shows the visual analysis of clinical variants with a portable small instrument, and Figures 6D-E show the results of analysis with a portable small instrument.

Detailed ways

Unless otherwise specified, all experimental materials, reagents and equipment in the examples can be obtained through conventional purchasing channels; unless otherwise specified, the experimental methods used in the examples are conventional methods.

Example 1 crRNA for detection of single base mutant RNA viruses

This embodiment provides crRNA for detection of single-base mutant RNA viruses. The design method is shown in Figure 1A. The specific steps include:

Step 1, according to the sequence of the single-base mutation RNA virus to be identified, set the site for identifying the single-base mutation at position 3 of the 3’ end of the crRNA;

Step 2, design the crRNA in any of the following ways:

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

2) Add n bases to the 3’ end of the crRNA and complementary pair with the first n bases at the 5’ end of the crRNA spacer to form a hairpin structure; the value of n is 8-10;

Step 3: Design the sequence of the remaining part of the crRNA based on the sequence of the single-base mutant RNA virus to be identified.

According to the above method, this example takes the characteristic mutations of different COVID-19 Omicron variants (as shown in FIG. 1B , Q493R, K444T, W152R, V83A, F486S, Δ69/70, N501Y) as an example, and designs a crRNA probe that can specifically identify the mutant strain. According to the method shown in FIG. 1C (SM1: the second position at the 5' end of the crRNA spacer is a mismatch; SM2: the fifth position at the 5' end of the crRNA spacer is a mismatch; DM: the second and fourth positions at the 5' end of the crRNA spacer are mismatches; TM: the second, fourth, and fifth positions at the 5' end of the crRNA spacer are mismatches; Hairpin-spacer: 8 bases are added to the 3' end of the crRNA, which are complementary to the first 8 bases at the 5' end of the crRNA spacer to form a hairpin structure), 6 crRNA sequences are designed as follows:

In addition, this example also detects the conserved region sequences of the N gene and O gene as a verification of the new coronavirus. The 2 crRNA sequences are as follows:

The sequences of the above 9 genes and their mutant types (MT is the mutant type and WT is the wild type) are shown in the following table:

To verify the ability of the Cas13a/crRNA complex composed of the above crRNA to recognize SNP, the method is as follows:

(1) Preparation of crRNA: Design the corresponding crRNA (SEQ ID NO.1-SEQ ID NO.9) according to the screened different mutant target RNA sequences, then order the transcription template containing the T7 promoter, T7 forward primer and reverse primer for PCR reaction, serving as a double-stranded DNA template for transcribing crRNA. Use HiscribeTM T7 Quick High Yield RNA Synthesis Kit (Hiscribe T7 Quick High Yield RNA Synthesis) and incubate overnight (more than 8 hours) at 37°C for in vitro transcription synthesis. Finally, crRNA was purified using the TRlzon method. The concentration of the obtained crRNA solution was measured using a Nanodrop nucleic acid quantifier and stored in a -80°C refrigerator to prevent degradation.

(2) Recognition of single base mutation sites by Cas13a/crRNA: Mix 2μL 100nM Cas13a, 1μL 100nMcrRNA (SEQ ID NO.1-SEQ ID NO.9), 2μL 100nM Target RNA, 2μL 1μM ssRNA reporter, 2μL 10× Mix HOLMES Buffer 2 and 11 μL DEPC-treated water on ice. The reaction system was incubated at 37°C for 30 minutes, and then a microplate reader was used to detect the fluorescence signal (excitation wavelength: 488nm, emission wavelength: 520nm). Among them, the nucleotide sequence of ssRNAreporter is: U(FAM)UUUU(BHQ-1).

Figure 1D-H shows the screening of crRNA at different mutation sites. The abscissa marked in red indicates that the crRNA modification method can effectively distinguish the mutant type and wild type of this site.

The above experimental results show that for different mutation targets of the new coronavirus, the structure of crRNA can be modified by adding mismatches, transforming it into a hairpin shape, etc. to effectively distinguish single-base mutations at the mutation site and the wild-type site. .

Example 2 CRISPR diagnostic kit and CRISPR diagnostic method for detection of single-base mutation RNA viruses

This embodiment provides a kit for detecting single-base mutation RNA viruses, and a CRISPR diagnostic method for detecting single-base mutation RNA viruses using the kit. The crRNA used in this embodiment is designed according to the method of Example 1. For example, taking the characteristic mutations (Q493R, K444T, W152R, V83A, F486S, Δ69/70, N501Y) of different Omicron variants of COVID-19 as an example, the designed crRNA sequences are shown in SEQ ID NO.1-SEQ ID NO.7.

1. Composition of the test kit (10 servings)

Components Specification crRNA 10uL/tube (10uM) Cas13a 10uL/tube (10pmol/uL) Topological DNA/RNA nanorings 50uL/tube (300pM) T4PNK 20uL/tube (10U/uL) phi29DNA polymerase 10uL/tube (10U/uL) 10000×SYBR Green I Nucleic Acid Dye 10uL/tube Pyrophosphatase 10uL/tube (0.1U/uL) 10×phi29buffer 50uL/tube 10×HOLMES Buffer 2 50uL/tube 10mMdTNP mix 50uL/tube DEPC treated water 1mL/tube

The topological DNA/RNA nanothrust is used to respond to the trans-cleavage activity and signal amplification of Cas13a. The topological DNA/RNA nanothrust is formed by the topological hybridization of two circular DNA chains. Among the topological DNA/RNA nano-rings, one serves as a recognition loop, containing 2-4 consecutive RNA bases U, for trans-cleavage by Cas13a; the other serves as a reporter loop, functioning as a template for RCA. After the recognition loop is cleaved by Cas13a, it becomes a chain and serves as a primer to use the reporter loop as a template for RCA to initiate the RCA reaction.

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):

CTCCCACTTGAGTTCCTGACTTTCCTATTGTATCCCGGACTGTTTTAUUUUTCATCATTTTCCATCACGCG

The schematic diagram of the synthesis process of topological DNA/RNA nano-rings is shown in Figure 2A, which includes the following steps:

(1) Preparation of topological DNA/RNA nano-rings: First, mix 2 μL 10 μM ^C DNA _u- ring (containing 4 bases "U" and can be trans-cleaved by Cas13a/crRNA), 3 μL 10 μM 5' phosphorylated ^C DNA _R was mixed in 1×T4 DNA ligase buffer to a final volume of 19.5 μL. Heating at 95°C for 5 minutes, then lowering to 16°C at a speed of 0.1°C/s, then adding 0.5 μL T4 DNA ligase and leaving it to react at room temperature for 8 hours to obtain topological DNA/RNA nano-rings. 1×T4 DNA ligase buffer: 40mM Tris-HCl, 10mM MgCl ₂ , 10mM DTT, 0.5mM ATP, pH 7.8, 25°C.

(2) Purification of topological DNA/RNA nano-rings: Use Exonuclease I (Exo I) and Exonuclease III (Exonuclease III, Exo III) to treat the ligation reaction product, and place it at 37°C for reaction 8 hours, after the reaction is completed, incubate at 70°C for 5 minutes to obtain topological DNA/RNA nano-rings, and store them at -20°C for later use.

The synthesized topological DNA/RNA nanothreads were characterized. The polyacrylamide gel electrophoresis results of the preparation and purification of topological DNA/RNA nanothreads are shown in Figure 3A. For the last lane on the left (double loop after exonuclease treatment) ) were extracted and verified. The upper band (red arrow) will not undergo RCA under the action of Phi29 enzyme. RCA will only occur after RNaseA treatment, indicating that it is the double ring intended for this study. .

The results of topological DNA/RNA nano-ring electrophoresis are shown in Figure 3B, lane 5: After exonuclease treatment, the upper band was recovered by cutting the gel to obtain the double-ring product of this experiment. The topological structure verification of the topological DNA/RNA nanoloop is shown in Figure 3C. When two specific primers for a single loop are given, the single loop can undergo RCA reaction, while the double loop will not undergo RCA with both primers, proving that the double loop structure is stability. The above experimental results show that the topological DNA/RNA nano-ring was successfully synthesized and has structural stability.

2. CRISPR diagnostic method

Prepare a one-pot detection solution system according to the following solution: 9.1μL DEPC treated water, 1μL 10×HOLMESBuffer 2, 1μL 10×Phi29 buffer and 1μL 10mM dNTP mix, then add 2μL 300pM DNA/RNA nanothread, 2μL 100nM Cas13a, 1 μL 100 nMcrRNA, 0.5 μL T4 PNK, 0.2 μL Phi29 DNA polymerase, 0.2 μL pyrophosphatase and 2 μL 10×SYBR Green I. The reaction system was prepared on ice throughout the entire process and pipetted to mix to prepare the detection solution. The formula of 10×Phi29 buffer is: 330mM Tris-HCl, 100mM MgCl2, 660mMKCl, 1% (v/v) Tween 20, 10mM DTT, pH 7.9, 25℃

Add the sample to be tested into the detection solution, incubate the reaction system at 37°C for 1 hour, and then use a microplate reader to detect the fluorescence signal (excitation wavelength: 488nm, emission wavelength: 520nm) to detect the sample to be tested.

Add the synthetic sample of the gene to be tested in a series of concentration gradient dilutions (the final concentration of the test is set to 10nM, 1nM, 100pM, 10pM, 1pM, 100fM, 10fM, 1fM, 100aM, 10aM, 1aM, 0.1aM) into the detection solution, and add the reaction system Incubate at 37°C for 1 hour, then use a microplate reader to detect the fluorescence signal (excitation wavelength: 488nm, emission wavelength: 520nm) to draw a standard curve.

Example 3 Investigation of detection limit of CRISPR diagnostic method

This example uses a pseudovirus to investigate the detection limit of the one-pot detection system of Example 2, and the specific steps are as follows:

1. Preparation of pseudovirus RNA: Place the pseudovirus (ordered from Fubaiao (Suzhou) Biomedical Technology Co., Ltd., item number: FNRV2593). The pseudovirus is a retrovirus outer membrane wrapping part of the ORF1a/b gene sequence and S gene 1.3kb. sequence, the complete sequence of E gene, M gene and N gene, without mutation sites), take it out from the -20℃ refrigerator and place it on ice to melt or melt naturally at 4℃. After it is completely melted, relevant experiments can be carried out. Operation: Put the amount of pseudovirus required for the current experiment into an EP tube and place it at 56°C for inactivation for 30 minutes; then use the viral RNA rapid extraction kit to quickly prepare viral RNA from the pseudovirus sample.

2. Drawing of the standard curve: Order the pseudovirus standard plasmid and use RT-qPCR to conduct a quantitative detection experiment to obtain a standard curve of the Ct value and copy number of the pseudovirus standard plasmid. Then, the prepared pseudovirus RNA was subjected to RT-qPCR to obtain its Ct value, which was then substituted into the standard curve to obtain the copy number of the pseudovirus.

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

ATGTCTGATAATGGACCCCAAAATCAGCGAAATGCACCCCGCATTACGTTTGGTGGACCCTCAGATTCAACTGGCAGTAACCAGAATGGAGAACGCAGTGGGGCGCGATCAAAACAACGTCGGCCCCAAGGTTTACCCAATAATACTGCGTCTTGGTTCACCGCTCTCACTCAACATGGCAAGGAAGACCTTAAATTCCCTCGAGGACAAGGCGTTCCAA.

3. Pseudovirus RNA detection limit: Perform serial concentration gradient dilutions of pseudoviral RNA with known copy numbers (the final concentrations of the detection are set to 5.3, 4.3, 3.3, 2.3, 1.3, 0.3 respectively, the unit is lg (copy/microliter) ). Then refer to the method in "Part 2" to prepare the detection solution. Among them, N-gene (SEQ ID NO. 24) synthetic sample was used as the synthetic sample of the gene to be tested, and the crRNA sequence is shown in SEQ ID NO. 8. Add different concentrations of pseudoviral RNA, incubate the reaction system at 37°C for 1 hour, and then use a microplate reader to detect the fluorescence signal (excitation wavelength: 488nm, emission wavelength: 520nm).

The detection limit of the N gene of the synthetic sample by the one-pot method is shown in Figure 4A, and the detection limit is 0.1aM; the extraction and detection limit of the pseudovirus by the one-pot method is shown in Figure 4B, and the detection limit is 10 ^{^0.3} copies/μL, that is, 1.99copies/ μL; the detection stability of the one-pot method for low-copy samples is shown in Figure 4C. The low-copy samples were tested repeatedly 20 times to examine the stability and reproducibility of the method. The 20 repeated experiments showed that low-copy samples can also be detected. Keep up the great detection analysis.

The above experimental results show that the one-pot detection system formula provided by this method has the detection advantages of high sensitivity and robustness.

Example 4 Feasibility study of identifying different mutant strains using a one-pot detection system based on Cas13a probe and topological DNA/RNA nanoloops

This example examines the feasibility of identifying RNA virus mutant strains using the one-pot detection system of Example 2, which includes the following steps:

Design the sequences of different variant plasmids: Design the sequences of different variant plasmids (SEQ ID NO.29-SEQ ID NO.37) based on the mutations of different variants at 9 sites in Figure A, and insert the 9 sites In the PUC57 plasmid, add 10nt sequences before and after each site, and add the T7 promoter sequence at the very beginning of the inserted sequence to construct different variants, and then use the T7 in vitro transcription kit for transcription and purification to obtain different variant RNAs. target. The purified variant RNA was quantified using an ultramicro-volume spectrophotometer and stored at -20 degrees until use.

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

wild(SEQ ID NO.29)：

TAATACGACTCACTATAGGGAGCCTAAAAAGGACAAAAAGAAGAAGGC

TGATGAAACTCAAGCCTTACTTGGTGCCACTTCTGCTGCTCTTCAACCT

GAAGAAGAGCAAGAAGAAGACTTTCCTTTACAATCATATGGTTTCCAA

CCCACTAATGGTGTGGTTTTGGTGTTGAAGGTTTTAATTGTTACTTTCCT

TTACAATCATATGGTTTCTTGGTTCCATGCTATACATGTCTCTGGGACCA

ATGGTACTAAGAGGTCGTTATAGCTTGGAATTCTAACAATCTTGATTCTA

AGGTTGGTGGTAATTTTGGGTGTTTATTACCACAAAAACAACAAAAGTT

GGATGGAAAGTGTGGACCAATGGTACTAAGAGGTTTGATAACCCTGT

CCTACCATTTAAGGCCGGTAGCACACCTTGTAATGGTGTTGAAGGTTTTAATTGTTACTT;

BA.2(SEQ ID NO.30):

TAATACGACTCACTATAGGGAGCCTAAAAAGGACAAAAAGAAGAAGGC

TGATGAAACTCAAGCCTTACTTGGTGCCACTTCTGCTGCTCTTCAACCT

GAAGAAGAGCAAGAAGAAGACTTTCCTTTACAATCATATGGTTTCCAA

CCCACTTATGGTGTTGGTTTGGTGTTGAAGGTTTTAATTGTTACTTTCCT

TTACGATCATATGGTTTCTTGGTTCCATGCTATACATGTCTCTGGGACCA

ATGGTACTAAGAGGTCGTTATAGCTTGGAATTCTAACAATCTTGATTCTA

AGGTTGGTGGTAATTTTGGGTGTTTATTACCACAAAAACAACAAAAGTT

GGATGGAAAGTGTGGACCAATGGTACTAAGAGGTTTGATAACCCTGT

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 mutant strains: 9 crRNAs (SEQ ID NO. 1-9) recognize different variant RNA targets transcribed, and examine whether different sites in the variants can be distinguished, as well as wild type and mutations between different variants. Differentiation of type sites. For the detection system, see the CRISPR diagnostic method in the second part of Example 2.

The schematic diagram of the combined coding to identify different mutant strains is shown in Figure 5A; the results of the one-pot detection system based on Cas13a probe and topological DNA/RNA nanoloop to identify different mutant strains are shown in Figure 5b-h, each column color in the figure Represents a type of crRNA (refer to the right image of Figure A). Take BA.2 as an example to explain the results. First, N gene and O gene, as conserved region sequences, can be identified and detected by the one-pot method; for the other 7 sites, BA.2 has two sites: N501Y and Q493R. There is a single base mutation, and the other five sites are wild-type. The detection results also show that the two sites N501Y and Q493R produce signals above the threshold, and the other five wild-type sites are background signals.

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

Example 5 Identification of different mutant strains in clinical samples using a one-pot detection system based on Cas13a probe and topological DNA/RNA nanoloop

This example examines the feasibility of the one-pot detection system in Example 2 to identify RNA virus mutant strains. Three new coronavirus samples from clinical sources were tested using a one-pot method using a microplate reader and a portable small instrument (MajorScience Co. ., Ltd, BluViewTransilluminator, MBE-300) for result analysis. Nine kinds of crRNA (SEQID NO. 1-9) were used to conduct one-pot identification and verification of clinical samples. The experimental results were determined with reference to the combined coding diagram to determine which type of variant the sample belonged to. At the same time, 3 clinical samples were tested for new coronavirus. RNA whole genome sequencing to further verify variant types.

The results of the analysis of the three samples using an ELISA instrument are shown in Figure 6A-C. The variant types corresponding to S1, S2, and S3 in the upper right corner are the results identified by whole genome sequencing, and the results of the one-pot method are consistent with them. The results of the analysis of the three samples using a portable small instrument are shown in Figure 6D-E. The small instrument directly reads the results visually through blue light irradiation, and the size of the instrument is similar to that of a mobile phone; the blue light irradiation results (E left) and the gel imager UV shooting results (right), the mutation sites identified by the one-pot method are all detected in the mutation at the site in the whole genome sequencing, and the 3*3 position is consistent with the right picture of Figure 5A.

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

As can be seen from the above examples, the present invention provides a new crRNA and CRISPR diagnostic kit, which can overcome the problem of insufficient single-base mutation detection capability of CRISPR diagnosis and achieve specific detection of single-base mutation RNA viruses such as COVID-19. The detection is highly sensitive and has good application prospects.

Claims (10)

1. A crRNA for single base mutation RNA detection, characterized in that: the site used to identify the single base mutation is located at the 3rd position at the 5' end of the crRNA spacer region; The crRNA also has any of the following designs: 1) Positions 2, 4 or 5 at the 5’ end of the crRNA spacer region include 1-3 mismatches; 2) Add n bases to the 3' end of the crRNA and complementary pair with the first n bases at the 5' end of the crRNA spacer to form a hairpin structure; the value of n is 8-10. 2. The crRNA for detecting single-base mutation RNA according to claim 1, characterized in that the crRNA has any of the following designs: The 2nd and 4th positions at the 5' end of the crRNA spacer region are mismatched, and the nucleotide sequence of the crRNA is as shown in SEQ ID NO.1; Or, the 5' end of the crRNA spacer region is a mismatch, and the nucleotide sequence of the crRNA is as shown in SEQ ID NO. 6; Or, positions 2, 4, and 5 at the 5' end of the crRNA spacer region are mismatched, and the nucleotide sequence of the crRNA is as shown in SEQ ID NO. 4; Or, 8 bases are added to the 3' end of the crRNA, which is complementary to the first 8 bases at the 5' end of the crRNA spacer to form a hairpin structure; the nucleotide sequence of the crRNA is such as SEQ ID NO. 2, SEQ Shown in either ID NO.3 or SEQ ID NO.5. 3. A design method for crRNA for single-base mutation RNA detection according to claim 1 or 2, characterized in that it includes the following steps: Step 1, according to the sequence of the single-base mutation RNA to be identified, set the site for identifying the single-base mutation at position 3 at the 5’ end of the crRNA spacer region; Step 2, design the crRNA in any of the following ways: 1) Positions 2, 4 or 5 at the 5’ end of the crRNA spacer region include 1-3 mismatches; 2) Add n bases to the 3’ end of the crRNA and complementarily pair with the first n bases at the 5’ end of the crRNA spacer region to form a hairpin structure; the value of n is 8-10; Step 3: Design the sequence of the remaining part of the crRNA based on the sequence of the single-base mutant RNA to be identified. 4. Use of the crRNA for single-base mutation RNA detection according to claim 1 or 2 in preparing a CRISPR diagnostic kit for single-base mutation RNA detection. 5. A CRISPR diagnostic kit for single-base mutation RNA detection, characterized in that the kit comprises Cas13a and the crRNA for single-base mutation RNA detection according to claim 1 or 2. 6. The CRISPR diagnostic kit according to claim 5, characterized in that the kit further includes: topological DNA/RNA nano-ring, T4PNK, phi29 DNA polymerase, nucleic acid dye, pyrophosphatase and reaction buffer . 7. The CRISPR diagnostic kit according to claim 6, characterized in that: the topological DNA/RNA nano-loop is used to respond to the trans-cleavage activity and signal amplification of Cas13a, and the topological DNA/RNA nano-loop is Two circular DNA strands are formed by topological hybridization, one of which contains 2-4 consecutive RNA bases U. 8. The CRISPR diagnostic kit according to claim 6, wherein the nucleic acid dye is selected from 1×SYBRGreenI. 9. The CRISPR diagnostic kit according to claim 6, characterized in that: 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. 10. The CRISPR diagnostic kit according to claim 5, characterized in that: the kit further includes a synthetic sample of the gene to be detected.