CN115927749A - Method and kit for detecting and screening L452R mutation and T478K mutation of Delta strain of new coronavirus - Google Patents

Abstract

A method and a kit for detecting and screening a Delta strain L452R mutation and a T478K mutation of a new coronavirus comprise the steps of extracting nucleic acid to obtain nucleic acid to be detected; carrying out amplification reaction by using the designed primer to obtain a nucleic acid amplification product; and (3) preparing CRISPR reaction mixed liquor, adding 2 mu L of nucleic acid amplification product into the first CRISPR reaction mixed liquor or the second CRISPR reaction mixed liquor, incubating for 30 minutes at 37 ℃, and reading a detection result through fluorescence. The invention has the advantages of high detection speed, high accuracy, low cost and multi-scene real-time detection.

Description

Method and kit for detecting and screening L452R mutation and T478K mutation of Delta strain of new coronavirus

Technical Field

The invention belongs to the technical field of biological detection, and relates to a method and a kit for detecting and screening L452R mutation and T478K mutation of a Delta strain of a novel coronavirus.

Background

The novel coronavirus (COVID-19), called new coronavirus for short, is a newly discovered single-stranded RNA virus, has a total length of 29903 nucleotides, is transmitted through respiratory tract and conjunctiva by droplets, has strong infectivity and wide transmission range, and is the seventh coronavirus which is known at present and causes diseases to human beings. Compared with acute symptoms caused by other coronaviruses, the novel coronaviruses have infection symptoms from mild symptoms, cough symptoms, fever symptoms to dangerous symptoms, the infection symptoms are similar to common respiratory diseases, the concealment is strong, and the infectivity is strong.

With the spread of new coronaviruses, thousands of mutations of new coronaviruses have been monitored globally. Most mutations do not result in changes in the properties of the virus, but specific mutations that are partially located on the S protein of the new coronavirus may make the virus more harmful. The S protein (spike protein) of the novel coronavirus is a component of the outermost structure of the virus, and the novel coronavirus recognizes the hACE2 receptor on the surface of a host cell through a surface protein to invade the cell. The human body also achieves viral immunity by recognizing viral surface antigens and generating corresponding antibodies. Most of the current new corona vaccines are also designed against the S protein of new corona virus. Thus mutations occurring in the S protein of the novel coronavirus may result in a change in the corresponding amino acid, thereby rendering the mode of virus entry more infectious. Amino acid changes may result in changes in viral epitopes that render vaccine-induced protective antibodies ineffective.

Delta (Delta) is a variant strain of the new coronavirus, which was first discovered in india 10 months earlier than 2020, and was named b.1.617.2 by the world health organization and was named with the greek letter Delta (Delta) on 31 days 5 months. The delta strain has the following remarkable characteristics: 1. the transmitting ability is strong and is improved by more than 40 percent compared with the transmitting ability of alpha strain found in the UK; 2. the latent period or the passage interval is shortened, and the virus propagation speed is accelerated; 3. high virus load: the PCR detection of the infected sample shows that the virus load is obviously increased. The Ct value of a patient is very low, and the lower the Ct value is, the higher the viral load in vivo is, and the time required for the nucleic acid of the patient to turn negative is prolonged. Delta variants include the RBD mutation L452R, the RBD mutation T478K, and the like. Delta variants are resistant to neutralization by some anti-NTD and anti-RBD monoclonal antibodies, for example, three antibodies, etesivimab, casirivimab and imdevimab, lose activity against Delta variants, and these antibodies show impaired binding to spike proteins. For mAb 7B8, immune escape occurred with the T478K variant; lower affinity for mAb 9g11, ll452r and E484; mutations L452R, T478K and E484Q were far from the binding site of mAb CB6, resulting in the development of immune escape. The neutralizing effect of the convalescent phase and the vaccine serum on the Delta variant is reduced, the convalescent phase plasma is reduced by 2.7 times, the pfeiri-BioNTech vaccine is reduced by 2.5 times, and the oxford-Alizecon vaccine is reduced by 4.3 times. This indicates that the variant may escape immunization and render the vaccine immune sera ineffective thereby breaking through the population of sexually infected vaccines. Therefore, the rapid identification of the Delta strain provides reference for the selection of an antibody treatment scheme and a technical tool for the accurate control of epidemic situations, and has great significance for avoiding large-scale breakthrough infection.

A Single Nucleotide Polymorphism (SNP) refers to a polymorphism of a nucleic acid sequence due to a change of a single nucleotide base. Single nucleotide polymorphism detection (SNP detection) is a detection technique used to detect DNA sequence polymorphisms at the genomic level caused by single nucleotide variations. The SNP detection methods are roughly classified into 3 types: (1) gel-based detection of known polymorphisms, including polymerase chain reaction, restriction fragment length polymorphism labeling, oligonucleotide ligation analysis, and mini-sequencing; (2) non-gel high-flux detection technology, including fluorescence energy resonance transfer detection method, mass spectrum technology and DNA chip; (3) conformation-based detection of unknown mutations, including single-stranded conformation polymorphism, chemical or enzymatic mismatch modification analysis, denaturing gradient gel electrophoresis, denaturing high performance liquid chromatography, and the like. At present, the monitoring of the new coronavirus mutation mainly relies on high-throughput sequencing to identify SNP, although the accuracy is high, the detection period is as long as 7 days, the cost is high, and the method cannot be suitable for rapid screening of the new coronavirus mutation. Therefore, the method for rapidly detecting and screening the mutation of the new coronavirus by the SNP has the advantages of high speed, high accuracy and low cost, and is significant.

CRISPR is an abbreviation for "Clustered regular interspersed short palindromic repeats" and refers to regularly Clustered, interspersed short palindromic repeats. Cas is the abbreviation for "CRISPR-associated", CRISPR-associated. The CRISPR/Cas system is an adaptive mechanism for resisting phage invasion developed by bacteria and archaea, and then discovered and developed into a technology for carrying out specific nucleic acid editing on a target gene by guiding a Cas nuclease by a guide RNA. The CRISPR/Cas system works on the principle that a crRNA (CRISPR-derived RNA) is combined with a tracrRNA (trans-activating RNA) through base pairing to form a tracrRNA/crRNA complex, and the complex guides a nuclease such as a Cas9 protein to cut double-stranded DNA at a sequence target site paired with the crRNA, so that the genome DNA sequence is edited; by artificially designing the two RNAs, a gRNA (guide RNA) with a guiding function can be transformed to be enough to guide the site-specific cleavage of the DNA by Cas 9. At present, the CRISPR-based rapid detection technology of nucleic acid is mainly divided into two main categories, namely a Cas12 a-dependent isothermal detection technology of nucleic acid developed by Jennifer Doudna in 2020, and a Cas13 a-dependent isothermal detection technology of nucleic acid developed by CRISPR patent owner. The principle is that a target fragment is amplified and enriched by a constant temperature amplification method such as RPA, the amplified target fragment can be guided and targeted recognized by Cas protein through a section of crRNA, the Cas protein is activated to become a DNA cutter (Cas 12 a) or an RNA cutter (Cas 13 a), and all nearby single-stranded DNA (Cas 12 a) or single-stranded RNA (Cas 13 a) is cut. This property is used for reporting the detection result by acting on the single-stranded nucleic acid fluorescent probe.

The cutting detection of the CRISPR/Cas system needs CRISPR target RNA (crRNA) to combine and recognize a target sequence and activate Cas protein, and the single base mutation (SNP) detection utilizes the base recognition specificity of the crRNA to ensure that the crRNA can only recognize the mutant sequence but not recognize an original sequence without the SNP by placing mutant bases at different positions of the crRNA or artificially introducing base mismatch, thereby realizing the mutation typing detection.

As shown in fig. 1, mode principles of discrimination of SNPs by LwaCas13a and LbaCas12a are shown, respectively. SNP differentiation of Cas12 a: PAM (Protospace adjacent motif) is a short fixed sequence (TTTN) near the crRNA target binding region (spacer). The PAM and a seed region (seed region) which is 1-6 bases close to the PAM are crucial for recognition and activation of the LbaCas12a, and the base mismatch at the PAM and the seed region can reduce the cleavage activity of the LbaCas12a protein by about 1000 times. SNP identification can be performed by greatly affecting the activation of LbaCas12a by placing the mutant bases in the PAM or seed region regions. SNP differentiation of Cas13 a: unlike the high sensitivity of LbaCas12a to single bases, lwaCas13a has no region similar to PAM and can be activated and detected for cleavage with one base mismatch, but LwaCas13a cannot be activated with two base mismatches. Therefore, the critical point for SNP detection can be achieved by artificially introducing an additional base mismatch to the crRNA of LwaCas13 a: when the SNP sequence is detected, only one human mismatch exists, and the crRNA activates the Cas protein to perform cleavage detection; when the original sequence without SNP is detected, because of two base mismatches, the crRNA can not activate the Cas protein to generate corresponding spatial structure change, and the detection is negative. The reported SNP typing of ZIKV virus by utilizing LwaCas13a in the literature tends to place a mutant base at the third base of a spacer and place an artificially synthesized base mismatch at the 5 th base of the spacer corresponding to crRNA for SNP differential detection. However, due to different base composition sequences of different detection sites, the influence on the spatial variation of the LwaCas13a protein after the combination of the detection sites and crRNA is different, and the SNP identification design work of others can only be used as a reference and cannot be applied. Therefore, when the Delta strain of the LwaCas13a is detected, the mutant bases are tried to be placed at different positions of the spacer, the artificial mismatch is tried to be placed at different positions of the crRNA, so that a plurality of sets of crRNAs are designed for screening, and finally, one crRNA sequence is screened, so that the Delta strain can be detected with the highest specificity and sensitivity.

Disclosure of Invention

The invention aims to provide a method and a kit for detecting and screening L452R mutation and T478K mutation of a Delta strain of a novel coronavirus.

In order to achieve the above objects and other related objects, the present invention provides the following technical solutions: a kit for detecting and screening for the L452R mutation and the T478K mutation of the Delta strain of a novel coronavirus, the kit comprising:

two sets of RT-RAA amplification primers:

Cas13-L452R-for：

5’-GAAATTAATACGACTCACTATAGGGtatagcttggaattctaacaatcttgattc-3’；

Cas13-L452R-rev：5’-accggcctgatagatttcagttgaaatatc-3’；

Cas12-T478K-for:5’-ttgtttaggaagtctaatctcaaacctttt-3’；

Cas12-T478K-rev:5’-gaaagtaacaattaaaaccttcaacaccat-3’.

two crrnas for CRISPR-specific detection:

L452R-Cas13-crRNA:

5’-ggauuuagacuaccccaaaaacgaaggggacuaaaacuauuccgguaauuauaauuaccaccaac-3’；

T478K-Cas12-crRNA：

5’-guaauuucuacuaaguguagauguaccggccugauagauuuc-3’；

two types of fluorescent probes:

RNA fluorescent probe: 5 '-FAM-mARURGrGrCmArArrGrCmA-BHQ 1-3';

wherein m represents an oxymethyl modification at position 2, r represents a ribonucleotide;

DNA fluorescent probe: 5'-VIC-TTATTATT-BHQ1-3'.

The preferable technical scheme is as follows: further comprising: HEPES buffer, mgCl ₂ The solution, 10 XNEB buffer2.1 buffer, rNTP mix, lwaCas13a, cas13-crRNA, lwaCas 12a, cas12-crRNA, RNase inhibitor, T7 RNA polymerase and RNase-free water.

In order to achieve the above objects and other related objects, the present invention provides the following technical solutions: a method for non-diagnostic purposes of testing and screening for the L452R mutation and the T478K mutation of the Delta strain of a novel coronavirus, comprising the steps of:

step 1: immersing a sample to be detected in virus preservation solution, and then performing nucleic acid extraction by using an RNA extraction kit to obtain nucleic acid to be detected;

step 2: adding 37.5 mu L of Buffer A solution, 2 mu L of Cas13-L452R-for,2 mu L of Cas13-L452R-rev,2 mu L of Cas12-T478K-for,2 mu L of Cas12-T478K-rev and 2 mu L of nucleic acid to be detected into a reaction tube containing protease freeze-dried powder, then adding 2.5 mu L of Buffer B solution, covering a tube cover of the reaction tube, and carrying out amplification reaction to obtain a nucleic acid amplification product;

two sets of RT-RAA amplification primers:

Cas13-L452R-for：

5’-GAAATTAATACGACTCACTATAGGGtatagcttggaattctaacaatcttgattc-3’；

Cas13-L452R-rev：5’-accggcctgatagatttcagttgaaatatc-3’；

Cas12-T478K-for:5’-ttgtttaggaagtctaatctcaaacctttt-3’；

Cas12-T478K-rev:5’-gaaagtaacaattaaaaccttcaacaccat-3’.

and step 3: preparing a CRISPR reaction mixed solution: mixing 0.4 μ L of HEPES buffer solution with concentration of 1M, 0.18 μ L of MgCl2 solution with concentration of 1M, 1.6 μ L of 10 XNEB buffer2.1 buffer solution, 0.8 μ L of rNTP mix with concentration of 25 μ M each, 2 μ L of LwaCas13a with concentration of 63.2ng/μ L,1 μ L of Cas13-crRNA with concentration of 5ng/μ L,1 μ L of LbaCas12a with concentration of 1 μ M, 1 μ L of Cas12-crRNA with concentration of 7.5ng/μ L,1 μ L of RNase inhibitor with concentration of 40U/μ L, 0.1 μ L of T7 RNA polymerer with concentration of 50U/μ L, 0.1 μ L of DNA fluorescent probe with concentration of 100 μ M, 0.1 μ L of RNA fluorescent probe with concentration of 100 μ M and 8.92 μ L of water-free enzyme;

two crrnas for CRISPR-specific detection:

L452R-Cas13-crRNA：

5’-ggauuuagacuaccccaaaaacgaaggggacuaaaacuauuccgguaauuauaauuaccaccaac-3’；

T478K-Cas12-crRNA：

5’-guaauuucuacuaaguguagauguaccggccugauagauuuc-3’；

two types of fluorescent probes:

RNA fluorescent probe: 5 '-FAM-mARUrGrCmAmArArArArrGrCmA-BHQ 1-3';

wherein m represents an oxymethyl modification at position 2, r represents a ribonucleotide;

DNA fluorescent probe: 5'-VIC-TTATTATT-BHQ1-3';

and 4, step 4: adding 2 mu L of the nucleic acid amplification product obtained in the step 2 into the CRISPR reaction mixed solution obtained in the step 3, and incubating for 30 minutes at 37 ℃; the judgment is carried out by the following method:

fluorescence values under FAM and VIC channels can be read using a fluorescence reader at the beginning of CRISPR reaction incubation, incubated for 20 cycles at 37 ℃, with 1.5 min intervals between each cycle, and fluorescence signals recorded once at the end of each cycle. Judging the detection results of the L452R and T478K mutations according to the final fluorescence signal intensity;

that is, the fluorescence value is more than 3000, which represents the positive detection of the corresponding site; if the fluorescence value of the FAM channel is more than 3000, the new coronavirus S site corresponding to the Cas13a is detected to be positive; the fluorescence value of the VIC channel is greater than 3000, namely the new coronavirus N site corresponding to Cas12a is detected to be positive; if the fluorescence value is less than 2000, the detection of the corresponding site is negative, if the fluorescence value is between 2000 and 3000, the detection is carried out again, and if the fluorescence value is still 2000 to 3000, the detection of the corresponding site is positive; the FAM channel is a fluorescence detection channel when a qPCR instrument reads fluorescence, and reads a fluorescence signal with the wavelength of 450nm-490 nm; the VIC channel read wavelength is 500-535nm.

The preferable technical scheme is as follows: the preparation method of the b mu ffer A solution comprises the following steps: adding 50mmol of Tris buffer solution, 100nmol of potassium acetate, 20g of polyethylene glycol powder and 2mmol of dithiothreitol into 1L of water; the B mu fferB solution is a magnesium acetate solution with the concentration of 280 mM.

Due to the application of the technical scheme, compared with the prior art, the invention has the advantages that:

the invention has the advantages of accurate detection of mutation sites, high detection speed, lower cost, low equipment dependence of a detection mode and capability of multi-scene real-time detection.

Drawings

FIG. 1 is a pattern diagram of discrimination of SNPs by LwaCas13a and LbaCas12 a.

FIG. 2 flow chart of CRISPR dual-channel simultaneous detection of two kinds of mutations of Delta strain

FIG. 3 shows the design and screening of crRNA of Cas13a at L452R site.

Figure 4 crRNA design and screening of the T478K site Cas12 a.

FIG. 5 CRISPR two-channel simultaneous detection of two mutations of Delta strain.

Detailed Description

The following description of the embodiments of the present invention is provided for illustrative purposes, and other advantages and effects of the present invention will become apparent to those skilled in the art from the present disclosure.

Please refer to fig. 1-5. It should be understood that the structures, ratios, sizes, and other elements shown in the drawings and described in the specification are only for the purpose of understanding and reading the present disclosure, and are not intended to limit the scope of the present disclosure, which is defined by the following claims. The following examples are provided for a better understanding of the present invention, and are not intended to limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The experimental materials used in the following examples were all purchased from a conventional biochemical reagent store unless otherwise specified.

Example 1: a method for detecting and screening a Delta strain of a new coronavirus L452R mutation and a T478K mutation and a kit thereof are characterized by comprising the following technical steps.

(1) Extraction of novel coronavirus nucleic acids

Immersing the specimen to be tested in a virus preserving fluid containing 2-3ml (isotonic saline solution can also be used), discarding the tail part, and screwing the tube cover. The sample should be extracted and detected as soon as possible, and the sample which can be detected within 24 hours can be stored at 4 ℃; samples that could not be detected within 24 hours should be stored at-70 ℃ or below. Nucleic acid extraction using RNA extraction kit

Mini Kit (QIAGEN, cat No. 74106) for example: mu.L of the virus preservation solution is taken and added with 350 mu.L of Buffer RLT to be blown and mixed evenly, and 550 mu.L of 70 percent absolute ethyl alcohol is added to precipitate the virus RNA. The resulting turbid suspension was centrifuged through a filter column at 12000rpm,2min,4 ℃. The impurities were eluted using Buffer RW1 followed by Buffer RPE, and finally 80. Mu.L of RNase-free water was added to the adsorption column and viral nucleic acids were eluted by centrifugation.

(2) RT-RAA primer design

RT-RAA (Reverse-Transcription-recombination-aid Amplification) is a Reverse Transcription-Recombinase mediated Amplification reaction. Namely, reverse transcriptase carries out reverse transcription on RNA into cDNA, and then target fragments are amplified at 37 ℃ under the mediation of a plurality of recombinase and specific RT-RAA primers. The RT-RAA reaction is the first step in the CRISPR detection method and plays a role in amplifying signals so as to improve the detection sensitivity.

The design of the RT-RAA primers follows the following principles: 1. the length of the primer is 30-35 bases; 2. the GC content of the primer is more than 30 percent and less than 70 percent; 3. the length range of the amplified product is between 100bp and 200 bp; 4. the amplified region requires a GC content of 40% < GC < 60% avoiding single repeat sequences as well as palindromic sequences. And 3 groups of RT-RAA primers at the upstream and the downstream are designed around the detection site, and the RT-RAA primers with the best amplification effect are screened out by using a complete CRISPR detection cleavage reaction.

In the invention, 3 groups of primers are respectively designed aiming at the L452R mutation and the T478K mutation of a novel coronavirus Delta strain, the optimal amplification primer is obtained through final screening, and compared with a Cas12a system, a T7 promoter recognition site (shown in capital letters) is added at the 5' end of an upstream primer in the Cas13a system and is used for RNA transcription during CRISPR detection.

Cas13-L452R-for：5’-GAAATTAATACGACTCACTATAGGGtatagcttggaattctaacaatcttgattc-3’

Cas13-L452R-rev：5’-accggcctgatagatttcagttgaaatatc-3’

Cas12-T478K-for：5’-ttgtttaggaagtctaatctcaaacctttt-3’

Cas12-T478K-rev：5’-gaaagtaacaattaaaaccttcaacaccat-3’

(3) RT-RAA nucleic acid amplification

And amplifying the extracted sample nucleic acid by using the Hangzhou mass RT-RAA nucleic acid amplification basic kit and a corresponding RT-RAA primer, and incubating for 20 minutes at 37 ℃. The specific operation steps are as follows:

50 mu L RT-RAA amplification system, a reaction tube filled with protease freeze-dried powder is taken, 37.5 mu L Buffer A solution (50mM Tris pH 7.9,100nM potassium acetate (Potasi mu M acetate), 5% polyethylene glycol (PEG), 2mM Dithiothreitol (DTT)), 2 mu L RT-RAA upstream primers (10 mu M), 2 mu L RT-RAA downstream primers (10 mu M) and 2 mu L nucleic acid to be detected are sequentially added into the reaction tube. Add 2.5. Mu.L of Buffer B solution (280 mM magnesium Acetate solution (Magnesi. Mu.M Acetate)) to the tube cap, and immediately remove the reaction tube after the tube cap is closed, thereby starting the amplification reaction. The reaction tube was incubated at 39 ℃ in a water bath for 20 minutes or directly held in the palm and incubated with body temperature for 20 minutes.

(4) CRISPR reaction system for rapid SNP detection and screening of Delta strain of new coronavirus

The invention designs 18 crRNAs for distinguishing and identifying novel coronavirus Delta strains, 7 crRNAs for detecting Delta strain T478K mutation by utilizing LwaCas 12a and 11 crRNAs for detecting Delta strain T478K mutation by utilizing LwaCas13 a. The LwaCas13a is designed by combining the mutant base at the 3,4,6 th base of the spacer and the artificial mismatch base at the 2,3,4,5,6 th base of the spacer corresponding to the crRNA. The strength of fluorescence signals when the crRNA sites are respectively detected for mutant strains and original strains is compared, and the designed crRNA sites are subjected to complete fluorescence cutting detection screening.

The invention finally screens 2 specific and sensitive crRNA sequences which can be used for identifying and distinguishing L452R and T478K mutations of Delta strains, and the invention is published as follows:

L452R-Cas13-crRNA (numbering O4)

5’-ggauuuagacuaccccaaaaacgaaggggacuaaaacuauuccgguaauuauaauuaccaccaac-3’；

T478K-Cas12-crRNA: (No. Q2)

5’-guaauuucuacuaaguguagauguaccggccugauagauuuc-3’；

Verification of the Effect of each crRNA in detecting the L452R mutation Using CRISPR/Cas13a System Screen

Screening and verification of effect of each crRNA on detection of T478K mutation by using CRISPR/Cas12a system

The CRISPR reaction was configured with the components, volumes and concentrations as described in the following table:

the preparation system of the mixed liquid for the CRISPR Cas13a reaction is as follows:

the CRISPR Cas12a reaction mixed liquor configuration system is as follows:

components	Concentration of	Volume of	Source
				LbaCas12a	1μM	1μL	NEB
crRNA	15ng/μL	1μL	In vitro transcription
				NEB buffer2.1	10×	2μL	NEB
RNase-free water	Is free of	13.9μL	Saimei fly
				DNA fluorescent probe	100μM	0.1μL	Nanjing engine
Total of		18μL

RNA fluorescent probe: m is an oxymethyl modification at the 2-position, r is RNA.

5’-FAM-mArArUrGrGrCmAmArArUrGrGrCmA-BHQ1-3’；

DNA fluorescent probe:

5’-VIC-TTATTATT-BHQ1-3’。

the effect of the CRISPR reaction system on SNP detection is mainly embodied in specificity, namely the specific base recognition characteristic of the CRISPR system endows the CRISPR reaction system with the capability of carrying out SNP detection. The crRNA provided by the invention can effectively recognize a sequence of a DELTA strain and activate a corresponding Cas protein, and the fluorescence report probe around the cleavage shows that the Delta strain is positive in detection. When the crRNA detects an original sequence without mutation, the corresponding Cas protein cannot be activated due to base mismatch, so that the DELTA strain is shown to be detected negatively.

(5) Fluorescence detection and result reading of Delta strains

And adding 2 mu L of nucleic acid amplification product into the prepared 18 mu L of CRISPR reaction mixed solution, and incubating for 30 minutes at 37 ℃. The fluorescence can be detected by incubation with a fluorescent quantitative PCR instrument or by direct water bath and visual observation of the change in fluorescence. According to different Cas proteases, the CRISPR reaction mixed liquor is divided into two kinds, namely CRISPR Cas13a reaction mixed liquor for mutation detection by using LwaCas13a effector protein and CRISPR Cas12a reaction mixed liquor for mutation detection by using LwaCas 12a effector protein.

Fluorescence values under FAM channels can be read at the beginning of CRISPR reaction incubation using a qPCR instrument (berle CFX 96) and incubated at 37 ℃ for 20 cycles, with 2min intervals between each cycle and a fluorescent signal recorded at the end of each cycle. And judging the detection result of the Delta strain according to the final fluorescence signal intensity, namely judging that the Delta strain is positive when the fluorescence value is more than 3000, the Delta strain is negative when the fluorescence value is less than 2000, detecting once again when the fluorescence value is between 2000 and 3000, and judging that the Delta strain is positive when the fluorescence value is still between 2000 and 3000.

The FAM channel is a fluorescence detection channel when a qPCR instrument reads fluorescence, and reads a fluorescence signal with the wavelength of 450nm-490 nm.

Establishment of Delta strain detection method

1. Materials and methods

1.1 materials

The RT-RAA amplification primers, crRNA and single-stranded probes were synthesized by Nanjing Ongbenaceae and Nanjing Kingsry. RT-RAA nucleic acid based amplification kit was purchased from Hangzhou Mass testing Biometrics. LbaCas12a protein was purchased from NEB. The LwaCas13a protein was purchased from tsry bio-corporation, tokyo. The pseudovirus containing the mutated sequence is obtained by packaging in a laboratory, or a pseudovirus containing the corresponding mutated sequence may be packaged by entrusted companies.

1.2 methods

1.2.1: design of RT-RAA primers

According to the original strain sequence of the new coronavirus published in GenBank, an RT-RAA amplification primer of a Delta strain site is designed. Designing an upstream primer according to the design requirements of RT-RAA, and finally screening an optimal amplification primer:

Cas13-L452R-for：5’-GAAATTAATACGACTCACTATAGGGtatagcttggaattctaacaatcttgattc-3’

Cas13-L452R-rev：5’-accggcctgatagatttcagttgaaatatc-3’

Cas12-T478K-for：5’-ttgtttaggaagtctaatctcaaacctttt-3’

Cas12-T478K-rev：5’-gaaagtaacaattaaaaccttcaacaccat-3’

1.2.2 design of LwaCas13a crRNA for detection of the Delta strain L452R mutation

This example uses LwaCas13a as an example to detect a mutation in Delta strain L452R.

The crRNA is a guide RNA, which is composed of a fixed framework (scaffold) and a region (spacer) complementary to a target sequence, and can tolerate the mismatching of one base on the spacer and successfully activate according to the existing report Cas13a, so when a Delta strain is detected, under the condition that one mutation mismatching exists, an artificial mismatching is additionally introduced on the spacer, namely the crRNA can only recognize the mutant sequence but not the original sequence, thereby achieving the purpose of mutation typing. After comprehensively analyzing the space structure of the Cas13a protein and the crRNA during the action, aiming at the mutation of L452R, the invention artificially introduces base mismatch in different regions of the spacer, and designs 11 crRNAs for subsequent screening. Specific sequences are shown in the following Table, and sequence display is shown in FIG. 3

TABLE 5

/>

1.2.3 design of LbaCas12a crRNA for detection of the Delta Strain T478K mutation

The T478K mutation of the Delta strain brings a PAM for the area to be specifically identified by the Cas12a, the PAM is a key sequence for identifying and activating the Cas12a, and 7 Cas12a crRNAs are designed for identifying the T478K mutation according to the characteristic. The sequences are shown in the following table, and the sequences are shown in FIG. 4:

1.2.4 screening of crRNA effective in discriminating L452R mutation Using the complete RT-RAA CRISPR/Cas13a reaction

A sample positive for virus mutation was simulated using a pseudovirus containing Delta strain sequences, and a pseudovirus containing the original sequence was used as a negative control. The 11 crRNAs designed above were screened with two pseudoviral samples at 100 cp/. Mu.L. Cas13a crRNA which can detect the L452R mutation but can not detect the L452R original sequence is screened out through the fluorescence intensity detected by RT-RAA-CRISPR Cas13a cutting.

1.2.5 screening crRNA which can effectively distinguish T478K mutation by using complete RT-RAA CRISPR Cas12a reaction.

A sample positive for virus mutation was simulated using a pseudovirus containing Delta strain sequences, and a pseudovirus containing the original sequence was used as a negative control. The 7 crRNAs designed above were screened with 100 cp/. Mu.L of two pseudovirus samples. Cas12a crRNA which can detect T478K mutation but can not detect T478K original sequence is screened out through fluorescence intensity detected by RT-RAA-CRISPR Cas12a cleavage.

1.2.6 simultaneous detection of the L452R and T478K mutations of the Delta strain using two selected crRNAs A pseudovirus carrying the corresponding mutations mimics the Delta strain, a pseudovirus carrying the normal sequence mimics the original strain of the new corona.

Performing nucleic acid extraction on pseudovirus samples with different concentration gradients, performing isothermal amplification on sample nucleic acids with the volume of 2ul, performing isothermal amplification on sample nucleic acids with the volume of 0cp/ul,10cp/ul,100cp/ul,1000cp/ul,10000cp/ul and 100000cp/ul, adding the amplified RT-RAA product with the volume of 2ul into a prepared 18ul CRISPR double-target detection system, specifically detecting crRNA by using a Delta strain L452R site and a T478K site in the system, incubating for 30 minutes at 37 ℃, and reading fluorescence values of FAM and VIC channels by using an instrument.

2. As a result, the

2.1CRISPR Cas13a CRRNA screening result for effectively distinguishing L452R

Effective crRNA was screened by RT-RAA-CRISPR Cas13a cleavage detection reaction using positive samples containing mutations and negative samples without mutations. The concentration of the pseudovirus sample is 100 cp/. Mu.L, two repeats are set, and nucleic acid is extracted by using a nucleic acid extraction kit. And isothermal amplification was performed using a mass-detection RT-RAA nucleic acid based amplification kit. Preparing an independent CRISPR reaction system for each crRNA, adding 2 mu L of RT-RAA amplification product into 18 mu L of CRISPR reaction system, incubating at 37 ℃ for 20 cycles, and collecting fluorescence through Brillo CFX96 in the whole incubation process. The mutation distinguishing capability of each crRNA is judged by comparing the fluorescence intensity of the mutant sample and the original sample. Cas13-L452R-6+4 (numbered O4) as shown in FIG. 3 can effectively detect the L452R mutation without detecting the original template.

2.2CRISPR Cas12a screening result of crRNA for effectively distinguishing T478K

And (3) screening the crRNA capable of being effective through an RT-RAA-CRISPR Cas12a cutting detection reaction by using a positive sample containing the mutation and a negative sample without the mutation. The concentration of the pseudovirus sample is 100 cp/. Mu.L, two repeats are set, and nucleic acid is extracted by using a nucleic acid extraction kit. And isothermal amplification was performed using a mass-detection RT-RAA nucleic acid based amplification kit. Preparing an independent CRISPR reaction system for each crRNA, adding 2 mu L of RT-RAA amplification product into 18 mu L of CRISPR reaction system, incubating at 37 ℃ for 20 cycles, and collecting fluorescence through Brillo CFX96 in the whole incubation process. The mutation distinguishing capability of each crRNA is judged by comparing the fluorescence intensity of the mutant sample and the original sample. As shown in FIG. 4, cas12-T478K-1st (accession number Q2) was able to efficiently detect the T478K mutation without detecting the original template.

2.3 Using the two selected crRNAs to simultaneously detect the L452R mutation and the T478K mutation of the Delta strain

Performing nucleic acid extraction on pseudovirus samples with different concentration gradients, performing isothermal amplification on sample nucleic acids with the volume of 2ul, performing isothermal amplification on sample nucleic acids with the volume of 0cp/ul,10cp/ul,100cp/ul,1000cp/ul,10000cp/ul and 100000cp/ul, adding the amplified RT-RAA product with the volume of 2ul into a prepared 18ul CRISPR double-target detection system, specifically detecting crRNA by using a Delta strain L452R site and a T478K site in the system, incubating for 30 minutes at 37 ℃, and reading fluorescence values of FAM and VIC channels by using an instrument. As shown in FIG. 5, the method can effectively detect different concentrations of Delta strain L452R and T478K mutation, and can be used for identifying the Delta strain.

FIG. 1: the principle of mutation detection by using CRISPR/Cas13a and CRISPR/Cas12a is shown respectively. I.e., the mutant strain is more matched with the guide RNA (crRNA), thereby activating the Cas protein for cleavage detection.

FIG. 2: the CRISPR dual-channel flow chart for simultaneously detecting two mutations of the Delta strain is characterized in that a sample is firstly processed to obtain nucleic acid, then two nucleic acid sites detected by two pairs of RT-RAA primers are simultaneously subjected to constant-temperature amplification, finally the obtained RT-RAA product is added into a prepared double-target CRISPR reaction system, incubation is carried out for one hour at 37 ℃, and fluorescence signals of FAM and VIC channels are detected, so that a detection result is obtained.

FIG. 3: the first partial base sequence diagram shows the designed 11 LwaCas13a crRNAs used to distinguish the L452R mutation of the Delta strain. By artificially introducing base mismatch on crRNA, the Cas effector protein can be identified and activated when detecting mutant strains and cannot be identified when detecting original strains. The second part shows a screening effect graph of detecting mutation of various crRNAs, and each designed crRNA is used for detecting a Delta strain sample and an original strain sample respectively, and the detection effect of the mutation is compared. The screening result shows that the mutation distinguishing capability of Cas13-L452R-6+4 (number O4) is strongest.

FIG. 4: the first partial base sequence diagram shows the T478K mutation designed for 7 LbaCas12a crrnas to distinguish Delta strains. The mutation site is placed in the PAM recognition region of Cas12a, so that the Cas effector protein can be recognized and activated when detecting a mutant strain, and cannot be recognized when detecting an original strain. The second part shows a screening effect graph of detecting mutation of various crRNAs, and each designed crRNA is used for detecting a DELTA strain sample and an original strain sample respectively, and the detection effect of the mutation is compared. The screening results show that Cas12-T478K-1st (number Q2) has the strongest capacity of differentiating mutation.

FIG. 5: the method for detecting the L452R mutation and the T478K mutation of the Delta strain is used for simultaneously detecting the L452R mutation and the T478K mutation of the Delta strain at different concentrations, for example, a FAM channel corresponds to the L452R mutation, and a VIC channel corresponds to the T478K mutation.

The foregoing is illustrative of the preferred embodiment of the present invention and is not to be construed as limiting thereof in any way, and any modifications or variations thereof that fall within the spirit of the invention are intended to be included within the scope thereof.

Claims (4)

1. A kit for detecting and screening a Delta strain L452R mutation and a T478K mutation of a new coronavirus is characterized in that: the kit comprises:

two sets of RT-RAA amplification primers:

Cas13-L452R-for：

5’-GAAATTAATACGACTCACTATAGGGtatagcttggaattctaacaatcttgattc-3’；

Cas13-L452R-rev：5’-accggcctgatagatttcagttgaaatatc-3’；

Cas12-T478K-for:5’-ttgtttaggaagtctaatctcaaacctttt-3’；

Cas12-T478K-rev:5’-gaaagtaacaattaaaaccttcaacaccat-3’.

two crrnas for CRISPR-specific detection:

L452R-Cas13-crRNA:

5’-ggauuuagacuaccccaaaaacgaaggggacuaaaacuauuccgguaauuauaauuaccaccaac-3’；

T478K-Cas12-crRNA：

5’-guaauuucuacuaaguguagauguaccggccugauagauuuc-3’；

two types of fluorescent probes:

RNA fluorescent probe: 5 '-FAM-mARUrGrCmAmArArArArrGrCmA-BHQ 1-3';

wherein m represents an oxymethyl modification at position 2, r represents a ribonucleotide;

DNA fluorescent probe: 5'-VIC-TTATTATT-BHQ1-3'.

2. The kit for detecting and screening the L452R mutation and the T478K mutation of the Delta strain of the novel coronavirus according to claim 1, which is characterized in that: further comprising: HEPES buffer, mgCl ₂ The solution, 10 XNEB buffer2.1 buffer, rNTP mix, lwaCas13a, cas13-crRNA, lwaCas 12a, cas12-crRNA, RNase inhibitor, T7 RNA polymerase and RNase-free water.

3. A method for non-diagnostic screening of the Delta strain L452R mutation and T478K mutation of a novel coronavirus, characterized in that: comprises the following steps:

step 1: immersing a sample to be detected in a virus preservation solution, and then performing nucleic acid extraction by using an RNA extraction kit to obtain nucleic acid to be detected;

step 2: adding 37.5 mu L of Buffer A solution, 2 mu L of Cas13-L452R-for,2 mu L of Cas13-L452R-rev,2 mu L of Cas12-T478K-for,2 mu L of Cas12-T478K-rev and 2 mu L of nucleic acid to be detected into a reaction tube containing protease freeze-dried powder, then adding 2.5 mu L of Buffer B solution, covering the tube cover of the reaction tube and carrying out amplification reaction to obtain a nucleic acid amplification product;

two sets of RT-RAA amplification primers:

Cas13-L452R-for：

5’-GAAATTAATACGACTCACTATAGGGtatagcttggaattctaacaatcttgattc-3’；

Cas13-L452R-rev：5’-accggcctgatagatttcagttgaaatatc-3’；

Cas12-T478K-for:5’-ttgtttaggaagtctaatctcaaacctttt-3’；

Cas12-T478K-rev:5’-gaaagtaacaattaaaaccttcaacaccat-3’.

and step 3: preparing a CRISPR reaction mixed solution: mixing 0.4 μ L of HEPES buffer solution with concentration of 1M, 0.18 μ L of MgCl2 solution with concentration of 1M, 1.6 μ L of 10 XNEB buffer2.1 buffer solution, 0.8 μ L of rNTP mix with concentration of 25 μ M each, 2 μ L of LwaCas13a with concentration of 63.2ng/μ L,1 μ L of Cas13-crRNA with concentration of 5ng/μ L,1 μ L of LbaCas12a with concentration of 1 μ M, 1 μ L of Cas12-crRNA with concentration of 7.5ng/μ L,1 μ L of RNase inhibitor with concentration of 40U/μ L, 0.1 μ L of T7 RNA polymerer with concentration of 50U/μ L, 0.1 μ L of DNA fluorescent probe with concentration of 100 μ M, 0.1 μ L of RNA fluorescent probe with concentration of 100 μ M and 8.92 μ L of water-free enzyme;

two crrnas for CRISPR-specific detection:

L452R-Cas13-crRNA：

5’-ggauuuagacuaccccaaaaacgaaggggacuaaaacuauuccgguaauuauaauuaccaccaac-3’；

T478K-Cas12-crRNA：

5’-guaauuucuacuaaguguagauguaccggccugauagauuuc-3’；

two types of fluorescent probes:

RNA fluorescent probe: 5 '-FAM-mARUrGrCmAmArArArArrGrCmA-BHQ 1-3';

wherein m represents an oxymethyl modification at position 2, r represents a ribonucleotide;

DNA fluorescent probe: 5'-VIC-TTATTATT-BHQ1-3';

and 4, step 4: adding 2 mu L of the nucleic acid amplification product obtained in the step 2 into the CRISPR reaction mixed solution obtained in the step 3, and incubating for 30 minutes at 37 ℃; the judgment is carried out by the following method:

fluorescence values under FAM and VIC channels can be read using a fluorescence reader at the beginning of CRISPR reaction incubation, incubated at 37 ℃ for 20 cycles, with 1.5 minutes intervals between each cycle, and the fluorescence signal recorded once at the end of each cycle. Judging the detection results of the L452R and T478K mutations according to the final fluorescence signal intensity;

that is, the fluorescence value is more than 3000, which represents the positive detection of the corresponding site; if the fluorescence value of the FAM channel is more than 3000, the new coronavirus S site corresponding to the Cas13a is detected to be positive; the fluorescence value of the VIC channel is more than 3000, namely the new coronavirus N site corresponding to the Cas12a is detected to be positive; if the fluorescence value is less than 2000, the detection of the corresponding site is negative, if the fluorescence value is between 2000 and 3000, the detection is carried out again, and if the fluorescence value is still 2000 to 3000, the detection of the corresponding site is positive; the FAM channel is a fluorescence detection channel when a qPCR instrument reads fluorescence, and reads a fluorescence signal with the wavelength of 450nm-490 nm; the VIC channel read wavelength is 500-535nm.

4. The method for the non-diagnostic purpose of detecting the screening of the L452R mutation and the T478K mutation of the Delta strain of the new coronavirus according to claim 3, characterized in that: the preparation method of the b mu ffer A solution comprises the following steps: adding 50mmol of Tris buffer solution, 100nmol of potassium acetate, 20g of polyethylene glycol powder and 2mmol of dithiothreitol into 1L of water; the B mu fferB solution is a magnesium acetate solution with the concentration of 280 mM.

Images