WO2022257659A1 - Crispr/cas system-based sars-cov-2 double-target rapid detection method and kit - Google Patents

Abstract

A CRISPR/Cas system-based SARS-CoV-2 double-target rapid detection method and kit. The method comprises: performing nucleic acid extraction to obtain nucleic acid to be detected; simultaneously amplifying two sites to be detected by using two groups of RT-RAA primers to obtain nucleic acid products, namely a Cas12a targeted N gene amplification product and a Cas13a targeted S gene amplification product; and preparing a CRISPR reaction mixed solution, adding 2 μL of a nucleic acid amplification product into the CRISPR reaction mixed solution, incubating at 37°C for 30 min, and reading fluorescence to obtain a detection result.

Description

A novel coronavirus dual-target rapid detection method and kit based on CRISPR/Cas system technical field

The invention belongs to the technical field of biological detection, and relates to a novel coronavirus dual-target rapid detection method and a kit based on a CRISPR/Cas system.

Background technique

Novel coronavirus (COVID-19), referred to as "new coronavirus", is a newly discovered single-stranded RNA virus with a total length of 29,903 nucleotides. It is transmitted through the respiratory tract and conjunctiva through droplets. It is highly infectious and spreads widely. , is currently the seventh coronavirus known to be pathogenic to humans. Compared with the acute symptoms caused by other coronaviruses, the symptoms of 2019-nCoV infection range from mild, cough, fever to critical illness. The symptoms of infection are similar to common respiratory diseases, and they are highly concealed and highly contagious.

At present, there is no effective therapeutic drug for the new crown virus. The development of the new crown vaccine requires a long period of time. After the development of the vaccine, the universal vaccination and the formation of immunity also require a huge vaccine production capacity and a very long vaccination cycle. Therefore, the most effective way to prevent and control the new coronavirus is still early detection and early isolation, through nucleic acid testing to find people infected with the new coronavirus, and isolate the infected people and related close contacts to block the spread of the virus. The gold standard for nucleic acid diagnosis of the new coronavirus is real-time fluorescent quantitative PCR (qPCR). This method amplifies the target fragment of the new coronavirus nucleic acid by PCR, and cuts and breaks a taq-man probe every time a target fragment is generated during amplification to generate a fluorescent signal . The generation of fluorescent signal is synchronized with the amplification product. Since the template increases exponentially during PCR amplification, there is a logarithmic relationship between the number of cycles (Ct) performed when it reaches the set threshold and the reciprocal of the initial template amount, and quantitative analysis can be performed based on this, thereby realizing the quantitative detection of the new coronavirus. However, qPCR requires expensive thermal cyclers and skilled operators, and the detection cycle is generally 3-4 hours long, which is difficult to meet the screening needs in some special scenarios, such as epidemic prevention and control in resource-poor areas such as remote rural areas.

Some existing nucleic acid rapid detection methods are by RT-LAMP (reverse transcription-loop-mediated isothermal amplification) and RT-RPA-exo probe method (reverse transcription-recombinase polymerase amplification exo probe method), such The method gets rid of the dependence on variable temperature equipment by sacrificing part of the accuracy and sensitivity. Under the mediation of some special constant temperature nucleic acid amplification enzymes, the target nucleic acid can be amplified and detected at a fixed temperature. Although the nucleic acid constant temperature amplification detection based on RT-LAMP or RT-RAA gets rid of the limitation of thermal cycle equipment, the detection accuracy and sensitivity have a huge drop compared with qPCR. The constant temperature detection based on the CRISPR/Cas system introduces an accurate and sensitive CRISPR system based on the constant temperature amplification method, which greatly improves the accuracy and sensitivity of the detection method.

CRISPR is the abbreviation of "Clustered regularly interspaced short palindromic repeats", which refers to regularly clustered interspaced short palindromic repeats. Cas is the abbreviation of "CRISPR-associated", which is related to CRISPR. The CRISPR/Cas system is an adaptive mechanism evolved by bacteria and archaea to resist the invasion of phages. It was discovered and developed into a technology in which guide RNA guides Cas nucleases to perform specific nucleic acid editing on targeted genes. The working principle of the CRISPR/Cas system is that crRNA (CRISPR-derived RNA) combines with tracrRNA (trans-activating RNA) through base pairing to form a tracrRNA/crRNA complex, which guides nucleases such as Cas9 proteins to sequences that pair with crRNA The double-stranded DNA is cut at the target site, so as to realize the editing of the genomic DNA sequence; and by artificially designing these two RNAs, it can be transformed into a guide RNA (guide RNA), which is enough to guide Cas9 to cut DNA at a specific point. At present, the rapid nucleic acid detection technology based on CRISPR is mainly divided into two categories, that is, the nucleic acid constant temperature detection technology called DETECTR developed by Jennifer Doudna, the winner of the 2020 Nobel Prize in Chemistry, which relies on Cas12a, and the technology developed by Zhang Feng, the owner of the CRISPR patent. A nucleic acid isothermal detection technology called SHERLOCK that relies on Cas13a. The principle is to amplify and enrich the target fragment through a constant temperature amplification method such as RPA. The amplified target fragment will be targeted and recognized by the Cas protein through a piece of crRNA guidance, and the Cas protein will be activated to become a DNA cutting machine (Cas12a). Or an RNA cutter (Cas13a), which cuts all nearby single-stranded DNA (Cas12a) or single-stranded RNA (cas13a). This feature acts on single-stranded nucleic acid fluorescent probes and can be used to report detection results.

The DETECTR system and the SHERLOCK system can only detect a single site at a time, and cannot detect double or even triple targets by adding taq-man probes of different target sites like qPCR. When encountering some low-copy clinical samples, the multiple-target detection method can avoid false negatives caused by the incomplete nucleic acid genome of the new coronavirus. However, the DETECTR and SHERLOCK systems based on the CRISPR/Cas system can only detect a single target at present. However, the experiment found that each different Cas protein can only cut the corresponding single-stranded DNA probe (LbaCas12a) or single-stranded RNA probe (LwaCas13a) after being activated, which makes the integration of the two CRISPR systems into a single system for dual-target detection. Simultaneous detection is possible.

Contents of the invention

The object of the present invention is to provide a novel coronavirus dual-target rapid detection method and kit based on the CRISPR/Cas system.

In order to achieve the above purpose and other related purposes, the technical solution provided by the present invention is: a CRISPR/Cas system-based kit for rapid double-target detection of novel coronavirus, said kit comprising:

Two sets of RT-RAA amplification primers:

Cas13-S-for: 5'-GAAATTAATACGACTCACTATAGGGgctatcatcttatgtccttccctcagtcag-3';

Cas13-S-rev: 5'-aatggcaggagcagttgtgaagttcttttc-3';

Cas12-N-for: 5'-aatgtcgcgcattggcatggaagtcaca-3';

Cas12-N-rev: 5'-gacttgatctttgaaatttggatctttg-3';

Two crRNAs specifically detected by CRISPR:

Cas12a-N-crRNA:

5'-uaauuucuacuaaguguagaauauggcaccuguuguaggucaa-3';

Cas13a-S-crRNA:

5'-gauuuagacuaccccaaaaacgaaggggacuaaaacCAUAAGUCACAAUGCAAGAAGACUACAC C-3';

RNA fluorescent probe: 5'-FAM-mArArUrGrGrGrCmAmArArUrGrGrGrCmA-BHQ1-3';

Among them, m represents the 2-position oxymethyl modification, and r represents ribonucleotides;

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

The preferred technical scheme is: also includes bufferA solution and BufferB solution; bufferA solution configuration method is: add 50mmol of Tris buffer solution, 100nmol of potassium acetate, 20g of polyethylene glycol powder and 2mmol of dithiothreitol to 1L of water; The BufferB solution is a magnesium acetate solution with a concentration of 280 mM.

The preferred technical scheme is: also includes: HEPES buffer solution, MgCl ₂ solution, 10×NEB buffer2.1 buffer solution, RNase inhibitor, T7 RNA polymerase, RNase-free water.

In order to achieve the above purpose and other related purposes, the technical solution provided by the present invention is: a double-target rapid detection method for novel coronavirus based on CRISPR/Cas system, comprising the following steps:

Step 1: Immerse the sample to be tested in the virus preservation solution, and then use the RNA extraction kit to extract the nucleic acid to obtain the nucleic acid to be tested;

Step 2: Add 37.5 μL of buffer A solution, 2 μL of Cas13-S-for, 2 μL of Cas13-S-rev, 2 μL of Cas12-N-for, 2 μL of Cas12-N-rev and 2 μL nucleic acid to be tested, then add 2.5 μL BufferB solution, cover the cap of the reaction tube and perform amplification reaction to obtain nucleic acid amplification product;

Cas13-S-for: 5'-GAAATTAATACGACTCACTATAGGGgctatcatcttatgtccttccctcagtcag-3';

Cas13-S-rev: 5'-aatggcaggagcagttgtgaagttcttttc-3';

Cas12-N-for: 5'-aatgtcgcgcattggcatggaagtcaca-3';

Cas12-N-rev: 5'-gacttgatctttgaaatttggatctttg-3';

Step 3: Prepare CRISPR reaction mixture: mix 0.4 μL of 1M HEPES buffer, 0.18 μL of 1M MgCl ₂ solution, 1.6 μL of 10×NEB buffer2.1 buffer, 0.8 μL of 25uM Each rNTPmix, 2 μL of LwaCas13a at a concentration of 63.2 ng/μL, 1 μL of Cas13-crRNA at a concentration of 10 ng/μl, 1 μL of LbaCas12a at a concentration of 1 uM, 1 μL of Cas12-crRNA at a concentration of 15 ng/μl, 1 μL RNase inhibitor with a concentration of 40U/μl, 0.1μL of T7RNApolymerase with a concentration of 50U/μl, 0.1μL of a DNA fluorescent probe with a concentration of 100uM, 0.1μL of an RNA fluorescent probe with a concentration of 100uM and 8.92μL of RNA-free enzyme water mix;

Cas12a-N-crRNA:

5'-uaauuucuacuaaguguagaauauggcaccuguuguaggucaa-3';

Cas13a-S-crRNA:

5'-gauuuagacuaccccaaaaacgaagggggacuaaaacCAUAAGUCACAAUGCAAGAAAGACUACACC;

RNA Fluorescent Probes:

5'-FAM-mArUrGrGrGrCmAmArArUrGrGrCmA-BHQ1-3';

Among them, m represents the 2-position oxymethyl modification, and r represents ribonucleotides;

DNA fluorescent probes:

5'-VIC-TTATTATT-BHQ1-3';

Step 4: Add 2 μL of the nucleic acid amplification product obtained in step 2 to the CRISPR reaction mixture prepared in step 3, and incubate at 37°C for 30 minutes; judge by the following methods:

1. Incubate with a fluorescent quantitative PCR instrument and detect the fluorescence at the same time, or directly observe the fluorescence change with the naked eye at the end of the water bath;

2. Use the qPCR instrument to read the fluorescence value under the FAM channel at the beginning of the CRISPR reaction incubation, and incubate at 37°C for 20 cycles, with an interval of 2 minutes between each cycle, record the fluorescence signal once at the end of each cycle, and judge the duality by the final fluorescence signal intensity. Target detection results, that is, the fluorescence value greater than 3000 indicates that the corresponding site is positive. For example, the fluorescence value of the FAM channel is greater than 3000, that is, the S site of the new coronavirus corresponding to Cas13a is detected positive, and the fluorescence value of the VIC channel is greater than 3000, that is, the corresponding site of Cas12a The detection of the N site of the new coronavirus is positive, and the fluorescence value is less than 2000, which means that the corresponding site is negative. If the fluorescence value is between 2000-3000, it will be tested again. If the fluorescence value is still 2000-3000, it is determined that the corresponding site is positive.

The preferred technical scheme is: bufferA solution configuration method is: add 50mmol of Tris buffer solution, 100nmol of potassium acetate, 20g of polyethylene glycol powder and 2mmol of dithiothreitol to 1L of water.

Owing to above-mentioned technical scheme uses, the advantage that the present invention has compared with prior art is:

The present invention overcomes the incompatibility between the DETECTR and SHERLOCK systems, and successfully combines the LwaCas13a (Leptotrichia wadei Cas13a) derived from Leptotrichia wadei Cas13a with the Cas13a derived from Streptomyces ND2006 without reducing the sensitivity and accuracy of the detection method. LbaCas12a (Lachnospiraceae bacterium ND2006 Cas12a) is unified to realize the simultaneous detection of two target sites using the CRISPR system.

Description of drawings

Figure 1. The process and principle of the CRISPR dual-target detection method.

Figure 2 Design of CRISPR detection sites.

Figure 3 Cross-reactivity validation.

Figure 4 Optimization of the reaction system of the CRISPR dual-target detection method.

Fig. 5 Optimization of metal ion pair reaction sensitivity and reaction stability.

Figure 6 Sensitivity verification of dual-target detection.

Figure 7 Actual sample verification.

Detailed ways

The implementation of the present invention will be illustrated by specific specific examples below, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

See Figure 1-7. It should be noted that the structures, proportions, sizes, etc. shown in the drawings attached to this specification are only used to match the content disclosed in the specification, for those who are familiar with this technology to understand and read, and are not used to limit the implementation of the present invention. Restricted conditions, so there is no technical substantive significance, any modification of structure, change of proportional relationship or adjustment of size. The following examples are provided for a better understanding of the invention, but not to limit the invention. The experimental methods in the following examples are conventional methods unless otherwise specified. Unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent stores.

Example 1: A novel coronavirus dual-target rapid detection method and kit based on CRISPR/Cas system

Kit includes:

Cas13-S-for: 5'-GAAATTAATACGACTCACTATAGGGgctatcatcttatgtccttccctcagtcag-3';

Cas13-S-rev: 5'-aatggcaggagcagttgtgaagttcttttc-3';

Cas12-N-for: 5'-aatgtcgcgcattggcatggaagtcaca-3';

Cas12-N-rev: 5'-gacttgatctttgaaatttggatctttg-3';

Cas12a-N-crRNA:

5'-uaauuucuacuaaguguagaauauggcaccuguuguaggucaa-3';

Cas13a-S-crRNA:

5'-gauuuagacuaccccaaaaacgaagggggacuaaaacCAUAAGUCACAAUGCAAGAAAGACUACACC;

RNA fluorescent probe: 5'-FAM-mArArUrGrGrGrCmAmArArUrGrGrGrCmA-BHQ1-3';

Wherein, m is 2-position oxygen methyl modification, r is RNA;

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

bufferA solution, BufferB solution, HEPES buffer, MgCl ₂ solution, 10×NEB buffer2.1 buffer, RNase inhibitor, T7 RNA polymerase and RNase-free water.

The preferred technical scheme is: the bufferA solution configuration method is: add 50mmol Tris buffer solution, 100nmol potassium acetate, 20g polyethylene glycol powder and 2mmol dithiothreitol to 1L water; BufferB solution is the acetic acid whose concentration is 280mM Magnesium solution.

As shown in Figure 1, the process and principle of CRISPR-based dual-target detection are shown. First extract the nucleic acid of the sample to be tested, and then perform constant temperature amplification of the extracted nucleic acid at two sites (A/B site) by RT-RAA to amplify the signal to be detected, and the obtained constant temperature amplification product is added to the configured In the CRISPR detection system, LbaCas12a and LwaCas13a recognize their respective target nucleic acids under the guidance of their respective crRNAs and activate the corresponding non-specific cleavage capabilities. crRNA is a guide RNA consisting of a fixed scaffold and a spacer complementary to the target sequence. As shown in the figure, LbaCas12a recognizes the A site through Cas12acrRNA and activates the non-specific cleavage ability to cleave the surrounding single-stranded DNA probe to generate a fluorescent signal (VIC) to report the detection result of the A site. LwaCas13a recognizes the B site through Cas13acrRNA and activates the non-specific cleavage ability to cleave the surrounding single-stranded RNA probe to generate a fluorescent signal (FAM) to report the detection result of the B site. The detection of double targets can be carried out in the same tube at the same time, and collected by the fluorescence collection instrument.

A novel coronavirus dual-target rapid detection method based on the CRISPR/Cas system comprises the following technical steps.

(1) Extract the nucleic acid of the new coronavirus

The collected samples are stored in the collection tube, the swab head is immersed in 2-3ml virus preservation solution (isotonic saline solution can also be used), the tail is discarded, and the cap of the tube is tightened. Specimens should be extracted and tested for nucleic acid as soon as possible. Specimens that can be detected within 24 hours can be stored at 4°C; specimens that cannot be detected within 24 hours should be stored at -70°C or below. Nucleic acid extraction using the RNA extraction kit, with the kit

Take the Mini Kit (QIAGEN, Cat No. 74106) as an example: take 200 μL of virus preservation solution, add 350 μL of BufferRLT to mix by pipetting, and then add 550 μL of 70% absolute ethanol to precipitate viral RNA. The obtained turbid suspension was centrifuged by column filtration, 12000 rpm, 2 min, 4°C. Use BufferRW1 and BufferRPE successively to elute impurities, and finally add 80 μL of RNase-free water to the adsorption column, and dissolve and elute the viral nucleic acid by centrifugation.

(2) RT-RAA primer design

RT-RAA (Reverse-Transcription-Recombinase-aid Amplification) is reverse transcription-recombinase-mediated amplification reaction. That is, reverse transcriptase reverse-transcribes RNA into cDNA, and then amplifies the target fragment at a constant temperature of 37°C under the guidance of multiple recombinases and specific RT-RAA primers. The RT-RAA reaction is the first step in the CRISPR detection method, which can amplify the signal and improve the detection sensitivity.

The design of 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 30%<GC<70%; 3. The length of the amplified product is between 100bp-200bp; 4. . The GC content of the amplified region needs to be 40%<GC<60%, avoiding single repetitive sequences and palindromic sequences. Design a set of upstream and downstream primers around the detection site.

Since the present invention is a dual-target nucleic acid detection, it is necessary to design two sets of RT-RAA amplification primers, one set of RT-RAA primers for the N gene targeted by Cas12a, and one set of RT-RAA primers for the S gene targeted by Cas13a primers. Since Cas13a recognizes RNA, the DNA fragment obtained by constant temperature amplification needs to be transcribed into RNA, so the RT-RAA primer of Cas13a adds a section of T7 promoter recognition site (indicated by capital letters) at the 5' end of the upstream primer. RNA transcription during CRISPR detection.

Cas13-S-for: 5'-GAAATTAATACGACTCACTATAGGGgctatcatcttatgtccttccctcagtcag-3';

Cas13-S-rev: 5'-aatggcaggagcagttgtgaagttcttttc-3';

Cas12-N-for: 5'-aatgtcgcgcattggcatggaagtcaca-3';

Cas12-N-rev: 5'-gacttgatctttgaaatttggatctttg-3'.

(3) RT-RAA dual-target nucleic acid amplification

Use Hangzhou Zhongce RT-RAA Nucleic Acid Amplification Basic Kit and corresponding RT-RAA primers to amplify the extracted sample nucleic acid, and incubate at 37°C for 20 minutes. The specific operation steps are as follows:

For 50 μL RT-RAA amplification system, take the reaction tube containing protease freeze-dried powder, and add 37.5 μL buffer A solution (50mM Tris pH 7.9, 100nM Potassium acetate, 5% polyethylene glycol (PEG ), 2mM dithiothreitol (DTT)), 2 μL (10 μM) of each of the two target site RT-RAA upstream primers, 2 μL (10 μM) of each of the two target site RT-RAA downstream primers, and 2 μL of the nucleic acid to be tested. Add 2.5 μL of Buffer B solution (280 mM magnesium acetate solution (Magnesium Acetate)) to the cap of the tube, cover the tube cap and spin off the reaction tube to start the amplification reaction. Incubate the reaction tube in a 39°C water bath for 20 minutes or hold it directly in the palm of your hand and incubate at body temperature for 20 minutes.

(4) Design of double-target detection method CRISPR loci

Design crRNA for detection: The detection of LbaCas12a requires a PAM region for recognition on the sequence to be recognized, and the sequence of the PAM region is TTTN. The crRNA design of LwaCas13a has no special requirements. Therefore, by comparing the genome sequence of the new coronavirus (NC_045512) with several other coronaviruses, including hCoV-229E (NC_002645), hCoV-HKU1 (NC_006577), hCoV-NL63 (NC_005831), hCoV-OC43 (NC_006213), SARS- CoV (NC_004718) and bat SARS-like coronavirus (MG772933), avoiding homologous sequences, finally designed detection sites for LbaCas12a and LwaCas13a.

The detection site of final design of the present invention is as follows:

Cas12a-N-crRNA:

5'-uaauuucuacuaaguguagaauauggcaccuguuguaggucaa-3';

Cas13a-S-crRNA:

5'-gauuuagacuaccccaaaaacgaaggggacuaaaacCAUAAGUCACAAUGCAAGAAGACUACCC-3'.

(5) Configure CRISPR reaction system for rapid detection of CRISPR dual targets

Since the Cas12a system and the Cas13a system have different reaction systems, different ions and ion concentrations are required to achieve the best sensitivity. In the case of ensuring the sensitivity and stability of the detection method, the present invention finally succeeded in combining LbaCas12a and LwaCas13a by adding different ions. It is integrated and can be used for rapid ultrasensitive dual-target detection of the new coronavirus.

Configure the CRISPR dual-target detection system according to the composition, volume and concentration as described in the following table:

The CRISPR reaction mixture configuration system is as follows:

components	concentration	volume	source
RNase-free water	-	8.92ul	Thermo Fisher
HEPES	1M	0.4ul	Thermo Fisher
Mgcl 2	1M	0.18ul	Thermo Fisher
NEB buffer2.1	10×	1.6ul	NEB
rNTPmix	25mMeach	0.8ul	NEB
LwaCas13a	63.2ng/μl	2ul	Nanjing KingScript

Cas13-crRNA	10ng/μl	1ul	in vitro transcription
LbaCas12a	1uM	1ul	NEB
Cas12-crRNA	15ng/μl	1ul	in vitro transcription
RNase inhibitor	40U/μl	1ul	NEB
T7 RNA polymerase	50U/μl	0.1ul	Lucigen
VIC-DNA probe	100uM	0.1ul	Nanjing Qingke
FAM-RNA probe	100uM	0.2ul	Nanjing Qingke
total	-	18μL	the

RNA fluorescent probe (FAM-RNA probe): (m is 2-position oxymethyl modification, r is RNA);

5'-FAM-mArUrGrGrGrCmAmArArUrGrGrCmA-BHQ1-3';

DNA fluorescent probe (VIC-DNA probe):

5'-VIC-TTATTATT-BHQ1-3'.

Take 18ul of the detection system, add 2ul volume of RT-RAA amplification product to it, incubate at 37°C for 30 minutes, perform fluorescence monitoring through FAM and VIC channels, and obtain the monitoring results.

(6) Fluorescence detection and result reading of CRISPR dual targets

Take 2 μL of the nucleic acid amplification product and add it to the prepared 18 μL CRISPR reaction mixture, and incubate at 37°C for 30 minutes. At the beginning of the CRISPR reaction incubation, the qPCR instrument (Bio-Rad CFX96) can be used to read the fluorescence values under the FAM and VIC channels, incubate at 37°C for 20 cycles, with an interval of 2.5 minutes between each cycle, and record the fluorescence signal once at the end of each cycle. Judging the result of dual-target detection by the final fluorescence signal intensity,

That is, a fluorescence value greater than 3000 indicates that the corresponding site is detected positively. If the fluorescence value of the FAM channel is greater than 3000, it means that the S site of the new coronavirus corresponding to Cas13a is detected positive. The fluorescence value of the VIC channel is greater than 3000, that is, the N site of the new coronavirus corresponding to Cas12a is detected positive. If the fluorescence value is less than 2000, it means that the detection of the corresponding site is negative. If the fluorescence value is between 2000-3000, it will be tested again. If the fluorescence value is still 2000-3000, it will be determined that the detection of the corresponding site is positive. The FAM channel is a fluorescence detection channel when the qPCR instrument reads fluorescence, and reads fluorescence signals with a wavelength of 450nm-490nm. The VIC channel reads at a wavelength of 500-535nm.

Establishment of CRISPR dual-target rapid detection method

1. Materials

RT-RAA amplification primers, crRNA and single-stranded probes were synthesized by Nanjing Qingke Biotechnology Co., Ltd. and Nanjing GenScript Biotechnology Co., Ltd. RT-RAA nucleic acid basic amplification kit was purchased from Hangzhou Zhongce Biological Company. LbaCas12a protein was purchased from NEB. LwaCas13a protein was purchased from Nanjing GenScript Biotechnology Co., Ltd.

2. Methods and results

2.1: Design of RT-RAA primers

According to the sequence of the original novel coronavirus strain (NC_045512.2) published in GenBank, design RT-RAA amplification primers for the S site and N site, and design the upstream primers corresponding to each target site according to the requirements of RT-RAA With the downstream primers, the specific sequences are as follows:

2.2 Design of LbaCas12acrRNA and LwaCas13acrRNA

In order to ensure the specificity of the design and avoid false positives caused by homologous fragments of the same type of virus, when designing crRNA, we compared and analyzed the novel coronavirus genome sequence (NC_045512) with several other coronaviruses, including hCoV- 229E(NC_002645), hCoV-HKU1(NC_006577), hCoV-NL63(NC_005831), hCoV-OC43(NC_006213), SARS-CoV(NC_004718) and batSARS-like coronavirus(MG772933), from which the unique sequences of the novel coronavirus were selected as crRNA sites. In addition, when designing the site, the detection of LbaCas12a requires a PAM region for recognition on the sequence to be recognized, and the sequence of the PAM region is TTTN. The crRNA design of LwaCas13a has no special requirements.

The final designed crRNA detection site of the present invention is as follows:

Cas12a-N-crRNA:

5'-uaauuucuacuaaguguagaauauggcaccuguuguaggucaa-3'

Cas13a-S-crRNA:

5’-gauuuagacuaccccaaaaacgaagggggacuaaaacCAUAAGUCACAAUGCAAGAAAGACUACCC

Its sequence alignment with various coronaviruses is shown in Figure 2

2.3 Experimental verification that the non-specific cleavage of LbaCas12a and LwaCas13a will not cross-react

Since LbaCas12a only cuts the surrounding single-stranded DNA after activation, and LwaCas13a only cuts the surrounding single-stranded RNA after activation, theoretically there will be no cross-reaction to cause LbaCas12a to cut single-stranded RNA and LwaCas13a to cut single-stranded DNA. This is the theoretical premise for integrating LbaCas12a and LwaCas13a into the same system and performing dual-target detection. Therefore, it is firstly verified by experiments that LbaCas12a and LwaCas13a will not cross-react with different probe substrates after activation. In a complete DETECTR experiment, add an RNA probe (FAM labeled); in a complete SHERLOCK experiment, add a DNA probe (VIC labeled) to see if cross-cleavage of the substrate occurs.

2.4 Attempts of different buffer systems

To integrate LbaCas12a and LwaCas13a into the same system, we must first explore the ionic components of the reaction system to obtain a reaction system that can take into account both LbaCas12a and LwaCas13a. In the present invention, on the basis of several components (HEPES, rNTPmix, T7 RNA polymerase and RNase inhibitor) required for the LwaCas13a reaction, different components of the reaction system of LbaCas12a such as Nacl, Tris-Hcl, Mgcl2 are mixed in different proportions , BSA, etc.

Use 10000cp/ul virus genome for system compatibility verification. The virus genome is first amplified by RT-RAA at constant temperature, and then added to the prepared multi-group CRISPR dual-target reaction system, and the optimal reaction system is judged by monitoring the fluorescence signal of the FAM/VIC channel. That is, under a fixed virus concentration, the monitoring result has the highest fluorescence value and the most stable combination of monitoring results. It is known from experiments that when 80% of the LbaCas12a reaction system is introduced, the efficiency and stability of dual-target detection are the best. Both sites were efficiently detected. As shown in Figure 4

2.5 System optimization of different divalent metal ions

Metal ions are prosthetic groups or activators of enzymes, which can help to stabilize the conformation, constitute the active center of the enzyme, or act as a link, serving as a bridge to integrate the enzyme and the substrate. Common enzymatic divalent metal ions include Mg, Ca, Mn, Ni, Zn, Co, Cu, etc. The present invention screens divalent ions such as Mg, Ca, Mn, and Ni, and finally obtains the most suitable metal ion and its concentration for the CRISPR dual-target detection method. In this example, under the buffer system that has been explored, by setting a single variable, the influence of different metal ions on the detection of dual-target CRISPR is verified. In this example, a total of 5 sets of variables are set, and 1mMMg2+, 10mMMg2+, and 1mMCa2+ are respectively explored. , 1mM Mn2+, 1mM Ni2+'s influence on detection sensitivity. The experimental results show that Mg2+ has the greatest improvement on the reaction sensitivity, and high concentration of Mg2+ can improve the detection sensitivity and stability more than low concentration of Mg2+. As shown in Figure 5.

2.6 Dual-channel Effective Sensitivity Verification

After determining the optimal reaction system components of the CRISPR dual-target detection method, use the in vitro transcribed SARS-CoV-2 S segment and N segment RNA templates to serially dilute to 8 different concentration gradients: S/N, 0cp/ul, 0.7/1cp /ul, 7/10cp/ul, 17.5/25cp/ul, 35/50cp/ul, 70/100cp/ul, 700/1000cp/ul, 7000/10000cp/ul. The sensitivity of the detection method was verified by detecting the diluted templates with different concentrations through the complete CRISPR dual-target detection method. It can be seen from the figure that the novel coronavirus CRISPR dual-target detection method in the present invention can stably detect the sensitivities of the two sites: Cas13a 7cp/ul, Cas12a 25cp/ul. As shown in Figure 6

2.7 Two-channel actual sample verification

Actual clinical sample verification: 24 new crown clinical samples were tested using the CRISPR dual-target detection method. Nucleic acid extraction was carried out on the 24 clinical samples of the new coronavirus collected, and the RT-RAA primers of the new coronavirus S site and N site mentioned in the present invention were used to perform constant temperature amplification on the sample nucleic acid of 2ul volume, and the amplified Add 2ul of the RT-RAA product into the prepared 18ul CRISPR dual-target detection system and incubate at 37°C for 1 hour. At the beginning of the incubation, the fluorescence values of the FAM and VIC channels were read by a fluorescence reader. Using the CRISPR dual-target detection method to detect 24 clinical samples, the results showed that 16 cases were positive and 8 cases were negative, which was consistent with the qPCR results. As shown in Figure 7

Figure 1: Process and principle of CRISPR dual-target detection method

That is, the sample is first processed to obtain nucleic acid, and then two pairs of RT-RAA primers are used to simultaneously perform constant temperature amplification on the two detected nucleic acid sites, and finally the obtained RT-RAA product is added to the prepared dual-target CRISPR reaction system , incubate at 37°C for one hour and detect the fluorescent signals of the FAM and VIC channels to obtain the detection results.

Figure 2: Design of CRISPR detection sites

In order to ensure the specificity of the detection method, avoid false positive detection due to sequence similarity. When designing crRNA, in addition to considering the sequence characteristics of SARS-CoV-2, the present invention also performs homologous alignment of the sequence of SARS-CoV-2 with various other coronaviruses, including human coronavirus 229E, HKU1, NL63 , OC43, SARS-CoV and bat SARS-CoV. The blue part in the figure is the aligned homologous sequence, and the red part is the difference sequence between the genomes.

Figure 3: Cross-reactivity validation:

It is proved that the two key CRISPR proteases LwaCas13a and LbaCas12a in the dual-channel detection method will not cross-cut their respective reaction substrates after activation, thus ensuring the theoretical feasibility of CRISPR dual-target detection.

Figure 4: Reaction system optimization of CRISPR dual-target detection method

In this embodiment, different ratios of Cas12a protein reaction system were introduced into the reaction system of Cas13a protein, namely 100%, 80%, 60%, 40% and 20%. Use a fixed concentration of the new coronavirus template (10000cp/ul), first undergo dual-site RT-RAA constant temperature amplification, and then take 2ul volumes of RT-RAA amplification products and add them to the CRISPR dual-target detection system with different component ratios , incubate at 37°C for 1 hour, and detect the fluorescence values of the FAM and VIC channels every 2 minutes throughout the incubation. Among them, LwaCas13a cuts the FAM-labeled RNA probe, and LbaCas12a cuts the VIC-labeled DNA probe, so FAM positive means that the gene locus detected by lwaCas13a is positive, and VIC positive means that the gene locus detected by LbaCas12a is positive.

Figure 5: Optimization of metal ion pair reaction sensitivity and reaction stability

Verify the improvement effect of different divalent ions such as Mg, Ca, Mn, Ni on the detection sensitivity of CRISPR dual targets

Figure 6: Sensitivity validation of dual-target assays

After determining the optimal reaction system components of the CRISPR dual-target detection method, the complete CRISPR dual-target detection method is used to detect the diluted templates at different concentrations to verify the sensitivity of the detection method. It can be seen from the figure that the novel coronavirus CRISPR dual-target detection method in the present invention can stably detect the sensitivities of the two sites: Cas13a 7cp/ul, Cas12a 25cp/ul.

Figure 7: Actual Sample Verification

Using the CRISPR dual-target detection method to simultaneously detect 24 new crown clinical samples, a total of 16 positive and 8 negative samples were detected, which was consistent with the fluorescence quantitative results.

Example 2: A novel coronavirus dual-target rapid detection method and kit based on CRISPR/Cas system

A kit for rapid detection of novel coronavirus double targets based on CRISPR/Cas system, said kit comprising:

Two sets of RT-RAA amplification primers:

Cas13-S-for: 5'-GAAATTAATACGACTCACTATAGGGgctatcatcttatgtccttccctcagtcag-3';

Cas13-S-rev: 5'-aatggcaggagcagttgtgaagttcttttc-3';

Cas12-N-for: 5'-aatgtcgcgcattggcatggaagtcaca-3';

Cas12-N-rev: 5'-gacttgatctttgaaatttggatctttg-3';

Two crRNAs specifically detected by CRISPR:

Cas12a-N-crRNA:

5'-uaauuucuacuaaguguagaauauggcaccuguuguaggucaa-3';

Cas13a-S-crRNA:

5'-gauuuagacuaccccaaaaacgaaggggacuaaaacCAUAAGUCACAAUGCAAGAAAGACUACCC-3';

RNA fluorescent probe: 5'-FAM-mArArUrGrGrGrCmAmArArUrGrGrGrCmA-BHQ1-3';

Among them, m represents the 2-position oxymethyl modification, and r represents ribonucleotides;

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

Also includes bufferA solution and BufferB solution; bufferA solution configuration method is: add 50mmol Tris buffer solution, 100nmol potassium acetate, 20g polyethylene glycol powder and 2mmol dithiothreitol to 1L water; BufferB solution is 280mM magnesium acetate solution.

Also includes: HEPES buffer, MgCl ₂ solution, 10×NEB buffer2.1 buffer, RNase inhibitor, T7 RNA polymerase, RNase-free water.

A novel coronavirus dual-target rapid detection method based on CRISPR/Cas system, comprising the following steps:

Step 1: Immerse the sample to be tested in the virus preservation solution, and then use the RNA extraction kit to extract the nucleic acid to obtain the nucleic acid to be tested; the sample to be tested is extracted from the doorknob.

Step 2: Add 37.5 μL of bufferA solution, 2 μL of Cas13-S-for, 2 μL of Cas13-S-rev, 2 μL of Cas12-N-for, 2 μL of Cas12 into a reaction tube containing protease lyophilized powder -N-rev and 2 μL of the nucleic acid to be tested, then add 2.5 μL of BufferB solution, cover the cap of the reaction tube and perform the amplification reaction to obtain the nucleic acid amplification product;

Cas13-S-for: 5'-GAAATTAATACGACTCACTATAGGGgctatcatcttatgtccttccctcagtcag-3';

Cas13-S-rev: 5'-aatggcaggagcagttgtgaagttcttttc-3';

Cas12-N-for: 5'-aatgtcgcgcattggcatggaagtcaca-3';

Cas12-N-rev: 5'-gacttgatctttgaaatttggatctttg-3';

Step 3: Prepare CRISPR reaction mixture: mix 0.4 μL of 1M HEPES buffer, 0.18 μL of 1M MgCl2 solution, 1.6 μL of 10×NEB buffer2.1 buffer, 0.8 μL of 25uM per rNTPmix, 2μL of LwaCas13a at a concentration of 63.2ng/μL, 1μL of Cas13-crRNA at a concentration of 10ng/μl, 1μL of LbaCas12a at a concentration of 1uM, 1μL of a concentration of 15ng/μl Cas12-crRNA, 1μL of RNase inhibitor at a concentration of 40U/μl, 0.1μL of T7RNApolymerase at a concentration of 50U/μl, 0.1μL of a DNA fluorescent probe at a concentration of 100uM, 0.1μL of an RNA fluorescent probe at a concentration of 100uM, and 8.92μL of RNase-free water mix;

Cas12a-N-crRNA:

5'-uaauuucuacuaaguguagaauauggcaccuguuguaggucaa-3';

Cas13a-S-crRNA:

5'-gauuuagacuaccccaaaaacgaagggggacuaaaacCAUAAGUCACAAUGCAAGAAAGACUACACC;

RNA Fluorescent Probes:

5'-FAM-mArUrGrGrGrCmAmArArUrGrGrCmA-BHQ1-3';

Among them, m represents the 2-position oxymethyl modification, and r represents ribonucleotides;

DNA fluorescent probes:

5'-VIC-TTATTATT-BHQ1-3';

Step 4: Add 2 μL of the nucleic acid amplification product obtained in step 2 to the CRISPR reaction mixture prepared in step 3, and incubate at 37°C for 30 minutes; judge by the following methods:

1. Incubate with a fluorescent quantitative PCR instrument and detect the fluorescence at the same time, or directly observe the fluorescence change with the naked eye at the end of the water bath;

2. At the beginning of the CRISPR reaction incubation, use the qPCR instrument to read the fluorescence value under the FAM channel, incubate at 37°C for 20 cycles, with an interval of 2 minutes between each cycle, record the fluorescence signal once at the end of each cycle, and judge the duality by the final fluorescence signal intensity. Target detection results, that is, the fluorescence value greater than 3000 indicates that the corresponding site is positive. For example, the fluorescence value of the FAM channel is greater than 3000, that is, the S site of the new coronavirus corresponding to Cas13a is detected positive, and the fluorescence value of the VIC channel is greater than 3000, that is, the corresponding site of Cas12a The N site of the new coronavirus is positive, and the fluorescence value is less than 2000, which means that the corresponding site is negative. If the fluorescence value is between 2000-3000, re-test. If the fluorescence value is still 2000-3000, the corresponding site is determined to be positive.

The above are only preferred embodiments for explaining the present invention, and are not intended to limit the present invention in any form. Therefore, any modification or change of the present invention made under the same spirit of the invention , should still be included in the scope of protection intended by the present invention.

Claims (5)

1. A kit for rapid detection of novel coronavirus double targets based on CRISPR/Cas system, characterized in that: said kit includes:

   Two sets of RT-RAA amplification primers:

   Cas13-S-for: 5'-GAAATTAATACGACTCACTATAGGGgctatcatcttatgtccttccctcagtcag-3';

   Cas13-S-rev: 5'-aatggcaggagcagttgtgaagttcttttc-3';

   Cas12-N-for: 5'-aatgtcgcgcattggcatggaagtcaca-3';

   Cas12-N-rev: 5'-gacttgatctttgaaatttggatctttg-3';

   Two crRNAs specifically detected by CRISPR:

   Cas12a-N-crRNA:

   5'-uaauuucuacuaaguguagaauauggcaccuguuguaggucaa-3';

   Cas13a-S-crRNA:

   5'-gauuuagacuaccccaaaaacgaaggggacuaaaacCAUAAGUCACAAUGCAAGAAAGACUACCC-3';

   RNA fluorescent probe: 5'-FAM-mArArUrGrGrGrCmAmArArUrGrGrGrCmA-BHQ1-3';

   Among them, m represents the 2-position oxymethyl modification, and r represents ribonucleotides;

   DNA fluorescent probe: 5'-VIC-TTATTATT-BHQ1-3'.
2. The kit for rapid detection of novel coronavirus double targets based on CRISPR/Cas system according to claim 1, characterized in that: it also includes bufferA solution and BufferB solution; bufferA solution configuration method is: add 50mmol Tris buffer to 1L water solution, 100nmol of potassium acetate, 20g of polyethylene glycol powder and 2mmol of dithiothreitol; BufferB solution is a magnesium acetate solution with a concentration of 280mM.
3. The kit for rapid detection of novel coronavirus double targets based on CRISPR/Cas system according to claim 1, characterized in that: it also includes: _HEPES buffer, MgCl solution, 10×NEB buffer2.1 buffer, RNase inhibitor , T7 RNA polymerase, RNase-free water.
4. A novel coronavirus dual-target rapid detection method based on CRISPR/Cas system for non-diagnostic purposes, characterized in that: comprising the following steps:

   Step 1: Immerse the sample to be tested in the virus preservation solution, and then use the RNA extraction kit to extract the nucleic acid to obtain the nucleic acid to be tested;

   Step 2: Add 37.5 μL of buffer A solution, 2 μL of Cas13-S-for, 2 μL of Cas13-S-rev, 2 μL of Cas12-N-for, 2 μL of Cas12-N-rev and 2 μL nucleic acid to be tested, then add 2.5 μL BufferB solution, cover the cap of the reaction tube and perform amplification reaction to obtain nucleic acid amplification product;

   Cas13-S-for: 5'-GAAATTAATACGACTCACTATAGGGgctatcatcttatgtccttccctcagtcag-3';

   Cas13-S-rev: 5'-aatggcaggagcagttgtgaagttcttttc-3';

   Cas12-N-for: 5'-aatgtcgcgcattggcatggaagtcaca-3';

   Cas12-N-rev: 5'-gacttgatctttgaaatttggatctttg-3';

   Step 3: Prepare CRISPR reaction mixture: mix 0.4 μL of 1M HEPES buffer, 0.18 μL of 1M MgCl ₂ solution, 1.6 μL of 10×NEB buffer2.1 buffer, 0.8 μL of 25uM Each rNTPmix, 2 μL of LwaCas13a at a concentration of 63.2 ng/μL, 1 μL of Cas13-crRNA at a concentration of 10 ng/μl, 1 μL of LbaCas12a at a concentration of 1 uM, 1 μL of Cas12-crRNA at a concentration of 15 ng/μl, 1 μL RNase inhibitor at a concentration of 40U/μl, 0.1μL of T7 RNA polymerase at a concentration of 50U/μl, 0.1μL of a DNA fluorescent probe at a concentration of 100uM, 0.1μL of an RNA fluorescent probe at a concentration of 100uM, and 8.92μL of RNase-free water mix;

   Cas12a-N-crRNA:

   5'-uaauuucuacuaaguguagaauauggcaccuguuguaggucaa-3';

   Cas13a-S-crRNA:

   5'-gauuuagacuaccccaaaaacgaagggggacuaaaacCAUAAGUCACAAUGCAAGAAAGACUACACC;

   RNA Fluorescent Probes:

   5'-FAM-mArUrGrGrGrCmAmArArUrGrGrCmA-BHQ1-3';

   Among them, m represents the 2-position oxymethyl modification, and r represents ribonucleotides;

   DNA fluorescent probes:

   5'-VIC-TTATTATT-BHQ1-3';

   Step 4: Add 2 μL of the nucleic acid amplification product obtained in step 2 to the CRISPR reaction mixture prepared in step 3, and incubate at 37°C for 30 minutes; judge by the following methods:

   1. Incubate with a fluorescent quantitative PCR instrument and detect the fluorescence at the same time, or directly observe the fluorescence change with the naked eye at the end of the water bath;

   2. Use the qPCR instrument to read the fluorescence value under the FAM channel at the beginning of the CRISPR reaction incubation, and incubate at 37°C for 20 cycles, with an interval of 2 minutes between each cycle, record the fluorescence signal once at the end of each cycle, and judge the duality by the final fluorescence signal intensity. Target detection results, that is, the fluorescence value greater than 3000 indicates that the corresponding site is positive. For example, the fluorescence value of the FAM channel is greater than 3000, that is, the S site of the new coronavirus corresponding to Cas13a is detected positive, and the fluorescence value of the VIC channel is greater than 3000, that is, the corresponding site of Cas12a The detection of the N site of the new coronavirus is positive, and the fluorescence value is less than 2000, which means that the corresponding site is negative. If the fluorescence value is between 2000-3000, it will be tested again. If the fluorescence value is still 2000-3000, it is determined that the corresponding site is positive.
5. The novel coronavirus dual-target rapid detection method based on CRISPR/Cas system according to claim 4, characterized in that: bufferA solution configuration method is: add 50mmol Tris buffer solution to 1L water, 100nmol potassium acetate, 20g poly Ethylene glycol powder and 2 mmol dithiothreitol.

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