CN113755646A

CN113755646A - CrRNA and CRISPR-Cas12a system for novel bunyavirus detection and application

Info

Publication number: CN113755646A
Application number: CN202111176800.5A
Authority: CN
Inventors: 沈继录; 唐浩; 徐华铭; 夏兆新; 杨文苏; 朱毅
Original assignee: Fourth Affiliated Hospital of Anhui Medical University
Current assignee: Fourth Affiliated Hospital of Anhui Medical University
Priority date: 2021-10-09
Filing date: 2021-10-09
Publication date: 2021-12-07

Abstract

The invention relates to the technical field of medical treatment, and provides a CrRNA for detecting a novel bunyavirus, wherein the base sequence of the CrRNA is shown as SEQ ID NO.1, and the CrRNA is used for guiding Cas12a protein to be combined to an LAMP amplification product and be cut in a trans-form manner, so that early detection of virus diseases is realized. The invention also provides a CRISPR-Cas12a system and a method for rapidly detecting novel bunyavirus based on CRISPR-Cas12 a. Meanwhile, the invention also provides application of the CRISPR-Cas12a system in preparation of a novel bunyavirus detection kit. The invention has the advantages that: the method is rapid, convenient and fast, has strong applicability, can be realized in areas with deficient medical resources, does not depend on large instruments such as a PCR instrument and the like, has short time consumption, high sensitivity, low cost and low technical requirement on operators, and can be applied in various places.

Description

CrRNA and CRISPR-Cas12a system for novel bunyavirus detection and application

Technical Field

The invention relates to the technical field of medical treatment, in particular to a CrRNA and CRISPR-Cas12a system for novel bunyavirus detection and application thereof.

Background

The novel bunyavirus (SFTSV) is a new virus isolated and identified by the chinese centers for disease prevention and control in 2010. Infection of humans with SFTSV causes fever with thrombocytopenia syndrome (SFTS), whose major clinical manifestations are high fever, thrombocytopenia, leukopenia, gastrointestinal symptoms and high mortality. Therefore, rapid diagnosis of the disease is of paramount importance.

Currently, the etiologic diagnosis of SFTS relies primarily on SFTSV virus isolation and nucleic acid detection. Wherein, the virus separation experiment period is long, the cytopathic effect is not obvious, and the method is not suitable for clinical rapid diagnosis; PCR has the characteristics of high speed and high sensitivity, but has high requirements on equipment and operation technology, and is not suitable for portable detection.

Since the SFTSV virus is mainly transmitted by tick bites, cases mainly occur in mountain areas with poor medical conditions. The lack of medical conditions combined with the lack of an early, rapid and convenient means of detection, patients in these mountain areas often die due to their worsening caused by delayed definitive diagnosis.

Therefore, in order to meet the requirements of rapid and convenient diagnosis, a CrRNA and CRISPR-Cas12a system designed based on the CRISPR-Cas12a technology is developed, and a rapid portable nucleic acid detection method for detecting a novel bunyavirus in a clinical sample is developed by coupling with an immunochromatographic strip, which is important for rapid clinical diagnosis.

Disclosure of Invention

The invention aims to provide a system of CrRNA and CRISPR-Cas12a for detecting novel bunyavirus and application thereof, and the system is suitable for quickly and conveniently diagnosing patients infected by the novel bunyavirus under various medical conditions, so that the high mortality caused by delayed diagnosis in regions with deficient medical conditions is fully solved.

The invention adopts the following technical scheme to solve the technical problems:

a CrRNA for detecting a novel bunyavirus, wherein a base sequence of 5 '-3' of the CrRNA is shown as SEQ ID NO.1 (in a sequence corresponding to SEQ ID NO.1, y ═ c/t).

As one of the preferred modes of the invention, the CrRNA is used for guiding the Cas12a protein to recognize and bind to the LAMP amplified sequence to cut the target sequence and simultaneously cut any single-stranded DNA in the reaction system.

An application of the CrRNA in the preparation of a novel bunyavirus detection product.

A CRISPR-Cas12a system for detecting a novel bunyavirus comprises the crRNA.

As one of the preferable modes of the invention, the kit specifically comprises a Cas12a protein, crRNA, fluorescein isothiocyanate-quencher dual-labeled probe and a LAMP amplification product, or comprises a Cas12a protein, crRNA, fluorescein-biotin dual-labeled probe and a LAMP amplification product; the amplification primers of the LAMP amplification product comprise a primer F3, a primer B3, a primer BIP, a primer FIP and a primer LF, wherein 5 '-3' sequences of the primers are respectively shown as SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6 ((in the corresponding sequences of SEQ ID NO.2-6, y is c/t, r is a/g, w is a/t, and s is g/c)).

As one of the preferable modes of the invention, the CRISPR-Cas12a system further comprises reagents required by a LAMP amplification system and a Cas12a cleavage reaction system.

In a preferred embodiment of the present invention, in the CRISPR-Cas12a system, the LAMP amplification system (25 μ L) is specifically as follows: 0.5. mu.L of primer F3 (10. mu.M), 0.5. mu.L of primer B3 (10. mu.M), 1.0. mu.L of primer BIP (40. mu.M), 1.0. mu.L of primer FIP (40. mu.M), 0.5. mu.L of primer LF (20. mu.M), 12.5. mu.L of 2 × reaction mix buffer, 0.5. mu.L of fluorescein isothiocyanate, 4. mu.L of target DNA template, 4.5. mu.L of ddH₂O; the LAMP amplification conditions are as follows: stage one: amplifying at 65 ℃ for 1 min; 50 cycles; and a second stage: keeping the temperature at 4 ℃.

In a preferred embodiment of the present invention, in the CRISPR-Cas12a system, the Cas12a cleavage reaction system (20 μ L) is specifically as follows: mu.L NEB 3.1 buffer, 2. mu.L crRNA (10. mu.M), 1. mu.L LbCas12a (40nM), 1. mu.L fluorescein isothiocyanate-quencher dual-labeled probe (5. mu.M), 3. mu.L LAMP amplification product, 11. mu.L ddH₂O; the cracking conditions are as follows: incubating at 37 ℃ for 30min, with 1 cycle every 1 minute for a total of 30 cycles;

alternatively, the Cas12a cleavage reaction system (20 μ L) is specifically as follows: mu.L NEB 3.1 buffer, 2. mu.L LcrRNA (10. mu.M), 1. mu.L LbCas12a (40nM), 1. mu.L fluorescein-biotin double-labeled probe (5. mu.M), 3ul LAMP amplification product, 11. mu.L ddH₂O; the cracking conditions are as follows: incubate at 37 ℃ for 30 min.

A method for rapidly detecting novel bunyavirus based on CRISPR-Cas12a is used for detecting a sample to be detected by using the CRISPR-Cas12a system for detecting the novel bunyavirus.

As one of the preferred modes of the invention, when the CRISPR-Cas12a system adopts Cas12a protein, crRNA, fluorescein isothiocyanate-quencher dual-labeled probe and LAMP amplification product, the detection result is read by observing the fluorescence value of the cleavage product after Cas12a cleavage reaction;

when the CRISPR-Cas12a system adopts Cas12a protein, crRNA, a fluorescein-biotin double-labeled probe and a LAMP amplification product, a detection result is read through coupling reaction of a cleavage product after Cas12a cleavage reaction and a colloidal gold immunochromatographic strip.

When the CRISPR-Cas12a system adopts a Cas12a protein, crRNA, a fluorescein isothiocyanate-quencher double-labeled probe and an LAMP amplification product, the kit is used for indoor fluorescence detection; when the CRISPR-Cas12a system adopts Cas12a protein, crRNA, a fluorescein-biotin double-labeled probe and a LAMP amplification product, the kit is used for quickly detecting the immunochromatographic strip under outdoor low-resource conditions.

Compared with the prior art, the invention has the advantages that:

(1) the invention uses the loop-mediated isothermal amplification technology to amplify the novel bunyavirus RNA, the technology does not need the fussy circulating temperature changing process, only needs a water bath kettle, controls the temperature at 65 ℃, and incubates for 50min at constant temperature; the amplification method can be realized in the areas with deficient medical resources, does not depend on large instruments such as a PCR instrument and the like, has short time consumption, high sensitivity and low technical requirements on operators, and can be applied to various places.

(2) According to the invention, a Cas12a cleavage process is added after LAMP amplification, LAMP amplification specificity is greatly ensured, the LAMP amplification specificity is combined with an immunochromatography technology, and the novel bunyavirus can be visually detected through immunochromatography test paper, so that the method is particularly friendly to nucleic acid detection in a low-resource environment.

(3) The CrRNA can guide the Cas12a protein to be combined on a target sequence for recognizing and cutting the target sequence, and simultaneously, a single-stranded DNA reporter molecule in a reaction system is cut; when the single-stranded DNA reporter molecule is a fluorescein isothiocyanate-quencher double-labeled probe, the single-stranded DNA reporter molecule can be matched with a fluorescence detector to directly read a detection result; when the single-stranded DNA reporter molecule is a fluorescein-biotin double-labeled probe, the single-stranded DNA reporter molecule can be matched with an immunochromatographic strip to read a detection result; accordingly, the present invention can be used as a method for clinical large-scale detection of human beings, and compared with the conventional diagnostic detection methods, the detection method of the present invention greatly shortens the detection time, does not require repetitive training of operators, and has a low consumption price.

(4) The LAMP amplification and CRISPR-Cas12a technology are combined, and the established new detection platform is innovative and can be further popularized and applied to detection of various pathogens and genes. The detection platform is rapid, sensitive, strong in specificity, low in cost and simple in operation, and can meet the clinical requirements for rapid detection of the novel bunyavirus. At present, no related similar research is carried out on the novel bunyavirus at home and abroad, so that the method has the innovation of market value; the basic research is shifted to clinical research, and the method has conversion innovation.

Drawings

Fig. 1 is a process diagram of a method based on CRISPR-Cas12a novel bunyavirus in example 6;

FIG. 2 is a graph of LAMP amplification fluorescence in example 6;

FIG. 3 is an agarose gel electrophoresis of the LAMP amplification product of example 6;

fig. 4 is a graph of CRISPR-Cas12a cleavage fluorescence in example 6;

FIG. 5 is a schematic diagram of the design of the immune colloidal gold test strip in example 6;

FIG. 6 is a view showing the state of a chromatographic strip in example 6 when a positive result is shown;

FIG. 7 is a view showing the state of a chromatographic strip in example 6 when a negative result is shown;

FIG. 8 is a state diagram of the final chromatographic strip in example 6.

Detailed Description

The following examples are given for the detailed implementation and specific operation of the present invention, but the scope of the present invention is not limited to the following examples.

Example 1

This example is a CrRNA for the detection of a novel bunyavirus.

The crux of the CRISPR-Cas12a detection method lies in CrRNA, so the selection of CrRNA is directly related to the effectiveness of the final detection method.

The base sequence of the CrRNA is shown as SEQ ID NO.1, the CrRNA is used for guiding the Cas12a protein to recognize and bind to the LAMP amplified sequence so as to cut a target sequence, and meanwhile, the Cas12a protein reversely cuts any single-stranded DNA in a reaction system.

Specifically, any single-stranded DNA in the reaction system is mainly an LAMP amplification primer in a CRISPR-Cas12a system, and comprises a primer F3, a primer B3, a primer BIP, a primer FIP and a primer LF, wherein the sequences (5 '-3') of the primers are respectively shown as SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5 and SEQ ID No. 6.

Example 2

The CRISPR-Cas12a system for detecting the novel bunyavirus comprises a Cas12a protein, crRNA, a fluorescein isothiocyanate-quencher double-labeled probe and a LAMP amplification product. Wherein, the amplification primer of the LAMP amplification product comprises: the sequences (5 '-3') of the primer F3, the primer B3, the primer BIP, the primer FIP and the primer LF are respectively shown as SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO. 6.

Meanwhile, the CRISPR-Cas12a system also comprises various reagents required by a LAMP amplification system and a Cas12a cleavage reaction system.

Specifically, the LAMP amplification system (25. mu.L) was as follows: 0.5. mu.L of primer F3 (10. mu.M), 0.5. mu.L of primer B3 (10. mu.M), 1.0. mu.L of primer BIP (40. mu.M), 1.0. mu.L of primer FIP (40. mu.M), 0.5. mu.L of primer LF (20. mu.M), 12.5. mu.L of 2 × reaction mix buffer, 0.5. mu.L of fluorescein isothiocyanate, 4. mu.L of LDNA template, 4.5. mu.L of LddH₂And O. LAMP amplification conditions: stage one: amplifying at 65 ℃ for 1 min; 50 cycles; and a second stage: keeping the temperature at 4 ℃.

Cas12a cleavage reaction (20 μ L) was as follows: mu.L NEB 3.1 buffer, 2. mu.L crRNA (10. mu.M), 1. mu.L LbCas12a (40nM), 1. mu.L fluorescein isothiocyanate-quencher dual-labeled probe (5. mu.M), 3. mu.L LAMP amplification product, 11. mu.L ddH₂And O. And (3) cracking conditions: incubate at 37 ℃ for 30min, cycle 1 every 1 minute for 30 cycles.

Example 3

In the method for rapidly detecting the novel bunyavirus based on the CRISPR-Cas12a, the CRISPR-Cas12a system of example 2 is used for detecting a sample to be detected, and the complete method is as follows:

firstly, extracting RNA of the novel bunyavirus;

(1) sample treatment: a1.5 mL centrifuge tube was charged with 0.2mL of the liquid virus sample.

(2) 0.6mL of Buffer Rlysis-VG was added to 0.2mL of the sample obtained in the first step, and the mixture was shaken for 30 seconds and then left at room temperature for 10min (Buffer Rlysis-VG was placed in a water bath at 65 ℃ before use to completely dissolve the precipitate).

(3) 0.6mL of absolute ethanol was added, the tube cap was capped and vortexed for 15 sec.

(4) After brief centrifugation, 700. mu.l of the solution was transferred to a centrifugal adsorption column and allowed to stand at room temperature for 2 min.

(5) Centrifuging at 12000rpm for 1min at room temperature, removing the permeate, and returning the adsorption column to the collection tube.

(6) And (4) centrifuging the residual solution for a short time, transferring the residual solution into the centrifugal adsorption column in the step (4), and standing at room temperature for 2 min.

(7) Centrifuging at 12000rpm for 1min at room temperature, removing the penetrating liquid, and placing the centrifugal adsorption column back into the collection tube.

(8) Adding 500 μ l RPE Solution into centrifugal adsorption column, centrifuging at 12000rpm for 1min at room temperature, removing the liquid, and placing the column back into the collection tube.

(9) Repeating the step (8) for 1 time.

(10) Centrifuge at 12000rpm for 2min at room temperature, and discard the tube containing the permeate.

(11) The column was inserted into a new, self-contained RNase-free 1.5mL centrifuge tube, and 50. mu.l DEPC-treated ddH was added to the middle of the filter of the column₂O, and then left at room temperature for 2 min.

(12) Centrifuging at 12000rpm for 2min at room temperature, and collecting the sample in the tube, namely the virus RNA, as a template for the subsequent LAMP reaction.

Secondly, designing and amplifying LAMP primers;

(1) sets of primers were designed using PrimeExplorer v.5(https:// PrimeExplorer. jp/e /) software and BLAST alignment analysis was performed. The best performing set of primers was selected for subsequent studies and primers were synthesized by the company.

(2) The LAMP reaction system (25. mu.L) was as follows (Table 1): 0.5. mu.L of primer F3 (10. mu.M), 0.5. mu.L of primer B3 (10. mu.M), 1.0. mu.L of primer BIP (40. mu.M), 1.0. mu.L of primer FIP (40. mu.M), 0.5. mu.L of primer LF (20. mu.M), 12.5. mu.L of 2 × reaction mix buffer, 0.5. mu.L of fluorescein isothiocyanate, 4. mu.L of LDNA template (novel bunyavirus RNA), 4.5. mu.L of LddH₂And O. Wherein, the sequences (5 '-3') of the primer F3, the primer B3, the primer BIP, the primer FIP and the primer LF are respectively shown as SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO. 6.

TABLE 1LAMP reaction System

(3) The LAMP amplification conditions are as follows:

stage one: amplifying at 65 ℃ for 1 min; 50 cycles;

and a second stage: keeping the temperature at 4 ℃;

(4) observing the condition of the fluorescence value;

the peak value of the fluorescence value is observed, namely the amplification is successful.

Thirdly, identifying LAMP amplification products;

performing agarose gel electrophoresis on the LAMP product;

adding 1.5% agarose gel, adding LAMP product (containing loading buffer) and DNA Marker into the sample adding hole, setting the voltage at 120V, and taking out the gel after electrophoresis for 35 min. And (3) placing the LAMP amplification product after amplification in a gel imaging system for imaging, taking a photo, and observing the existence of a ladder-shaped strip in agarose, namely the LAMP amplification is successful.

Fourthly, the Cas12a protein cuts the amplified target sequence under the guidance of CrRNA, and meanwhile, the Cas12a protein cuts a fluorescein isothiocyanate-quencher double-labeled probe;

cas12a cleavage reaction (20 μ L): mu.L NEB 3.1 buffer, 2. mu.L LcrRNA (10. mu.M), 1. mu.L LLbCas12a (40nM), 1. mu.L fluorescein isothiocyanate-quencher ditag probe (5. mu.M) and LAMP amplification product 3ul, 11. mu.L ddH₂O (Table 2).

TABLE 2CRISPR-Cas12a cleavage reaction System (fluorescein isothiocyanate-quencher dual-labeled probe)

The above system was added to a fluorescent quantitative PCR tube and incubated at 37 ℃ for 30 min. Once every minute for 30 cycles. And observing the fluorescence value of the cut probe by using an ABI7500 fluorescence quantitative PCR instrument. A significant increase in fluorescence was positive and indicated the presence of the gene of interest. The result of no increase in fluorescence was negative, indicating that the gene of interest was not contained.

Example 4

The CRISPR-Cas12a system for detecting the novel bunyavirus comprises a Cas12a protein, crRNA, a fluorescein-biotin double-labeled probe and a LAMP amplification product; wherein the amplification primer of the LAMP amplification product comprises a primer F3, a primer B3, a primer BIP, a primer FIP and a primer LF, and the sequences (5 '-3') of the primers are respectively shown as SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO. 6.

Cas12a cleavage reaction (20 μ L) was as follows: mu.L NEB 3.1 buffer, 2. mu.L LcrRNA (10. mu.M), 1. mu.L LbCas12a (40nM), 1. mu.L fluorescein-biotin double-labeled probe (5. mu.M), 3ul LAMP amplification product, 11. mu.L ddH₂O; the cracking conditions are as follows: incubate at 37 ℃ for 30 min.

Example 5

In the method for rapidly detecting the novel bunyavirus based on the CRISPR-Cas12a, the CRISPR-Cas12a system of example 4 is used for detecting a sample to be detected, and the complete method is as follows:

firstly, extracting RNA of the novel bunyavirus;

the same as in example 3.

Secondly, designing and amplifying LAMP primers;

the same as in example 3.

Thirdly, identifying LAMP amplification products;

the same as in example 3.

Fourthly, the Cas12a protein cuts the amplified target sequence under the guidance of CrRNA, and meanwhile, the Cas12a protein cuts a fluorescein-biotin double-labeled probe;

cas12a cleavage reaction (20 μ L) was as follows: 2 μ L NEB 3.1 buffer, 2 μ LcrRNA (10. mu.M), 1. mu.L of LbCas12a (40nM), 1. mu.L of fluorescein-biotin double-labeled probe (5. mu.M), 3ul of LAMP amplification product, 11. mu.L ddH₂O (Table 3).

TABLE 3 CRISPR-Cas12a cleavage reaction System (fluorescein-biotin double-labeled probe)

The above system was loaded into a centrifuge tube and incubated in a 37 ℃ water bath for 30 minutes.

Preparing colloidal gold nanoparticles and coupling substances;

(1) 350mL (1mM) gold chloride solution (HAuCl4) was boiled.

(2) After boiling, stir and add 3.5mL of 1% sodium citrate solution.

(3) After addition of the sodium citrate solution, this mixture formed a colorless solution which turned wine-red after boiling for 10 minutes and could be used after cooling at room temperature.

(4) Next, AuNP-avidin conjugate was prepared, and 1mL of the prepared colloidal gold nanoparticle solution was collected by first centrifugation (12000 Xg, 25min) and concentrated 4 times.

(5) Then mixed with 100 μ L of suspension buffer (10% sucrose, 0.1% NaN3, 5% BSA, 0.25% tween-20, 20mM Na3PO 4). Finally, 0.5mg/mL of anti-biotin antibody was added.

(6) Free anti-biotin antibody was removed by gently shaking at 4 ℃ for 3h, centrifuging (12000 Xg, 25min), and then washing three times with suspension buffer. The red particles were resuspended in 100. mu.L of suspension buffer and then dispensed onto the conjugate pad.

Sixthly, preparing a colloidal gold immunochromatographic strip;

(1) the sample pad, the conjugate pad and the nitrocellulose membrane constitute the main three parts of the immunochromatographic strip. anti-FAM and anti-mouse IgG antibodies were coated on nitrocellulose strips (25mm wide), respectively, to form a control zone and a test zone, respectively, and the distance between the test line and the control line was maintained to be greater than 5 mm.

(2) Part of the test strip material was dried at 22 ℃ for 12 hours and stored in a desiccant container until use, also to protect the nitrocellulose membrane from moisture.

(3) To assemble the complete test strip, the sample pad (16mm wide) was immersed in sample pad buffer (1% Triton, 1% BSA, 2% glucose, 50mm boric acid, pH 8.0), dried and stored. All mats were assembled in sequence along the bonded portions of the nitrocellulose membrane, overlapped by 2mm, and then cut into 0.4cm wide strips.

(4) The home-made sample pad and absorbent pad were soaked in a pH 8.0 buffer consisting of 1% Triton, 1% BSA, 2% glucose, 50mM boric acid, dried and stored in a dry container. The conjugate pad and the absorbent pad were used without a buffering treatment and then assembled in sequence along the nitrocellulose membrane.

Seventhly, capturing the cutting product by a colloidal gold immunochromatographic strip;

(1) after the Cas12a cleavage reaction is finished, adding a diluent into the reaction product, determining the dilution times according to the concentration of a target substance, then taking 80ul of the supernatant, slowly dripping the supernatant on a sample adding hole of a test strip, and judging the result within 5-10 minutes.

(2) When the detection line is observed to have strips, or the control line and the detection line have strips simultaneously, the detection result is positive; when only the control line is striped, the result is negative.

Example 6

Referring to fig. 1, validation of the CRISPR-Cas12 a-based novel bunyavirus method in one of the above embodiments of the present embodiment.

Firstly, extracting RNA of the novel bunyavirus;

the same as in example 3.

Secondly, designing and amplifying LAMP primers;

(1) the procedure of (3) is the same as in example 3.

(4) Observing the condition of fluorescence value;

the results are shown in FIG. 2. According to FIG. 2 (positive sample, negative sample, no peak), a peak in fluorescence value was observed, indicating successful amplification.

Thirdly, identifying LAMP amplification products;

placing the amplified LAMP amplification product in a gel imaging system for imaging, taking a photo, and observing the presence of a ladder-shaped strip in agarose, as shown in figure 3; according to FIG. 3 (the positive product shows a ladder-shaped band after agarose gel electrophoresis, and the negative product has no characteristic band), LAMP amplification is completed.

Fourthly, the amplified fragment is cut by the Cas12a protein, and simultaneously, the fluorescein isothiocyanate-quencher double-labeled probe is cut;

(1) loading the designed CrRNA, a Cas12a protein, a target sequence, a fluorescein isothiocyanate-quencher double-labeled probe and the like, and constructing a Cas12a cleavage reaction system;

cas12a cleavage reaction (20 μ L): mu.L NEB 3.1 buffer, 2. mu.L LcrRNA (10. mu.M), 1. mu.L LLbCas12a (40nM), 1. mu.L fluorescein isothiocyanate-quencher ditag probe (5. mu.M) and LAMP amplification product 3ul, 11. mu.L ddH₂O。

(2) And adding the reaction system after the sample addition into a fluorescent quantitative PCR tube, and incubating for 30min at 37 ℃. Once every minute for 30 cycles. The fluorescence value of the cleaved probe was observed by ABI7500 fluorescence quantitative PCR instrument, and the result is shown in FIG. 4 (the FAM fluorescence intensity of the positive sample was increased, and the negative sample was unchanged). Since fluorescein fluoresces without being affected by the quencher as the probe is cleaved, the intensity of the fluorescent signal increases significantly, and the result in fig. 4 shows a positive result, i.e., it indicates that the sample contains the target gene.

Therefore, the CRISPR-Cas12a system containing the fluorescein isothiocyanate-quencher dual-labeled probe and the corresponding detection method can accurately and rapidly detect the novel bunyavirus, and have high feasibility.

Fifthly, the amplified fragment is cut by the Cas12a protein, and simultaneously, a fluorescein-biotin double-labeled probe is cut;

(1) loading the designed CrRNA, Cas12a protein, target sequence, fluorescein-biotin double-labeled probe and the like to construct a Cas12a cleavage reaction system;

cas12a cleavage reaction (20 μ L) was as follows: 2 μ L NEB 3.1 buffer, 2 μ L crRNA (10 μ M), 1 μ L LbCas12a (40 μ M)nM), 1. mu.L fluorescein-biotin double-labeled probe (5. mu.M), 3ul LAMP amplification product, 11. mu.L ddH₂O。

(2) And adjusting the water bath to 37 ℃, putting the Cas12a cleavage reaction system subjected to sample addition into the water bath, and incubating for 30min to obtain a cleavage product for later use.

Sixthly, preparing colloidal gold nanoparticles and coupling substances;

the same as in example 5.

Preparing a colloidal gold immunochromatographic strip;

the same as in example 5.

Eighthly, capturing the cutting product by a colloidal gold immunochromatographic strip;

(2) The schematic design of the immune colloidal gold plate is shown in FIG. 5 (positive sample T-line developed, negative sample T-line not developed). Specifically, when a band appears on the detection line, or a band appears on both the control line and the detection line, the detection result is positive, which indicates that the sample contains the novel bunyavirus gene, as shown in fig. 6; when only the control line shows a band, the result is negative, which indicates that the sample does not contain the new bunyavirus gene, as shown in FIG. 7.

(3) The detection result of the present embodiment is shown in fig 8,

accordingly, the CRISPR-Cas12a system containing the fluorescein-biotin double-labeled probe and the corresponding detection method can accurately and rapidly detect the novel bunyavirus, and have high feasibility.

Among them, it should be noted that, due to the difference in the requirements of the detection conditions, the "CRISPR-Cas 12a system containing a fluorescein isothiocyanate-quencher dual-labeled probe and the corresponding detection method" are suitable for indoor environment detection, while the "CRISPR-Cas 12a system containing a fluorescein-biotin dual-labeled probe and the corresponding detection method" are more suitable for rapid detection under outdoor low-resource conditions.

In conclusion, the method for rapidly detecting the novel bunyavirus based on the CRISPR-Cas12a technology and the application thereof can effectively, rapidly and conveniently detect the novel bunyavirus nucleic acid, are suitable for various places, and have strong practicability. The invention is converted into a diagnostic kit which can be used for clinical early diagnosis, thereby improving the survival rate of a patient infected by the novel bunyavirus.

Meanwhile, preliminary verification proves that the kit has extremely high sensitivity and specificity and good detection performance.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.

SEQUENCE LISTING

<110> fourth subsidiary hospital of medical university of Anhui

<120> CrRNA and CRISPR-Cas12a system for novel bunyavirus detection and application

<130> 2021

<160> 6

<170> PatentIn version 3.3

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yggagccagc aagacagarg ttgacagagt tcacagcwgc at 42

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Claims

1. A CrRNA for detecting a novel bunyavirus is characterized in that a base sequence is shown as SEQ ID NO. 1.

2. The CrRNA for the detection of a novel bunyavirus according to claim 1, wherein the CrRNA is used for guiding the recognition and binding of Cas12a protein to the LAMP-amplified sequence to cut a target sequence and simultaneously cut any single-stranded DNA in a reaction system.

3. Use of CrRNA according to any one of claims 1-2 in the preparation of a novel bunyavirus assay product.

4. A CRISPR-Cas12a system for detecting a novel bunyavirus, comprising the crRNA of any of claims 1-2.

5. The CRISPR-Cas12a system for detecting the novel bunyavirus according to claim 4, which specifically comprises a Cas12a protein, crRNA, fluorescein isothiocyanate-quencher dual-labeled probe and LAMP amplification product, or comprises a Cas12a protein, crRNA, fluorescein-biotin dual-labeled probe and LAMP amplification product; the amplification primers of the LAMP amplification product comprise a primer F3, a primer B3, a primer BIP, a primer FIP and a primer LF, and the sequences of the primers are respectively shown as SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO. 6.

6. The CRISPR-Cas12a system for detecting the novel bunyavirus according to claim 5, wherein the CRISPR-Cas12a system further comprises reagents required by a LAMP amplification system and a Cas12a cleavage reaction system.

7. The CRISPR-Cas12a system for detecting the novel bunyavirus according to claim 6, wherein in the CRISPR-Cas12a system, the LAMP amplification system is as follows: 0.5. mu.L of 10. mu.M primer F3, 0.5. mu.L of 10. mu.M primer B3, 1.0. mu.L of 40. mu.M primer BIP, 1.0. mu.L of 40. mu.M primer FIP, 0.5. mu.L of 20. mu.M primer LF, 12.5. mu.L of 2 × reaction mix buffer, 0.5. mu.L of fluorescein isothiocyanate, 4. mu.L of target DNA template, and ddH added thereto₂O to a final volume of 25. mu.L; the LAMP amplification conditions are as follows: stage one: amplifying at 65 ℃ for 1 min; 50 cycles; and a second stage: keeping the temperature at 4 ℃;

in the CRISPR-Cas12a system, a Cas12a cleavage reaction system is as follows: mu.L of NEB 3.1 buffer, 2. mu.L of 10. mu.M crRNA, 1. mu.L of 40nM LbCas12a, 1. mu.L of 5. mu.M fluorescein isothiocyanate-quencher double-labeled probe, 3ul of LAMP amplification product, and ddH added₂O to a final volume of 20. mu.L; the cracking conditions are as follows: incubating at 37 ℃ for 30min, with 1 cycle every 1 minute for a total of 30 cycles;

alternatively, the Cas12a cleavage reaction system is specifically as follows: mu.L NEB 3.1 buffer, 2. mu.L 10. mu.McrRNA, 1. mu.L 40nM LbCas12a, 1. mu.L 5. mu.M fluorescein-biotin double-labeled probe, 3ul LAMP amplification product, and ddH added₂O to a final volume of 20. mu.L; the cracking conditions are as follows: incubate at 37 ℃ for 30 min.

8. A method for rapidly detecting a novel bunyavirus based on CRISPR-Cas12a, which is characterized in that a CRISPR-Cas12a system for detecting the novel bunyavirus as claimed in any one of claims 5 to 7 is used for detecting a sample to be detected.

9. The method for rapidly detecting the novel bunyavirus based on CRISPR-Cas12a according to claim 8, wherein when the CRISPR-Cas12a system adopts Cas12a protein, crRNA, fluorescein isothiocyanate-quencher dual-labeled probe and LAMP amplification product, the detection result is read by observing the fluorescence value of the cleavage product after Cas12a cleavage reaction;

10. Use of the CRISPR-Cas12a system for detecting a novel bunyavirus according to any one of claims 5-7 in the preparation of a novel bunyavirus detection kit, wherein when the CRISPR-Cas12a system adopts a Cas12a protein, crRNA, a fluorescein isothiocyanate-quencher dual-labeled probe and a LAMP amplification product, the kit is used for indoor fluorescence detection; when the CRISPR-Cas12a system adopts Cas12a protein, crRNA, a fluorescein-biotin double-labeled probe and a LAMP amplification product, the kit is used for quickly detecting the immunochromatographic strip under outdoor low-resource conditions.