DNA fragment for detecting hantavirus SEOV subtype based on RPA-CRISPR / Cas12a system, recombinant vector, reagent combination, kit and application

Abstract

The invention discloses a DNA fragment, a recombinant vector, a reagent combination and a kit for detecting hantavirus SEOV subtypes based on an RPA-CRISPR / Cas12a system, and application of the kit to the non-diagnostic purpose of detecting the hantavirus SEOV subtypes. The SEOV detection accuracy can reach 100%, the target substance can be accurately detected, and the false positive or false negative probability is reduced; meanwhile, the specificity is high, only the Seoul virus can be detected, and the Xinjiang hemorrhagic fever virus, the Marburg virus and the Ebola virus which are in genetic relationship with the SEOV virus cannot be detected; the method has the characteristics of high efficiency, economical efficiency, convenience and environmental protection of detection at the same time.

Description

Technical Field

[0001] The present invention relates to the field of Hantavirus detection, and in particular to a DNA fragment, a recombinant vector, a reagent combination and a kit for detecting the SEOV subtype of Hantavirus based on an RPA-CRISPR / Cas12a-LFD system. Background Art

[0002] Hemorrhagic fever with renal syndrome (HFRS) is a major infectious disease caused by Hantavirus (HV) and spread by rodents (mainly rats), which seriously endangers the health of the people. As a Class B infectious disease that is under key monitoring in my country, HFRS has the characteristics of rapid onset and high mortality. It is reported that the number of HFRS cases reported each year is about 100,000.

[0003] With the development of modernization, the overexploitation of natural resources has intensified deforestation, resulting in a large number of rodents entering human settlements, increasing the possibility of Hantavirus infection, and the number of cases is expected to continue to increase. There are four types of HV that cause HFRS: Hantaan virus (HTNV), SEOV virus (SEOV), Puumala virus (PUUV) and Dobrava-Belgrade virus (DOBV). The clinical manifestations of diseases caused by different subtypes of viruses are also different. HTNV and DOBV usually cause more severe HFRS, SEOV causes moderate HFRS, and PUUV causes milder HFRS. Therefore, early identification and detection of the four subtypes of HFRS can not only help clinicians better assess the patient's condition, formulate treatment strategies, and improve treatment effects; it can also help predict the patient's prognosis to provide more accurate prognostic guidance and management. In addition, the infection and prevalence of viruses between different subtypes have geographical distribution, and HFRS in my country is mainly caused by infection with two types of viruses, HTNV and SEOV. Among them, the SEOV subtype has a higher carrying rate in rodents and is more likely to cause the spread and prevalence of the disease. Therefore, establishing an accurate, efficient and portable detection method for SEOV plays an important role in the early treatment of HFRS patients and controlling the epidemic outbreak of the disease.

[0004] At present, laboratory tests for SEOV mainly include hematological examination and pathogen nucleic acid detection. Hematological examination mainly detects anti-hantavirus IgG or IgM antibodies in patient serum through enzyme-linked immunosorbent assay, but this detection method has low sensitivity, poor specificity, and is prone to missed detection, which can not make early diagnosis and delay treatment. Real-time fluorescence quantitative PCR is currently the gold standard for rapid pathogen detection of Hantavirus, but it requires expensive professional instruments, is complicated to operate, takes a long time to detect, and has cumbersome result interpretation, so it cannot be widely popularized and applied in primary medical and health institutions.

[0005] As a new type of constant temperature amplification technology, recombinase polymerase amplification (RPA) mainly simulates DNA amplification in vivo through recombinase, single-strand binding protein and polymerase, and can quickly, sensitively, efficiently and specifically amplify the target fragment under constant temperature conditions. This method has the advantages of high sensitivity, strong specificity, short reaction time and high amplification efficiency. The CRISPR / Cas system is an acquired immune system of prokaryotes that can resist the invasion of exogenous phages or plasmid genetic elements. It has been widely used in the fields of genetic engineering and nucleic acid detection. CRISPR-Cas12a is a variant of the CRISPR-Cas system, also known as Cpf1. The Cas12a enzyme has DNA enzyme cutting function and can cut specific single-stranded DNA probes, thereby realizing rapid nucleic acid detection. Under the guidance of crRNA, Cas12a can cut the double-stranded template DNA and fluorescent labeled probes amplified by RPA. The probe is hydrolyzed to release fluorescent signals, and the presence or absence of target nucleic acid fragments can be determined by detecting the strength of the fluorescent signals. At the same time, it can also be combined with flow filtration test strips (LFD) to observe the results more accurately and intuitively. However, to date, there has been no report on the application of the CRISPR system in the detection and diagnosis of Hantavirus. Summary of the invention

[0006] Based on the above technical problems, one of the objects of the present invention is to provide a DNA fragment for detecting the SEOV subtype of Hantavirus based on the RPA-CRISPR / Cas12a system.

[0007] The second purpose of the present invention is to provide a recombinant vector for detecting the SEOV subtype of Hantavirus based on the RPA-CRISPR / Cas12a system.

[0008] The third object of the present invention is to provide a reagent combination for detecting Hantavirus SEOV subtype based on the RPA-CRISPR / Cas12a system.

[0009] A fourth object of the present invention is to provide a kit for detecting the SEOV subtype of Hantavirus based on the RPA-CRISPR / Cas12a system.

[0010] The fifth object of the present invention is to provide a kit for detecting Hantavirus SEOV subtypes based on the RPA-CRISPR / Cas12a system for the non-diagnostic purpose of detecting virus SEOV subtypes.

[0011] The technical solution provided by the present invention is: A DNA fragment for detecting the SEOV subtype of Hantavirus based on the RPA-CRISPR / Cas12a system, wherein the DNA fragment is a DNA fragment containing a base sequence shown in SEQ ID NO.1.

[0012] A recombinant vector for detecting the SEOV subtype of Hantavirus based on the RPA-CRISPR / Cas12a system, wherein the recombinant vector comprises a DNA fragment consisting of a base sequence shown in SEQ ID NO.1.

[0013] Preferably, the plasmid used in the recombinant vector is PUC57.

[0014] A reagent combination for detecting the SEOV subtype of Hantavirus based on the RPA-CRISPR / Cas12a system, the reagent combination comprising a primer set and crRNA for RPA amplification to obtain a DNA fragment containing a base sequence shown in SEQ ID NO.1; Wherein, the primer set includes an upstream primer sequence as shown in SEQ ID NO.2 and a downstream primer sequence as shown in SEQ ID NO.3; and the crRNA is an RNA fragment composed of a base sequence as shown in SEQ ID NO.4.

[0015] Preferably, it further comprises a ssDNA probe; The 5' end of the ssDNA probe is labeled with FAM and the 3' end is labeled with BHQ1; or the 5' end of the ssDNA probe is labeled with FAM and the 3' end is labeled with Biotin.

[0016] Preferably, it further comprises a ssDNA probe; Wherein, the nucleotide sequence of the ssDNA probe is 5'-FAM-TTATT-BHQ1-3'; or the nucleotide sequence of the ssDNA probe is 5'-FAM-TTTTTTTATTTTTTT-Biotin-3'.

[0017] A kit for detecting Hantavirus SEOV subtypes based on the RPA-CRISPR / Cas12a system, comprising a reagent combination for detecting Hantavirus SEOV subtypes based on the RPA-CRISPR / Cas12a system as described in claims 4-6.

[0018] Preferably, it also includes: RPA amplification reagent, CRISPR / Cas12a recognition and cleavage reagent and probe.

[0019] A kit for detecting Hantavirus SEOV subtypes based on an RPA-CRISPR / Cas12a system is used for non-diagnostic purposes of detecting virus SEOV subtypes, and the kit for detecting Hantavirus SEOV subtypes based on an RPA-CRISPR / Cas12a system is used.

[0020] Preferably, the method comprises the following steps: Step 1: extracting DNA from the sample to be tested as template DNA; Step 2: Add the template DNA obtained in step 1 to the RPA reaction system and mix thoroughly, and perform RPA amplification reaction using primer pairs; Among them, the RPA reaction system and RPA reaction conditions of the RPA amplification reaction are as follows: RPA reaction system: add 29.5μL Primer Free Rehydration buffer, 2.4μ of the upstream primer and downstream primer in the primer pair, 12.2μ of ddH2O, 1μ of 100ng / μL template DNA, and finally add 2.5μ of MgOAc activator on the lid. RPA reaction conditions: The RPA reaction system was fully mixed and amplified at 37°C to 41°C for 10 to 35 minutes; Step 3: Mix crRNA, ssDNA probe and Cas12a enzyme with the RPA amplification product obtained in step 2, and perform Cas12a fluorescence detection reaction; Among them, the Cas12a fluorescence detection reaction system is as follows: In a sterile PCR tube, 3 μ of 10×Culease Buffer, 10 nM~50 nM LbCas12a Nuclease, 300 nM~700 nM ssDNA-FQ, 10 nM~70 nM crRNA, and 16 μ of DEPCH2O were added in sequence, mixed, and 2 μ of RPA amplification product was added. After mixing and centrifugation, the mixture was placed in a fluorescence quantitative PCR instrument and incubated at 39°C. The fluorescence signal was collected every 30 s, and a total of 90 cycles were set. The reaction results were judged by visually observing the fluorescence intensity of the reaction solution under ultraviolet light and statistically analyzing the fluorescence value in the fluorescence quantitative PCR instrument; or 3 μ of 10×Culease Buffer, 10 nM~50 nM LbCas12a Nuclease, 2 μM ssDNA-FB 6 μ, 10 nM~70 nM crRNA, and DEPC H2O were added in sequence in a sterile PCR tube. 16μ, add 2μ of RPA amplification product after mixing, centrifuge and mix, place in a 35℃~41℃ warm bath for 20min, add high pressure ultrapure water to the reaction tube to make up to 50μl, insert the Cas12 / 13 special nucleic acid detection test strip into the reaction tube, let it stand for 1-2min, and then observe the results; Among them, the initial concentration of the LbCas12a Nuclease is 1 μM, and the Cas12a fluorescence detection reaction system is constructed after dilution to a final concentration of 10 nM~50 nM; the initial concentration of the ssDNA-FQ is 2 μM, and the Cas12a fluorescence detection reaction system is constructed after dilution to a final concentration of 300 nM~700 nM; the initial concentration of the crRNA is 100 μM, and the Cas12a fluorescence detection reaction system is constructed after dilution to a final concentration of 10 nM~70 nM.

[0021] The beneficial effects of the present invention are: 1. The reagent combination and kit for detecting the SEOV subtype of Hantavirus based on the RPA-CRISPR / Cas12a system provided by the present invention have accurate detection results, and the detection accuracy of the SEOV virus can reach 100%, which can accurately detect the target substance and reduce the probability of false positive or false negative; at the same time, it also has the characteristics of strong specificity, and can only detect the SEOV virus, but cannot detect the Xinjiang hemorrhagic fever virus, Marburg virus, and Ebola virus that are related to the SEOV virus; 2. The reagent combination and kit for detecting the SEOV subtype of Hantavirus based on the RPA-CRISPR / Cas12a system provided by the present invention are highly efficient during detection. The kit is easy to operate, does not require professional operators, and has a fast test speed. The results can be interpreted by observing the fluorescence intensity under ultraviolet light or the test strip band with the naked eye, which can speed up the detection process and save time and human resources. 3. The reagent combination and kit for detecting the SEOV subtype of Hantavirus based on the RPA-CRISPR / Cas12a system provided by the present invention are economical when used for non-diagnostic applications of detecting the SEOV subtype of the virus. Compared with traditional detection methods, the kit of the present invention does not require the use of large-scale instruments and equipment, has a lower cost, and is conducive to mass production and popularization and application; 4. The reagent combination and kit for detecting the SEOV subtype of Hantavirus based on the RPA-CRISPR / Cas12a system provided by the present invention are easy to use, and the kit is compact in design, easy to carry and store, and is suitable for use in hospitals, clinics and outdoor large-scale screening; 5. When the reagent combination and kit for detecting the SEOV subtype of Hantavirus based on the RPA-CRISPR / Cas12a system provided by the present invention are used, the synthesis method is more environmentally friendly, the raw materials used are non-toxic, and the risk of environmental pollution is reduced. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 a is a schematic diagram of the gel electrophoresis bands for RPA primer verification according to the present invention, Figure 1 b is a schematic diagram of the gel electrophoresis bands when the temperature of the RPA reaction system of the present invention is optimized, Figure 1 c is a fluorescence value analysis diagram of the RPA reaction system when the temperature is optimized according to the present invention, Figure 1 d is a schematic diagram of the fluorescence intensity of each reaction temperature under ultraviolet light when the temperature of the RPA reaction system of the present invention is optimized. Figure 1 e is a schematic diagram of the gel electrophoresis bands of the RPA amplification time optimization of the present invention, Figure 1 f is a fluorescence value analysis chart collected by the qPCR instrument of the present invention, Figure 1 g is a schematic diagram of the fluorescence intensity of each reaction tube of the present invention observed by naked eyes under ultraviolet light; Figure 2 a is a fluorescence value analysis diagram at each reaction temperature when the temperature of the CRISPR / Cas12a system of the present invention is optimized, Figure 2 b is a schematic diagram of fluorescence intensity observed by naked eyes under ultraviolet light at various reaction temperatures when the temperature of the CRISPR / Cas12a system of the present invention is optimized, Figure 2 c is a fluorescence value analysis diagram of the qPCR instrument when the LbCas12a enzyme concentration is optimized according to the present invention, Figure 2d is a schematic diagram of the fluorescence intensity of the reaction tube under ultraviolet light with optimized LbCas12a enzyme concentration according to the present invention, Figure 2 e is a fluorescence value analysis diagram of the crRNA concentration optimization of the present invention, Figure 2 f is the fluorescence intensity diagram under ultraviolet for the crRNA concentration optimization of the present invention, Figure 2 g is a fluorescence value analysis diagram of the optimized dosage of the reporter probe described in the present invention, Figure 2 h is a fluorescence intensity diagram under ultraviolet light with optimized dosage of the reporter probe of the present invention; Figure 3 a is a fluorescence value analysis diagram of each copy number reaction tube in the sensitivity detection of the present invention, Figure 3 b is a schematic diagram of the fluorescence intensity of each copy number reaction tube under ultraviolet light in the sensitivity detection of the present invention, Figure 3 c is a schematic diagram of the sensitivity of each copy number reaction tube combined with the flow chromatography test strip according to the present invention, Figure 3 d is the amplification curve diagram of each copy number during the qPCR reaction sensitivity detection described in the present invention, Figure 3 e is a standard curve diagram of each copy number during the qPCR reaction sensitivity detection of the present invention, Figure 3 f is the amplification curve diagram of the specific verification of the qPCR method described in the present invention, Figure 3 g is a fluorescence value analysis diagram of each plasmid template during the specific detection described in the present invention, Figure 3 h is a schematic diagram of the fluorescence intensity of each plasmid and the negative control tube under ultraviolet light of the present invention, Figure 3 i is a schematic diagram of the interpretation results of the specific test strips of each plasmid and the negative control tube under ultraviolet light described in the present invention ( Figure 3 a~ Figure 3 The corresponding copy numbers of the SEOV plasmids in c are 1 × 10 9 copies / μL, 1 × 10 8 copies / μL, 1 × 10 7 copies / μL, 1 × 10 6 copies / μL, 1× 10 5 copies / μL, 1 × 10 4 copies / μL, 1 × 10 3 copies / μL, 1 × 10 2 copies / μL, 1 × 10 1 copies / μL, 1copy / μL; Figure 3 d~ Figure 3 The corresponding copy numbers in e are: 1 × 10 9copies / μL, 1 × 10 8 copies / μL, 1 × 10 7 copies / μL, 1 × 10 6 copies / μL, 1 × 10 5 copies / μL, 1 × 10 4 copies / μL, 1 × 10 3 copies / μL, 1 × 10 2 copies / μL, 1× 10 1 copies / μL, 1copy / μL). DETAILED DESCRIPTION

[0023] The present invention is further described in detail below in conjunction with the accompanying drawings so that those skilled in the art can implement the invention with reference to the description.

[0024] The present invention provides a DNA fragment for detecting the SEOV subtype of Hantavirus based on the RPA-CRISPR / Cas12a system, wherein the DNA fragment is a DNA fragment composed of a base sequence shown in SEQ ID NO.1.

[0025] The present invention also provides a recombinant vector for detecting the SEOV subtype of Hantavirus based on the RPA-CRISPR / Cas12a system, the recombinant vector comprises a DNA fragment consisting of a base sequence as shown in SEQ ID NO.1, and the plasmid used by the recombinant vector is PUC57.

[0026] The present invention also provides a reagent combination for detecting Hantavirus SEOV subtypes based on the RPA-CRISPR / Cas12a system, which includes a primer set, crRNA and ssDNA probe for RPA amplification to obtain a DNA fragment containing a base sequence shown in SEQ ID NO.1; wherein the primer set includes an upstream primer sequence shown in SEQ ID NO.2 and a downstream primer sequence shown in SEQ ID NO.3, crRNA is an RNA fragment composed of a base sequence shown in SEQ ID NO.4, the 5' end of the ssDNA probe is labeled with FAM and the 3' end is labeled with BHQ1, or the 5' end of the ssDNA probe is labeled with FAM and the 3' end is labeled with Biotin.

[0027] In another embodiment, the nucleotide sequence of the ssDNA probe is 5'-FAM-TTATT-BHQ1-3' or 5'-FAM-TTTTTTTATTTTTTT-Biotin-3'.

[0028] The present invention also provides a kit for detecting Hantavirus SEOV subtypes based on the RPA-CRISPR / Cas12a system, comprising a reagent combination for detecting Hantavirus SEOV subtypes based on the RPA-CRISPR / Cas12a system, an RPA amplification reagent, a CRISPR / Cas12a recognition and cleavage reagent, and a probe.

[0029] The present invention also provides a kit for detecting Hantavirus SEOV subtypes based on the RPA-CRISPR / Cas12a system for non-diagnostic purposes of detecting virus SEOV subtypes, which specifically includes the following steps: Step 1: extracting DNA from the sample to be tested as template DNA; Step 2: Add the template DNA obtained in step 1 to the RPA reaction system and mix thoroughly, and perform RPA amplification reaction using primer pairs; Among them, the RPA reaction system and RPA reaction conditions of the RPA amplification reaction are as follows: RPA reaction system: add 29.5μL Primer Free Rehydration buffer, 2.4μ of the upstream primer and downstream primer in the primer pair, 12.2μ of ddH2O, 1μ of 100ng / μL template DNA, and finally add 2.5μ of MgOAc activator on the lid. RPA reaction conditions: The RPA reaction system was fully mixed and amplified at 37°C to 41°C for 10 to 35 minutes; Step 3: Mix crRNA, ssDNA probe and Cas12a enzyme with the RPA amplification product obtained in step 2, and perform Cas12a fluorescence detection reaction; Among them, the Cas12a fluorescence detection reaction system is as follows: 3 μ of 10×Culease Buffer, 10 nM~50 nM LbCas12a Nuclease, 300 nM~700 nM ssDNA-FQ, 10 nM~70 nM crRNA, and 16 μ of DEPCH2O were added to a sterile PCR tube in sequence. 2 μ of RPA amplification product was added after mixing. After mixing and centrifugation, it was placed in a fluorescent quantitative PCR instrument and incubated at 39 ° C. The fluorescence signal was collected every 30 s, and a total of 90 cycles were set. The reaction results were interpreted by visually observing the fluorescence intensity of the reaction solution under ultraviolet light and statistically analyzing the fluorescence value in the fluorescent quantitative PCR instrument. In this embodiment, the initial concentration of LbCas12a Nuclease was 1 μM, and the Cas12a fluorescence detection reaction system was constructed after dilution to a final concentration of 10 nM~50 nM; the initial concentration of ssDNA-FQ was 2 μM, and it was diluted to a final concentration of 300 nM~700 nM to construct the Cas12a fluorescence detection reaction system; the initial concentration of crRNA was 100 μM, and it was diluted to a final concentration of 10 nM~70 nM to construct the Cas12a fluorescence detection reaction system.

[0030] In another embodiment, in step three, the Cas12a fluorescence detection reaction system is as follows: 3 μ of 10 × Culease Buffer, 10 nM ~ 50 nM LbCas12a Nuclease, 2 μM ssDNA-FB 7.5 μ, 10 nM ~ 70 nM crRNA, DEPC H2O16 μ were added to a sterile PCR tube in sequence, 2 μ of RPA amplification product was added after mixing, centrifuged and mixed in a 35 ° C ~ 41 ° C warm bath for 20 min, high pressure ultrapure water was added to the reaction tube to make up to 50 μl, and the Cas12 / 13 special nucleic acid detection test strip was inserted into the reaction tube and allowed to stand for 1-2 min to observe the results; in this embodiment, the initial concentration of LbCas12a Nuclease was 1 μM, and the Cas12a fluorescence detection reaction system was constructed after dilution to a final concentration of 10 nM ~ 50 nM; the initial concentration of crRNA was 100 μM, and the Cas12a fluorescence detection reaction system was constructed after dilution to a final concentration of 10 nM ~ 70 nM.

[0031] The "recombinant vector" in the present invention refers to an expression vector to which a gene has been connected, and the terms "recombinant plasmid" and "recombinant vector" can be used interchangeably. Unless otherwise specified, the reagents, enzymes, etc. in the embodiments of the present invention can be purchased through commercial channels. Example

[0032] 1. Instruments and reagents RT-RPA DNA Amplification Kit , Plasmid extraction kit (Shanghai Biotechnology Co., Ltd.), DNA extraction kit (Shanghai Biotechnology Co., Ltd.), DNA Marker (Shanghai Biotechnology Co., Ltd.), agarose (Shanghai Biotechnology Co., Ltd.), Green-2-Go 2X qPCR ROX premix (Shanghai Biotechnology Co., Ltd.), LbCas12a Nuclease (Guangzhou Aidi Gene Co., Ltd.), LbCas12a Cleavage Buffer (Guangzhou Aidi Gene Co., Ltd.), ssDNA-FQ type and ssDNA-FB type (Guangzhou Aidi Gene Co., Ltd.), Cas12 / 13 special nucleic acid detection test strips (Guangzhou Aidi Gene Co., Ltd.), ultra-micro spectrophotometer NanoDrop2000, 7500 fluorescence quantitative PCR instrument, MyCycler PCR instrument, WD-9403F UV instrument, BIO-RAD gel imaging analyzer.

[0033] 2. Sample The present invention uses a total of four recombinant plasmids, including SEOV, Ebola virus (EBOV), Marburg virus (MARV), and Xinjiang hemorrhagic fever virus (XHFV); the target sequence of SEOV is the full sequence of the S segment, the vector is PUC57, it has a potassium iodide resistance gene, and the host bacteria is Escherichia coli; the target sequences of EBOV and MARV are NP protein genes, the target sequence of SEOV is the full sequence of the S segment, the plasmid vector is PUC57, it has an ampicillin resistance gene, and the host bacteria is Escherichia coli; the four recombinant plasmids are all preserved in this laboratory; EBOV, MARV, and XHFV are used for specific verification of SEOV.

[0034] 3. Design and synthesis of primers and crRNA The SEOV (GenBank: KU204960.2) S fragment mRNA was searched on the NCBI website for sequence comparison with the mRNAs of multiple other strains, and the conservative fragments were selected to design specific RPA primers, which were synthesized by Shanghai Biotechnology Co., Ltd. and the primer specificity was verified. The specific crRNA (sequence number: SEQ ID NO.4), fluorescent probe and test strip probe that guide the Cas12a enzyme were designed based on the DNA fragment amplified by RPA (sequence number: SEQ ID NO.1). The crRNA, fluorescent reporter probe and test strip probe were all synthesized by Guangzhou Aidi Gene Co., Ltd., and the sequences are shown in Table 1.

[0035] Table 1 Sequence Listing 4. Primer synthesis method Solid phase phosphoramidite triester method: The phosphoramidite triester method is used to synthesize DNA fragments, which has the characteristics of high efficiency, rapid coupling and relatively stable starting reactants. The phosphoramidite triester method is to fix DNA on a solid phase carrier to complete the synthesis of the DNA chain. The direction of synthesis is from the 3' end of the primer to be synthesized to the 5' end, and adjacent nucleotides are connected by 3'→5' phosphodiester bonds; Step 1: reacting the active group-protected nucleotide pre-connected to the solid phase carrier CPG with trichloroacetic acid to remove the DNA protecting group (DMT) of the 5'-hydroxyl group to obtain a free 5'-hydroxyl group; Step 2, raw materials for synthesizing DNA: Phosphoramidite-protected nucleotide monomers are mixed with the activating agent tetrazole to obtain a nucleoside phosphorous acid activated intermediate, whose 3' end is activated and the 5'-hydroxyl group is still protected by DMT, and condensation reaction occurs with the free 5'-hydroxyl group in the solution: Step 3: Capping reaction. There may be very few 5'-hydroxyl groups that do not participate in the condensation reaction (less than 2%). The reaction continues after termination with acetic anhydride and 1-methylimidazole. This short fragment can be separated during purification. Step 4: Under the action of the oxidizing agent iodine, the phosphite form is converted into a more stable phosphate triester.

[0036] After the above four steps, a deoxynucleotide is connected to the nucleotide of the solid phase carrier. Then use trichloroacetic acid to remove the protective group DMT on its 5'-hydroxyl group, and repeat the above steps until all the bases required to be synthesized are connected. During the synthesis process, the color of the TCA treatment stage can be observed to determine the synthesis efficiency. The primer connected to CPG is cut off by high-temperature treatment with ammonia water, and the primer is purified by PAGE and other means. The finished primer is concentrated, desalted, and precipitated with C18. The precipitated primer is suspended in water, OD260 is measured for quantification, and packaged.

[0037] 5. Template preparation Use a DNA extraction kit to extract the novel Bunyavirus and hemorrhagic fever with renal syndrome virus nucleic acid, and use a plasmid extraction kit to extract SEOV, XHFV, EOBV and MARV plasmids, strictly following the instructions of the kit.

[0038] 6. Construction of reaction system Step 1: Construction of RPA reaction system Prepare the reaction premix in a clean EP tube: Primer Free Rehydration buffer 29.5 μL, Forward Primer (upstream primer as shown in SEQ ID NO.2) 2.4 μ, Reverse Primer (downstream primer as shown in SEQ ID NO.3) 2.4 μ, ddH2O 12.2 μ, mix well and add to the EP tube containing freeze-dried enzyme, add 1 μ template DNA (100 ng / μL), and finally add 2.5 μ MgOAc activator on the lid, shake and mix well, centrifuge, and heat in a 37 ℃ ~ 41 ℃ water bath for 10 ~ 35 min. The amplified product (DNA fragment as shown in SEQ ID NO.1) is purified and the experimental results are observed by gel electrophoresis; Step 2: Construction of RPA-CRISPR / Cas12a system Establishment of RPA-CRISPR / Cas12a fluorescence system: 3μ of 10×Culease Buffer, 10 nM~50 nM LbCas12a Nuclease, 300 nM~700 nM fluorescent reporter probe (ssDNA-FQ), 10 nM~70 nM crRNA (RNA fragment as shown in SEQ ID NO.4), and 16μ of DEPCH2O were added to two sterile PCR tubes in sequence. After the reaction system was shaken and mixed, 2μ of RPA product was added to one of the PCR tubes, and 2μ of DEPC H2O was added to the other tube as a negative control. After mixing and centrifugation, the mixture was placed in a fluorescence quantitative PCR instrument and incubated at 39°C. The fluorescence signal was collected every 30s, and a total of 90 cycles were set. The reaction results can be interpreted by observing the fluorescence intensity of the reaction solution under ultraviolet light with the naked eye and statistically analyzing the fluorescence value in the fluorescence quantitative PCR instrument; in the process of constructing the RPA-CRISPR / Cas12a fluorescence system, the initial concentration of LbCas12a Nuclease used is 1 μM, which is diluted to a final concentration of 10 nM~50 nM for construction and use, the initial concentration of ssDNA-FQ used is 2 μM, which is diluted to a final concentration of 300 nM~700 nM for construction and use, and the initial concentration of crRNA used is 100 μM, which is diluted to a final concentration of 10 nM~70 nM for construction and use.

[0039] Establishment of RPA-CRISPR / Cas12a-LFD system: Add 3μ of 10×Culease Buffer, 10 nM~50 nM LbCas12a Nuclease, 6μ of 2μM fluorescent reporter probe (ssDNA-FB type), 10 nM~70 nM crRNA (RNA fragment as shown in SEQ ID NO.4), 16μ of DEPC H2O, mix well, add 2μ of RPA amplification product, centrifuge and mix well, place in a 35℃~41℃ warm bath for 20min, add high pressure ultrapure water to the reaction tube to make up to 50μl. Insert the Cas12 / 13 special nucleic acid detection test strip into the reaction tube and let it stand for 1-2min before observing the results. Interpretation criteria: the appearance of red bands on the test line (T line) indicates a positive result; the appearance of red bands on the quality control line (C line) and the absence of color on the test line (T line) indicate a negative result; in the process of constructing the RPA-CRISPR / Cas12a-LFD system, the initial concentration of LbCas12a Nuclease used was 1 μM, which was diluted to a final concentration of 10 nM~50 nM for construction and use; the initial concentration of crRNA used was 100 μM, which was diluted to a final concentration of 10 nM~70 nM for construction and use.

[0040] 7. Optimization of reaction conditions (1) RPA reaction temperature optimization The temperature of the reaction system will affect the amplification efficiency of the target fragment. In the temperature range of 37 ℃ to 41 ℃, set an experimental tube every 2 ℃ to optimize the RPA reaction system. The temperature selection is based on the amplification efficiency, and the results are judged by observing the gel electrophoresis bands and CRISRP-Cas12a fluorescence brightness after the reaction.

[0041] (2) RPA response time optimization RPA amplification time will affect the amplification efficiency of the reaction to a certain extent. Within a certain range, the gel electrophoresis bands and the fluorescence values ​​of the CRISPR-Cas12a system will increase with the extension of the RPA amplification time. At the selected optimal temperature, 10, 15, 20, 25, 30 and 35 min were set for RPA amplification of the SEOV plasmid, and the amplified product was used as a template and ddH2O as a negative control for CRISPR / Cas12a reaction. The optimal amplification time was screened by observing the gel electrophoresis bands of the RPA product, the fluorescence value of the qPCR instrument, and the fluorescence intensity observed by naked eyes.

[0042] (3) CRISPR / Cas12a reaction temperature optimization The incubation temperature of the CRISPR / Cas12a system affects the activity of crRNA and LbCas12a enzymes, and thus affects the efficiency of probe cutting. To facilitate the visual observation of the results, this study optimized the temperature of the CRISPR / Cas system. Four reaction tubes at 35°C, 37°C, 39°C, and 41°C and a negative control tube were set up. The RPA-CRISPR / Cas12a fluorescence system was constructed according to the construction method of the RPA-CRISPR / Cas12a system, and the optimal reaction temperature was screened by observing the fluorescence intensity of each reaction tube under ultraviolet light and the fluorescence value in the PCR instrument.

[0043] (4) Optimization of LbCas12a enzyme concentration The RPA-CRISPR / Cas12a fluorescence detection method constructed by the construction method of the RPA-CRISPR / Cas12a system was used to optimize the LbCas12a enzyme concentration in the reaction system, and the enzyme concentrations in the reaction system were set to 10nM, 30nM, and 50nM, respectively, and a negative control tube was set for comparative analysis. The cutting reaction was carried out at the optimal reaction temperature selected under the CRISPR / Cas12a reaction temperature optimization conditions. After the reaction was completed, the fluorescence intensity of the reaction solution was observed under ultraviolet light and the fluorescence values ​​at each concentration in the PCR instrument were compared and analyzed to select the optimal enzyme concentration.

[0044] (5) Optimization of crRNA concentration The crRNA was set to four concentration gradients: 70nM, 50nM, 30nM, and 10nM. The RPA-CRISPR / Cas12a fluorescence system was constructed according to the construction method of the RPA-CRISPR / Cas12a system using the optimized enzyme concentration, and the cleavage reaction was performed at the optimal temperature. The results were determined by the fluorescence value and fluorescence intensity of the qPCR instrument.

[0045] (6) Optimization of ssDNA Reporter Concentration After the above operations, the optimal reaction temperature, optimal Cas12a enzyme and crRNA concentrations were obtained, and the amount of reporter probe in the reaction system was optimized. ssDNA-FQ was set to the following five concentration gradients: 300nM, 400nM, 500nM, 600nM, 700nM. The reaction tube was placed in a qPCR instrument, and the fluorescence signal was collected every 30s at 39°C, and 90 cycles were set to count the fluorescence values ​​at each concentration.

[0046] 8. Sensitivity verification The SEOV plasmid was diluted to 1 mg / mL and then diluted to 10 9 ~10 0SEOV primers were added to the reaction tubes for RPA amplification. The amplified products of the reaction tubes at each concentration were used as templates to construct the RPA-CRISPR / Cas12a fluorescence system and the test strip system according to the construction method of the RPA-CRISPR / Cas12a system. The fluorescence intensity, fluorescence value and positive nucleic acid test strip of each copy number reaction tube were observed and compared with the qPCR results to evaluate the sensitivity of the method. The qPCR reaction was determined by drawing the RT-PCR standard curve to determine the detection limit, and finally the sensitivity difference between the two methods was determined.

[0047] 9. Specificity Verification Using the extracted SEOV, XHFV, MARV and EOBV plasmids as templates, SEOV primers were added for recombinase polymerase amplification, and the amplified products were used as templates to construct the CRISPR / Cas12a fluorescence system and CRISPR / Cas12a-LFD system, respectively. The results can be interpreted by observing the fluorescence brightness with the naked eye, detecting the fluorescence value with a qPCR instrument, and the negative and positive status of the test strips. The specificity of this method can be evaluated by comparing it with the qPCR method.

[0048] Test Case 1. Verification of RPA primers and determination of the optimal temperature of the reaction system like Figure 1 As shown in a, the designed and synthesized RPA primers were added to the constructed RPA reaction system. After the product was purified, gel electrophoresis showed that the primers successfully amplified the target fragment. After determining the reaction primers, the reaction temperature of the RPA system was optimized. The SEOV plasmid was RPA amplified at 37°C, 39°C, and 41°C for 20 minutes. The amplified products at different temperatures were used to construct the RPA-CRISPR-Cas12a fluorescence system. The optimal reaction temperature was determined by observing the gel electrophoresis bands, the fluorescence intensity after the probe was cut by naked eye, and the fluorescence value of the qPCR reaction. Figure 1 b~ Figure 1 As shown in Figure d, at 39°C, the electrophoresis bands are more obvious and the fluorescence intensity observed by naked eyes and the fluorescence value in the instrument are the largest. Therefore, 39°C is selected as the optimal reaction temperature.

[0049] 2. Determination of the optimal amplification time of the RPA system Set up an experimental tube every 5 minutes between 10 and 35 minutes, and perform constant temperature amplification of the RPA system at 39°C according to the constructed RPA reaction system. Figure 1 e~ Figure 1 As shown in Figure g, when the RPA reaction time is 30 min, the gel electrophoresis band is the clearest and the fluorescence value detected by the fluorescence quantitative PCR instrument reaches the maximum, so the constant temperature amplification can achieve the maximum amplification efficiency at 30 min.

[0050] 3. Determination of the optimal reaction temperature of the CRISPR / Cas12a system In the present invention, a reaction tube is set at 35°C to 41°C every 2°C to perform the fluorescent probe cutting reaction. Figure 2 a. Figure 2 As shown in b, when incubated at 39°C, the fluorescence brightness under UV and the fluorescence value in the instrument both reached the maximum, so 39°C was selected as the optimal temperature for the CRISPR / Cas12a cutting system.

[0051] 4. Selection of LbCas12a enzyme dosage like Figure 2 c. Figure 2 As shown in Figure d, the dosage of Cas12a enzyme in the reaction system was optimized. When the enzyme concentration was 30 nM, the fluorescence intensity under ultraviolet light was significantly higher than that of the other two concentrations, and the PCR instrument showed that the light intensity value reached the maximum, so 30 nM was selected as the optimal concentration of LbCas12a enzyme.

[0052] 5. Selection of crRNA dosage Set a concentration gradient of 20 nM every 10 nM between 10 nM and 70 nM, and place three reaction tubes in total. Replace the template with DEPC H2O as a negative control group. Observe the fluorescence intensity and fluorescence value of the reaction tubes with different concentrations to determine the most suitable amount. Figure 2 e. Figure 2 As shown in FIG. 5 , when the crRNA concentration is 50 nM, the fluorescence brightness and fluorescence value observed by naked eyes are higher than those at other concentrations.

[0053] 6. ssDNA-FQ concentration optimization Based on the determined optimal Cas12a enzyme and crRNA concentrations, the fluorescent reporter probe concentration was optimized, and a reaction tube was set up every 100 nM between 300-700 nM to screen out the most suitable amount through comparative analysis. Figure 2 g. Figure 2 As shown in Figure 3, the fluorescence intensity gradually increased with the increase of probe concentration between 300-600 nM. When the concentration reached 700 nM, the fluorescence value measured by the naked eye and the instrument decreased slightly, so 600 nM was selected as the optimal probe concentration.

[0054] 7. Sensitivity test Using SEOV genomes with different copy numbers as templates, RPA-CRISPR / Cas12a fluorescence systems were constructed for detection. The lowest detection line of SEOV virus in the RPA-CRISPR / Cas12a fluorescence system was determined by observing the fluorescence intensity under ultraviolet light with the naked eye and detecting the fluorescence value with a qPCR instrument. At the same time, the reaction system was combined with a flow chromatographic test strip to construct the RPA-CRISPR / Cas12a-LFD system, and its sensitivity was explored based on its portability and easy interpretation of the results. The qPCR reaction determined the detection limit by drawing the RT-PCR standard curve, and the two results were compared. Figure 3 As shown in a~3f, the detection limits of the SEOV plasmid RPA-CRISPR / Cas12a fluorescence detection method and qPCR method were both 1 copies / μL; the minimum detection limit of the RPA-CRISPR / Cas12a-LFD method was 10 copies / μL.

[0055] 8. Specificity evaluation EBOV, MARV, and XHFV plasmids were extracted, and SEOV RPA primers were added to perform recombinase polymerase amplification. The amplified products were used as templates for subsequent CRISPR / Cas12a cleavage reactions. The reaction results were observed by combined flow chromatographic test strips. SEOV standard plasmids were set as positive controls and DEPC H2O as negative controls. The results were compared with qPCR reactions to evaluate the specificity of the method. Figure 3 As shown in g~3i, the qPCR, fluorescence value and RPA-CRISPR / Cas12a-LFS results of EBOV, MARV and XHFV were all negative, and the specificity of both methods was 100%. This result shows that the fluorescence value interpretation method and the test strip interpretation method of RPA-CRISPR / Cas12a have good specificity.

[0056] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation modes, and they can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and the illustrations shown and described herein.

Claims

1. A method for detecting a DNA fragment of a Hantavirus SEOV subtype based on an RPA-CRISPR / Cas12a system, characterized in that: The DNA fragment is a DNA fragment containing the base sequence shown in SEQ ID NO.

1.

2. A recombinant vector for detecting Hantavirus SEOV subtype based on RPA-CRISPR / Cas12a system, characterized in that: The recombinant vector comprises a DNA fragment consisting of the base sequence shown in SEQ ID NO.

1.

3. The recombinant vector for detecting Hantavirus SEOV subtype based on the RPA-CRISPR / Cas12a system as claimed in claim 2, characterized in that: The plasmid used in the recombinant vector is PUC57.

4. A reagent combination for detecting Hantavirus SEOV subtype based on RPA-CRISPR / Cas12a system, characterized in that: The reagent combination includes a primer set and crRNA for RPA amplification to obtain a DNA fragment containing a base sequence shown in SEQ ID NO.1; Wherein, the primer set includes an upstream primer sequence as shown in SEQ ID NO.2 and a downstream primer sequence as shown in SEQ ID NO.3; and the crRNA is an RNA fragment composed of a base sequence as shown in SEQ ID NO.

4.

5. The reagent combination for detecting Hantavirus SEOV subtype based on the RPA-CRISPR / Cas12a system as claimed in claim 4, characterized in that: Also included are ssDNA probes; The 5' end of the ssDNA probe is labeled with FAM and the 3' end is labeled with BHQ1; or the 5' end of the ssDNA probe is labeled with FAM and the 3' end is labeled with Biotin.

6. The reagent combination for detecting Hantavirus SEOV subtype based on the RPA-CRISPR / Cas12a system as claimed in claim 4, characterized in that: Also included are ssDNA probes; Wherein, the nucleotide sequence of the ssDNA probe is 5'-FAM-TTATT-BHQ1-3'; or the nucleotide sequence of the ssDNA probe is 5'-FAM-TTTTTTTATTTTTTT-Biotin-3'.

7. A kit for detecting Hantavirus SEOV subtype based on RPA-CRISPR / Cas12a system, characterized in that: It includes a reagent combination for detecting Hantavirus SEOV subtype based on the RPA-CRISPR / Cas12a system as described in claims 4-6.

8. The kit for detecting Hantavirus SEOV subtype based on the RPA-CRISPR / Cas12a system as claimed in claim 7, characterized in that Also includes: RPA amplification reagents, CRISPR / Cas12a recognition and cleavage reagents and probes.

9. A kit for detecting Hantavirus SEOV subtype based on RPA-CRISPR / Cas12a system for non-diagnostic purposes of detecting virus SEOV subtype, characterized in that: A kit for detecting Hantavirus SEOV subtype based on the RPA-CRISPR / Cas12a system as described in claim 7 or 8 is used.

10. The use of the kit for detecting Hantavirus SEOV subtype based on the RPA-CRISPR / Cas12a system as claimed in claim 9 for non-diagnostic purposes of detecting virus SEOV subtypes, characterized in that: The steps include: Step 1: extracting DNA from the sample to be tested as template DNA; Step 2: Add the template DNA obtained in step 1 to the RPA reaction system and mix thoroughly, and perform RPA amplification reaction using primer pairs; Among them, the RPA reaction system and RPA reaction conditions of the RPA amplification reaction are as follows: RPA reaction system: add 29.5μL Primer Free Rehydration buffer, 2.4μ of the upstream primer and downstream primer in the primer pair, 12.2μ of ddH2O, 1μ of 100ng / μL template DNA, and finally add 2.5μ of MgOAc activator on the lid. RPA reaction conditions: The RPA reaction system was fully mixed and amplified at 37°C to 41°C for 10 to 35 min; Step 3, crRNA, ssDNA probe and Cas12a enzyme are mixed with the RPA amplification product obtained in the step 2, and Cas12a fluorescence detection reaction is performed; Among them, the Cas12a fluorescence detection reaction system is as follows: 3μ of 10×Culease Buffer, 10 nM~50 nM LbCas12aNuclease, 300nM~700nM ssDNA-FQ, 10 nM~70 nM crRNA, and 16μ of DEPC H2O were added to a sterile PCR tube in sequence, and 2μ of RPA amplification product was added after mixing. After centrifugation, the mixture was incubated at 39°C in a fluorescence quantitative PCR instrument, and the fluorescence signal was collected every 30s. A total of 90 cycles were set, and the reaction results were interpreted by visually observing the fluorescence intensity of the reaction solution under ultraviolet light and statistically analyzing the fluorescence value in the fluorescence quantitative PCR instrument; or 3μ of 10×Culease Buffer, 10 nM~50 nM LbCas12a Nuclease, 2μM ssDNA-FB 6μ, 10 nM~70nM crRNA, and DEPC H2O were added to a sterile PCR tube in sequence. 16μ, add 2μ of RPA amplification product after mixing, centrifuge and mix, place in a 35℃~41℃ warm bath for 20min, add high pressure ultrapure water to the reaction tube to make up to 50μl, insert the Cas12 / 13 special nucleic acid detection test strip into the reaction tube, let it stand for 1-2min, and then observe the results; Among them, the initial concentration of the LbCas12a Nuclease is 1 μM, and the Cas12a fluorescence detection reaction system is constructed after dilution to a final concentration of 10 nM~50 nM; the initial concentration of the ssDNA-FQ is 2 μM, and the Cas12a fluorescence detection reaction system is constructed after dilution to a final concentration of 300 nM~700 nM; the initial concentration of the crRNA is 100 μM, and the Cas12a fluorescence detection reaction system is constructed after dilution to a final concentration of 10 nM~70 nM.

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