DNA fragment for detecting hantavirus HTNV 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 HTNV subtypes based on an RPA-CRISPR / Cas12a system, and application of the kit to the non-diagnostic purpose of detecting the hantavirus HTNV subtypes. The detection accuracy of the Hantaan virus can reach 100%, a target substance can be accurately detected, and the probability of false positive or false negative is reduced; meanwhile, the specificity is high, only the Hantaan virus can be detected, and Xinjiang hemorrhagic fever virus, Marburg virus and Ebola virus which have genetic relationship with the Hantaan 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 Hantavirus HTNV subtypes 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, and my country is the main epidemic area of ​​HFRS, accounting for 90% of the total number of cases worldwide, ranking first in the world. Jilin Province is the main epidemic area of ​​HFRS.

[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), Seoul virus (SEOV), Puumala virus (PUUV) and Dobrava-Belgrade virus (DOBV). The clinical manifestations of diseases caused by different subtypes of viruses are also different. HFRS caused by HTNV and DOBV are usually more severe. Therefore, the 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 also helps to 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 two types of virus infections, HTNV and SEOV. Among them, HFRS caused by HTNV infection is the most serious, with a mortality rate of 9%-12%. Therefore, establishing an accurate, efficient and portable detection method for HTNV 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 HTNV mainly include hematological examinations and pathogen nucleic acid detection. Hematological examinations mainly detect 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 lead to early diagnosis and delayed treatment. Real-time fluorescence quantitative PCR is currently the gold standard for rapid pathogen detection of Hantavirus, but it requires expensive professional instruments, is complex to operate, takes a long time to detect, and has cumbersome result interpretation, so it cannot be widely used 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-stranded 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 a DNA enzyme cutting function and can cut specific single-stranded DNA probes, thereby realizing rapid nucleic acid detection. RPA amplified double-stranded DNA, under the guidance of crRNA, Cas12a can cut double-stranded template DNA and fluorescent labeled probes. The probe is hydrolyzed to release fluorescent signals, and the presence or absence of target nucleic acid fragments is determined by detecting the strength of the fluorescent signals. At the same time, it can also be combined with a flow chromatographic test strip (LFD) to observe the results more accurately and intuitively. However, to date, there have been no reports on the application of CRISPR systems in the detection and diagnosis of Hantavirus. This study will develop a simple, rapid and easy-to-operate HTNV detection method, increase the sensitivity of the reaction through RPA, and use the cleavage activity of Cas12a to exclude false positive results of the RPA reaction, and establish an RPA-CRISPR Cas12a-LFD detection system to achieve on-site detection of HTNV. 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 Hantavirus HTNV subtypes based on the RPA-CRISPR / Cas12a system.

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

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

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

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

[0011] The technical solution provided by the present invention is:

[0012] A DNA fragment for detecting Hantavirus HTNV subtype based on an RPA-CRISPR / Cas12a system, wherein the DNA fragment is a DNA fragment containing a base sequence shown in SEQ ID NO.1.

[0013] A recombinant vector for detecting Hantavirus HTNV subtypes 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.

[0014] Preferably, the plasmid used in the recombinant vector is PUC57.

[0015] A reagent combination for detecting Hantavirus HTNV subtypes based on an 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;

[0016] 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

[0017] The crRNA is an RNA fragment composed of the base sequence shown in SEQ ID NO.4.

[0018] Preferably, it further comprises a ssDNA probe;

[0019] Wherein, the 5' end of the ssDNA probe is labeled with FAM and the 3' end is labeled with BHQ1; or

[0020] The ssDNA probe is labeled with FAM at the 5' end and Biotin at the 3' end.

[0021] Preferably, it further comprises a ssDNA probe;

[0022] Wherein, the nucleotide sequence of the ssDNA probe is 5'-FAM-TTATT-BHQ1-3'; or

[0023] The nucleotide sequence of the ssDNA probe is

[0024] 5'-FAM-TTTTTTTTATTTTTTT-Biotin-3'.

[0025] A kit for detecting Hantavirus HTNV subtypes based on the RPA-CRISPR / Cas12a system, comprising the reagent combination for detecting Hantavirus HTNV subtypes based on the RPA-CRISPR / Cas12a system.

[0026] Preferably, it also includes: RPA amplification reagent, CRISPR / Cas12a recognition and cleavage reagent and probe.

[0027] A kit for detecting Hantavirus HTNV subtypes based on an RPA-CRISPR / Cas12a system is used for non-diagnostic purposes of detecting virus HTNV subtypes, and the kit for detecting Hantavirus HTNV subtypes based on an RPA-CRISPR / Cas12a system is used.

[0028] Preferably, the method comprises the following steps:

[0029] Step 1: extracting DNA from the sample to be tested as template DNA;

[0030] 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;

[0031] Among them, the RPA reaction system and RPA reaction conditions of the RPA amplification reaction are as follows:

[0032] 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.

[0033] RPA reaction conditions: The RPA reaction system was fully mixed and amplified at 37°C to 41°C for 10 to 35 minutes;

[0034] 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;

[0035] Among them, the Cas12a fluorescence detection reaction system is as follows:

[0036] In a sterile PCR tube, add 3μ of 10×Culease Buffer, 10nM~50nM LbCas12a Nuclease, 300nM~700nM ssDNA-FQ, 10nM~50nM crRNA, and 16μ of DEPCH2O in sequence, mix well, add 2μ of RPA amplification product, mix well, centrifuge, and incubate at 39°C in a fluorescence quantitative PCR instrument, collect fluorescence signals every 30s, and set a total of 90 cycles. The reaction results are 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

[0037] Add 3μ of 10×Culease Buffer, 10nM~50nM LbCas12a Nuclease, 2μM ssDNA-FB 7.5μ, 10nM~50nM crRNA, and 16μ of DEPC H2O to a sterile PCR tube, 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, let stand for 1-2min, and observe the results;

[0038] 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 10nM to 50nM; 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 300nM to 700nM; the initial concentration of the crRNA is 500nM, and the Cas12a fluorescence detection reaction system is constructed after dilution to a final concentration of 10nM to 50nM.

[0039] The beneficial effects of the present invention are:

[0040] 1. The reagent combination and kit for detecting Hantavirus HTNV subtypes based on the RPA-CRISPR / Cas12a system provided by the present invention have accurate detection results, and the detection accuracy of Hantavirus 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 has the characteristics of strong specificity, and can only detect Hantavirus, but cannot detect Xinjiang hemorrhagic fever virus, Marburg virus, and Ebola virus that are related to Hantavirus;

[0041] 2. The reagent combination and kit for detecting Hantavirus HTNV subtypes 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.

[0042] 3. The reagent combination and kit for detecting Hantavirus HTNV subtypes based on the RPA-CRISPR / Cas12a system provided by the present invention are economical when used for non-diagnostic applications of detecting virus HTNV subtypes. 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;

[0043] 4. The reagent combination and kit for detecting Hantavirus HTNV subtypes 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;

[0044] 5. When the reagent combination and kit for detecting Hantavirus HTNV subtypes 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

[0045] 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;

[0046] 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 2 d 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;

[0047] 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 fluorescence intensity of the negative control tube and the interpretation result of the specificity test strip according to the present invention ( Figure 3 a~ Figure 3 The corresponding copy numbers of HTNV plasmids in c are 1×10 9 copies / μL, 1×10 8 copies / μL, 1×10 7 copies / μL, 1×10 6copies / 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 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). DETAILED DESCRIPTION

[0048] 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.

[0049] The present invention provides a DNA fragment for detecting Hantavirus HTNV subtypes based on an RPA-CRISPR / Cas12a system, wherein the DNA fragment is a DNA fragment composed of a base sequence shown in SEQ ID NO.1.

[0050] The present invention also provides a recombinant vector for detecting Hantavirus HTNV subtypes based on the RPA-CRISPR / Cas12a system, the recombinant vector comprising 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.

[0051] The present invention also provides a reagent combination for detecting Hantavirus HTNV 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.

[0052] In another embodiment, the nucleotide sequence of the ssDNA probe is 5'-FAM-TTATT-BHQ1-3' or 5'-FAM-TTTTTTTATTTTTTT-Biotin-3'.

[0053] The present invention also provides a kit for detecting Hantavirus HTNV subtypes based on the RPA-CRISPR / Cas12a system, comprising a reagent combination for detecting Hantavirus HTNV subtypes based on the RPA-CRISPR / Cas12a system, an RPA amplification reagent, a CRISPR / Cas12a recognition and cleavage reagent, and a probe.

[0054] The present invention also provides a kit for detecting Hantavirus HTNV subtypes based on the RPA-CRISPR / Cas12a system for non-diagnostic purposes of detecting virus HTNV subtypes, which specifically includes the following steps:

[0055] Step 1: extracting DNA from the sample to be tested as template DNA;

[0056] 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;

[0057] Among them, the RPA reaction system and RPA reaction conditions of the RPA amplification reaction are as follows:

[0058] 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.

[0059] RPA reaction conditions: The RPA reaction system was fully mixed and amplified at 37°C to 41°C for 10 to 35 minutes;

[0060] Step 3: Mix crRNA, ssDNA probe and Cas12a enzyme with the RPA amplification product obtained in step 2, and perform Cas12a fluorescence detection reaction;

[0061] Among them, the Cas12a fluorescence detection reaction system is as follows:

[0062] 3 μ of 10×Culease Buffer, 10 nM to 50 nM LbCas12a Nuclease, 300 nM to 700 nM ssDNA-FQ, 10 nM to 50 nM crRNA, and 16 μ of DEPCH2O were added to a sterile PCR tube in sequence, and 2 μ of RPA amplification product was added after mixing. 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 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 this embodiment, LbCas12a The initial concentration of Nuclease was 1 μM, and the Cas12a fluorescence detection reaction system was constructed after dilution to a final concentration of 10 nM to 50 nM; the initial concentration of ssDNA-FQ was 2 μM, and the Cas12a fluorescence detection reaction system was constructed after dilution to a final concentration of 300 nM to 700 nM; the initial concentration of crRNA was 500 nM, and the Cas12a fluorescence detection reaction system was constructed after dilution to a final concentration of 10 nM to 50 nM.

[0063] In another embodiment, in step three, the Cas12a fluorescence detection reaction system is as follows:

[0064] 3 μ of 10 × Culease Buffer, 10 nM to 50 nM LbCas12a Nuclease, 2 μM ssDNA-FB 7.5 μ, 10 nM to 50 nM crRNA, DEPC H2O16 μ were added to a sterile PCR tube in sequence, and 2 μ of RPA amplification product was added after mixing, and the mixture was centrifuged and placed in a 35 ° C to 41 ° C warm bath for 20 min, and ultrapure water was added to the reaction tube after high pressure 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 to 50 nM; the initial concentration of crRNA was 500 nM, and the Cas12a fluorescence detection reaction system was constructed after dilution to a final concentration of 10 nM to 50 nM.

[0065] 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.

[0066] Example

[0067] 1. Instruments and reagents

[0068] RT-RPA DNA Amplification Kit ( Basic), 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.), LbCas12aCleavage 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 strip (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.

[0069] 2. Sample

[0070] The invention uses 4 kinds of recombinant plasmids, including HTNV, Ebola virus (EBOV), Marburg virus (MARV), and Xinjiang hemorrhagic fever virus (XHFV); the target sequence of HTNV is the whole sequence of 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 XHFV is the whole sequence of S segment, the plasmid vector is PUC57, it has an ampicillin resistance gene, and the host bacteria is Escherichia coli; the 4 kinds of recombinant plasmids are all stored in this laboratory; EBOV, MARV, and XHFV are used for the specific verification of HTNV.

[0071] 3. Design and synthesis of primers and crRNA

[0072] The HTNV (GenBank: KT885049.1) S fragment mRNA was queried 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 according to 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.

[0073] Table 1 Sequence Listing

[0074]

[0075] 4. Primer synthesis method

[0076] 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;

[0077] 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;

[0078] 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:

[0079] 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.

[0080] Step 4: Under the action of the oxidizing agent iodine, the phosphite form is converted into a more stable phosphate triester.

[0081] 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.

[0082] 5. Template preparation

[0083] 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 HTNV, XHFV, EOBV and MARV plasmids, strictly following the instructions of the kit.

[0084] 6. Construction of reaction system

[0085] Step 1: Construction of RPA reaction system

[0086] Prepare the reaction premix in a clean EP tube: Primer Free Rehydration buffer 29.5μL, Forward Primer (such as the upstream primer shown in SEQ ID NO.2) 2.4μ, Reverse Primer (such as the downstream primer shown in SEQ ID NO.3) 2.4μ, ddH2O 12.2μ, mix well and add to the EP tube containing freeze-dried enzyme, add 1μ template DNA (100ng / μL), and finally add 2.5μ MgOAc activator on the lid, shake and mix well, centrifuge, and heat in a 37℃~41℃ water bath for 10~35min. After purification, the amplified product (such as the DNA fragment shown in SEQ ID NO.1) is observed by gel electrophoresis;

[0087] Step 2: Construction of RPA-CRISPR / Cas12a system

[0088] Establishment of RPA-CRISPR / Cas12a fluorescence system: 3μ of 10×Culease Buffer, 10nM~50nM LbCas12aNuclease, 300nM~700nM fluorescent reporter probe (ssDNA-FQ), 10nM~50nM crRNA (RNA fragment as shown in SEQ ID NO.4), and 16μ DEPC H2O 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 DEPC H2O was added to the other tube as a negative control. After mixing and centrifugation, it was placed in a fluorescent quantitative PCR instrument and incubated at 39°C. The fluorescent 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; wherein, in the process of constructing the RPA-CRISPR / Cas12a fluorescence system, the initial concentration of LbCas12aNuclease used is 1 μM, which is diluted to a final concentration of 10 nM to 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 to 700 nM for construction and use, and the initial concentration of crRNA used is 500 nM, which is diluted to a final concentration of 10 nM to 50 nM for construction and use.

[0089] Establishment of RPA-CRISPR / Cas12a-LFD system: 3μ of 10×Culease Buffer, 10nM~50nM LbCas12aNuclease, 2μM fluorescent reporter probe (ssDNA-FB type) 7.5μ, 10nM~50nM crRNA (RNA fragment as shown in SEQ ID NO.4), DEPC H2O16μ were added in sequence in a sterile PCR tube, and 2μ of RPA amplification product was added after mixing, centrifuged and mixed, and placed in a 35℃~41℃ warm bath for 20min, and ultrapure water was added to the reaction tube after high pressure to make up to 50μl. Insert the Cas12 / 13 special nucleic acid detection test strip into the reaction tube and observe the results after standing for 1-2min. 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 LbCas12aNuclease used was 1 μM, which was diluted to a final concentration of 10 nM to 50 nM for construction and use, and the initial concentration of crRNA used was 500 nM, which was diluted to a final concentration of 10 nM to 50 nM for construction and use.

[0090] 7. Optimization of reaction conditions

[0091] (1) RPA reaction temperature optimization

[0092] The temperature of the reaction system will affect the amplification efficiency of the target fragment. In the temperature range of 37°C to 41°C, set an experimental tube every 2°C 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.

[0093] (2) RPA response time optimization

[0094] 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, 20 and 35 min were set for RPA amplification of the HTNV 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.

[0095] (3) CRISPR / Cas12a reaction temperature optimization

[0096] 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.

[0097] (4) Optimization of LbCas12a enzyme concentration

[0098] 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.

[0099] (5) Optimization of crRNA concentration

[0100] The crRNA was set to three concentration gradients: 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.

[0101] (6) Optimization of ssDNA Reporter Concentration

[0102] 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.

[0103] 8. Sensitivity verification

[0104] The HTNV plasmid was diluted to 1 mg / mL and then diluted to 109 ~10 0 , add HTNV primers to the reaction tube for RPA amplification, and use the amplified products of each concentration reaction tube as templates to construct the RPA-CRISPR / Cas12a fluorescence system and test strip system according to the construction method of the RPA-CRISPR / Cas12a system. Observe the fluorescence intensity, fluorescence value and nucleic acid test strip positivity of each copy number reaction tube, and compare with the qPCR results to evaluate the sensitivity of the method. The qPCR reaction determines the detection limit by drawing the RT-PCR standard curve, and finally judges the sensitivity difference between the two methods.

[0105] 9. Specificity Verification

[0106] Using the extracted HTNV, XHFV, MARV and EOBV plasmids as templates, HTNV primers were added for recombinase polymerase amplification, and the amplified products were used as templates to construct 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 the 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.

[0107] Test Case

[0108] 1. Verification of RPA primers and determination of the optimal temperature of the reaction system

[0109] 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 HTNV plasmid was amplified by RPA 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.

[0110] 2. Determination of the optimal amplification time of the RPA system

[0111] Set up an experimental tube every 5 minutes between 10-35 minutes, and perform constant temperature amplification of the RPA system at 39°C according to the constructed RPA reaction system. Figure 1 e~ Figure 1As shown in Figure g, when the RPA reaction time is 30 minutes, the gel electrophoresis band is the clearest and the fluorescence value detected by the fluorescence quantitative PCR instrument reaches the maximum. Therefore, the maximum amplification efficiency can be achieved at 30 minutes of constant temperature amplification.

[0112] 3. Determination of the optimal reaction temperature of the CRISPR / Cas12a system

[0113] The present invention sets a reaction tube every 2°C between 35°C and 41°C to perform 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.

[0114] 4. Selection of LbCas12a enzyme dosage

[0115] 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.

[0116] 5. Selection of crRNA dosage

[0117] Set a concentration gradient of 20 nM every 10-50 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 30 nM, the fluorescence brightness and fluorescence value observed by naked eyes are higher than those at other concentrations.

[0118] 6. ssDNA-FQ concentration optimization

[0119] 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 increases with the increase of probe concentration between 300-600 nM. When the concentration reaches 600 nM and 700 nM, there is no obvious difference between the fluorescence values ​​measured by the naked eye and the instrument. In order to save reagent costs, 600 nM was selected as the optimal probe concentration.

[0120] 7. Sensitivity test

[0121] Using HTNV genomes with different copy numbers as templates, the RPA-CRISPR / Cas12a fluorescence system was constructed for detection. The lowest detection line of HTNV 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 HTNV plasmid RPA-CRISPR / Cas12a fluorescence detection method and qPCR method are both 1 copies / μL; the minimum detection limit of RPA-CRISPR / Cas12a-LFD method is 10 copies / μL.

[0122] 8. Specificity evaluation

[0123] EBOV, MARV, and XHFV plasmids were extracted, and HTNV 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. HTNV 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 to 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.

[0124] 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 HTNV 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 HTNV subtypes based on the 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 HTNV subtypes 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 HTNV subtypes based on the 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 the base sequence shown in SEQ ID NO.

4.

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

6. The reagent combination for detecting Hantavirus HTNV subtypes 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 HTNV subtypes based on the RPA-CRISPR / Cas12a system, characterized in that: It includes a reagent combination for detecting Hantavirus HTNV subtypes based on the RPA-CRISPR / Cas12a system as described in claims 4-6.

8. The kit for detecting Hantavirus HTNV subtypes 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 HTNV subtypes based on the RPA-CRISPR / Cas12a system for non-diagnostic purposes of detecting virus HTNV subtypes, characterized in that: A kit for detecting Hantavirus HTNV subtypes 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 HTNV subtypes based on the RPA-CRISPR / Cas12a system as claimed in claim 9 for non-diagnostic purposes of detecting virus HTNV 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 minutes; 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: Add 3μ of 10×Culease Buffer, 10nM~50nM LbCas12aNuclease, 300nM~700nM ssDNA-FQ, 10nM~50nM crRNA, and 16μ of DEPC H2O to a sterile PCR tube, mix well, add 2μ of RPA amplification product, mix well, centrifuge, and incubate at 39°C in a fluorescence quantitative PCR instrument. Collect fluorescence signals every 30s, set a total of 90 cycles, and interpret the reaction results by visually observing the fluorescence intensity of the reaction solution under ultraviolet light and statistically analyzing the fluorescence values ​​in the fluorescence quantitative PCR instrument; or In a sterile PCR tube, add 3μ of 10×Culease Buffer, 10nM~50nM LbCas12aNuclease, 2μM ssDNA-FB 7.5μ, 10nM~50nM crRNA, and DEPC H2O 16μ in sequence, 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, let it stand for 1-2min, and 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 10nM to 50nM; 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 300nM to 700nM; the initial concentration of the crRNA is 500nM, and the Cas12a fluorescence detection reaction system is constructed after dilution to a final concentration of 10nM to 50nM.

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