Method for detecting PRRSV-1 based on CRISPR-Cas13d system

Abstract

The invention discloses a method for detecting PRRSV-1 based on a CRISPR-Cas13d system, and belongs to the technical field of molecular biology diagnosis. The method can effectively detect a clinical positive PRRSV-1 sample, can realize on-site visual detection of PRRSV-1 by combining RNA fluorescent reporter molecules, and can judge a detection result by only using a blue light transmission instrument to irradiate and observing whether a fluorescent signal is generated in a reaction system or not in the detection process. In addition, the method also supports quantitative detection by using a qPCR (quantitative polymerase chain reaction) instrument, and the result can be quickly judged within one hour. According to the method, the diagnosis cost is reduced, the diagnosis time is shortened, the PRRSV-1 detection capacity in the live pig breeding industry is improved through visual detection of the result, and the biological safety risk of a breeding farm is reduced.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biological diagnosis, and in particular relates to a method for detecting PRRSV-1 based on a CRISPR-Cas13d system. Background Art

[0002] Porcine reproductive and respiratory syndrome (PRRS) is a highly contagious disease caused by porcine reproductive and respiratory syndrome virus (PRRSV), which seriously affects the global pig farming industry. Porcine reproductive and respiratory syndrome virus (PRRSV) is an enveloped single-stranded positive-sense RNA virus belonging to the family Arteriviridae and the order Nidovirales. The PRRSV genome is approximately 15 kb in length and consists of a 5′ and a 3′ polyadenylated untranslated region (UTR) and at least 11 open reading frames (ORFs), including ORF1a, ORF1b, ORF2a, ORF2b, ORF5a, ORF3-7, and ORF2TF. The virus is mainly transmitted through the respiratory tract, and infecting pigs can cause severe respiratory diseases and reproductive problems, such as abortion, premature birth, stillbirth, and high mortality in piglets. PRRSV is divided into two types according to its genetic characteristics: PRRSV-1 (European type) and PRRSV-2 (North American type), which have only 60% nucleotide homology at the whole genome level. PRRSV-2 has attracted much attention due to its rapid transmission speed and high pathogenicity, and in recent years, the pathogenicity of PRRSV-1 has tended to increase. Therefore, monitoring of PRRSV-1 is necessary.

[0003] At present, in addition to diagnosis through intuitive clinical symptoms, the methods for PRRSV 1 detection mainly include virus isolation (VI), indirect immunofluorescence assay (IFA), immunoperoxidase monolayer assay (IPMA), serum sickness PCR (dPCR), recombinase polymerase amplification (RPA), loop-mediated isothermal amplification (LAMP), metagenomic next-generation sequencing (mNGS) and enzyme-linked immunosorbent assay (ELISA). However, these methods often require complex equipment and long detection time, and cannot meet the needs of rapid on-site diagnosis. CRIPSR / Cas13d, as a relatively new detection technology, has become a popular research field in molecular biology detection technology after its feasibility, high specificity, high sensitivity and reliability have been proven. If CRIPSR / Cas13d technology is used for the detection of PRRSV 1, it will strengthen our monitoring and control of the virus.

[0004] CRISPR / Cas13d (Clustered Regularly interspaced short palindromicrepeat) is an emerging gene editing and diagnostic tool, and is part of the CRISPR family. The CRISPR system was originally discovered as an adaptive immune system of bacteria and archaea, and can resist foreign genetic material through an RNA-guided DNA cutting mechanism. The CRISPR-Cas system consists of the CRISPR locus and the Cas gene, and the CRISPR locus includes a leader sequence, a repeat sequence, and a spacer sequence. In the system, the Cas gene encodes multiple CRISPR-related proteins, such as Cas9, Cas12a, and Cas13d, a total of about dozens of species. In recent years, CRISPR technology has developed rapidly and has been widely used in gene editing, biomedical research, and molecular diagnosis. Cas13d is a newly discovered effector protein in the CRISPR family. Unlike the more well-known Cas9, Cas13d targets RNA rather than DNA. It is characterized by efficient RNA recognition and cutting capabilities, and its relatively small molecular weight makes it easier to express in cells. In addition, Cas13d activates collateral activity after cutting the target RNA, causing the surrounding non-specific RNA to be cut as well. This feature is cleverly used in molecular diagnostic technology. In the CRISPR / Cas13d system, the guide RNA (gRNA) guides the Cas13d protein to specifically recognize and bind to the target RNA molecule. After binding, Cas13d activates its RNase activity and cuts the target RNA. At the same time, due to the collateral activity, it can cut the surrounding reporter RNA (usually a fluorescently labeled RNA fragment), thereby generating an easily detectable fluorescent signal. This CRISPR / Cas13d-based detection method has high specificity and sensitivity, and can achieve rapid detection without the need for complex equipment. Compared with traditional molecular diagnostic techniques, the CRISPR / Cas13d system can provide results in a shorter time, is easy to operate, and has a lower cost, making it suitable for on-site detection and large-scale screening applications.

[0005] PRRSV-1 (Porcine reproductive and respiratory syndrome virus type 1) is a major infectious disease facing the global pig industry. Currently, many countries have reported highly pathogenic PRRSV-1. The object of detection in the authorized patent CN110656123B is PRRSV-2, not PRRSV-1. Currently, the detection of PRRSV-1 mainly relies on RT-PCR and RT-qPCR technology. This method requires instrument support and is cumbersome to operate. There may be missed detection during the detection. Summary of the invention

[0006] The present invention utilizes CRISPR / Cas13d and RAA technologies to develop a detection kit for detecting porcine reproductive and respiratory syndrome virus type 1 (PRRSV-1) that is easy to operate, highly sensitive, and has intuitive results, thereby achieving efficient and visual detection of PRRSV-1, and providing an innovative technical means for on-site rapid diagnosis and disease prevention and control.

[0007] The present invention provides a crRNA for detecting PRRSV-1 based on the CRISPR-Cas13d system, and the sequence of the crRNA molecule is shown in SEQ ID NO.9.

[0008] The present invention also provides a product for detecting PRRSV-1 based on the CRISPR-Cas13d system, wherein the product contains the above-mentioned crRNA molecule.

[0009] Preferably, the product is a reagent or a kit.

[0010] More preferably, the kit also contains Cas13d protein, ssRNA fluorescent reporter probe, and upstream primers and downstream primers for recombinase polymerase nucleic acid amplification.

[0011] More preferably, the ssRNA fluorescent reporter probe is 5′-FAM-UUUUU-BHQ1-3′.

[0012] The present invention also provides the use of the above-mentioned crRNA molecule in the preparation of a product for detecting PRRSV-1.

[0013] Preferably, the product is a kit.

[0014] More preferably, the kit also contains Cas13d protein, ssRNA fluorescent reporter probe, and upstream primers and downstream primers for recombinase polymerase nucleic acid amplification.

[0015] More preferably, the method for using the kit is:

[0016] (1) Using the cDNA of the sample to be tested as a template to amplify the product, and transcribe in vitro to obtain ssRNA;

[0017] (2) reacting the ssRNA, Cas13d protein, ssRNA fluorescent reporter probe, and crRNA molecule obtained in step (1) in a CRISPR-Cas13d reaction system;

[0018] (3) After the reaction in step (2) is completed, the reaction system is placed under blue light or ultraviolet light to observe the detection results.

[0019] More preferably, the sequences of the upstream primer and downstream primer for recombinase polymerase nucleic acid amplification are shown as SEQ ID NO.3 and SEQ ID NO.6, respectively.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The present invention provides a kit for on-site visual detection of porcine reproductive and respiratory syndrome virus type 1 (PRRSV-1), which is based on recombinase polymerase amplification technology (RAA) and CRISPR-Cas13d technology. This method can effectively detect clinically positive PRRSV-1 samples and realize on-site visual detection through RNA fluorescent reporter molecules. The detection only requires irradiation with a blue light transilluminator, and the test result can be determined by observing whether an amplification product appears in the reaction system. In addition, the kit also supports quantitative detection using a qPCR instrument, so that the results can be quickly obtained within 1 hour.

[0022] With its unique advantages, this invention significantly reduces the economic cost and time consumption in the diagnosis process, while getting rid of the dependence on expensive laboratory equipment. Its key elements, primers and crRNA, simplify the operating procedures of the test kit, greatly improve the detection speed, and make the results intuitive and visual due to their excellent specificity and sensitivity. This detection method based on RAA and CRISPR-Cas13d technology not only realizes the on-site visual detection of PRRSV-1, but also brings unprecedented convenience to the pig farming industry. The introduction of this technical means will undoubtedly greatly enhance the detection ability of PRRSV-1, thereby effectively reducing the biosafety risks of farms, and will have a far-reaching impact on the healthy development of the pig farming industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Detection results of RAA amplification products of different RAA primer combinations in Example 1, M is the marker lane, and the rest are lanes of RAA amplification products of different RAA primer combinations.

[0024] Figure 2 The target gene amplification results of the positive plasmid constructed in Example 1, M is the marker lane, 1 is the PCR product lane, and 2 is the negative control lane.

[0025] Figure 3 The detection results of colony PCR in Example 1, M is the marker lane; 1 is the colony PCR amplification product lane; 2 is the negative control lane.

[0026] Figure 4The results of SDS-PAGE experiments using 50% elution buffer (3 tubes) and 75% (7 tubes) for the expression and purification of CRISPR-Cas13d protein in Example 1 are shown in Figure 1. From left to right, the results of SDS-PAGE experiments using 50% elution buffer (3 tubes) and 75% (7 tubes) are shown.

[0027] Figure 5 The Western-blot experimental results for the His tag of CRISPR-Cas13d protein in Example 1, M is the protein Marker lane, 1 is the negative control lane, and 2 is the purified protein lane.

[0028] Figure 6 The SDS-PAGE experimental results of the protein sample after the CRISPR-Cas13d protein was concentrated and stored in Example 1, M is the marker lane, 1 is the concentrated protein lane, and 2 is the negative control lane.

[0029] Figure 7 It is a fluorescence amplification graph for verification of crRNA and Cas13d protein activity in Example 1; the red curve (fluorescence value is about 52500 at the highest) is crRNA-1, the orange curve (fluorescence value is about 42500 at the highest) is crRNA-2, the blue curve (fluorescence value is about 35000 at the highest) is crRNA-3, and the green curve (fluorescence value does not change) is the negative control.

[0030] Figure 8 It is a specific fluorescence amplification diagram of the combination of RAA and CRISPR-Cas13d in Example 1; the red curve (the highest fluorescence value is 52000) is a PRRSV-1 clinical sample.

[0031] Fig. 9 This is the sensitivity fluorescence amplification diagram of combining RAA and CRISPR-Cas13d in Example 1. The light green curve (maximum fluorescence value is 58000) has a template copy number of 10 8 Gray curve (maximum fluorescence value is 54000) template copy number is 10 7 ; Blue curve (maximum fluorescence value is about 45000) template copy number is 10 6 ; Yellow curve (maximum fluorescence value is about 43000) template copy number is 10 5 ; Light orange curve (maximum fluorescence value is about 37500) template copy number is 10 4 ; Pink curve (maximum fluorescence value is about 34500) template copy number is 10 3 ; Fluorescent blue curve (maximum fluorescence value is about 31000) template copy number is 10 2; Black curve (maximum fluorescence value is about 25000) template copy number is 10 1 ; Black curve (maximum fluorescence value is about 24500) template copy number is 10 0 ; The green curve (fluorescence value did not change) is the negative control.

[0032] Fig.10 This is the naked eye fluorescence of the product under ultraviolet light / blue light conditions after the sensitive reaction combining RAA and CRISPR-Cas13d in Example 1. DETAILED DESCRIPTION

[0033] Example 1

[0034] 1. Design and synthesize RT-RAA primers

[0035] Multiple PRRSV gene sequences (including PRRSV-1 and PRRSV-2) were downloaded from the GenBank database, and these gene sequences were analyzed using MEGA 7.0 software. Primers for PRRSV-1 were designed based on their conserved sequences and RT-RAA primer design principles, and a T7 promoter sequence was added to the 5' end of the upstream primer for in vitro transcription. The designed primer sequences are detailed in Table 1. The designed primer sequences were sent to Sangon Biotech (Shanghai) Co., Ltd. for synthesis.

[0036] The principles of primer design are as follows: ① The length of RAA primers is usually 30 to 35 bases. If the primer is too short, it will affect its binding efficiency with the recombinase, resulting in the inability to quickly amplify the target fragment; if the primer is too long, it is easy to form primer dimers or secondary structures during the amplification process. ② The 5' end of the primer sequence should avoid repeated G and C bases, and the 3' end should preferably contain G or C bases, and the total content of G and C bases should be between 40% and 50%. ③ The optimal amplification product size is 100 to 300 bases. ④ The GC content and single base repeat sequence at the 5' end of the primer should not exceed 5. ⑤ The 3' end of the primer should be G and C bases as much as possible to provide a stable "clamp" target.

[0037] DNAMAN 6.0 software was used to design RT-RAA primers. In order to transcribe the RT-RAA amplification product into target ssRNA, a T7 promoter sequence (SEQ ID NO. 14: 5'-TAATACGACTC ACTATAGGG-3') was added to the upstream 5' end of each primer. After the design was completed, the primer sequence was sent to Shanghai Sangon Biotech Co., Ltd. for synthesis.

[0038] Table 1 PRRSV-1 ORF6 gene T7-RT-RAA primers

[0039]

[0040] 2. RT-RAA amplification and RAA primer screening

[0041] The RAA nucleic acid amplification kit provided by Jiangsu Qitian Gene Biotechnology Co., Ltd. was used, the primers listed in Table 1 were used, and the PRRSV-1 genome provided by the Animal Biotechnology Center of Sichuan Agricultural University was used as a template to perform RT-RAA reaction. The reaction program was set at 37°C and the duration was 20 minutes. After the reaction was completed, 5 μL of the reaction product was taken for agarose gel electrophoresis analysis. The results were as follows Figure 1 According to the results, the most suitable RAA primer is F2+R3.

[0042] Table 2 RT-RAA body reaction system

[0043] Components Volume (μL) Upstream and downstream primers (10 μM) 2.1 each template 2 Buffer 25 Starter 3 <![CDATA[RNase-freedH2O]]> Replenish to 50

[0044] 3. T7 in vitro transcription

[0045] The double-stranded DNA template obtained by RT-RAA amplification was transcribed in vitro to generate target single-stranded RNA (ssRNA). The in vitro transcription reaction was carried out at 37°C for 5h. Subsequently, the remaining DNA template was eliminated using RNase-free DNase I, and the RNA was purified using a column RNA purification kit provided by Shanghai Shenggong Biotechnology Co., Ltd. After purification, the concentration of RNA was determined and stored in aliquots at -80°C. The reaction systems for in vitro amplification, DNase I in vitro digestion, and T7 RNA in vitro transcription are listed in Tables 3, 4, and 5, respectively.

[0046] Table 3 DNaseⅠ digestion system

[0047] Components Volume(uL) DNaseⅠ 2 ReactionBuffer 2 Ribonuclease inhibitor 1 RNA ALL <![CDATA[RNase-freeddH2O]]> Replenish to 30

[0048] Table 4 T7 in vitro transcription system

[0049]

[0050]

[0051] 4. Construction of standard positive plasmid

[0052] Using the PRRSV-1 ORF6 gene as a template, RT-PCR primers F: TGCCATGATCCTACTGCC (SEQ ID NO.7), R: ACCACTCCTCGTTTGACAG (SEQ ID NO.8) were selected for amplification using high-fidelity Gflex enzyme, and the amplified fragment size was 476 bp. The reaction system is as follows:

[0053] Table 5 Positive plasmid target gene amplification reaction system

[0054] Components Volume(uL) 2xGflexBuffer 10 Upstream and downstream primers (10 μM) 0.4 each template 0.8 Gflex enzyme 0.4 <![CDATA[ddH2O]]> Up to 20

[0055] The PCR reaction program was as follows: pre-denaturation at 95°C for 4 min, denaturation at 95°C for 30 s, annealing at 56°C for 30 s, extension at 72°C for 30 s, 35 cycles, and extension at 72°C for 10 min.

[0056] After the reaction is completed, the reaction product is subjected to 1% agarose gel electrophoresis to detect the amplification result (such as Figure 2 After confirming that the size of the amplified fragment is correct, the amplified product is purified by gel recovery using the Agarose Gel DNA Kit of Kangwei Reagents. The purified sample is stored at -20℃ for future use.

[0057] Take a small amount of purified sample for sequencing. After sequencing, compare the splicing results in the BLAST database of NCBI to check whether the results are correct. Since the PCR amplification product using Gflex enzyme is a flat-end DNA fragment without an A tail, it cannot be directly cloned by TA, so a terminal Taq DNA polymerase plus A reaction is required. The reaction system is shown in Table 6:

[0058] Table 6 Positive plasmid target gene plus A reaction system

[0059] Components Volume(uL) Ex-TaqBuffer 2.5 dNTPMixture 2 PCR products 20.25 Ex-Ta enzyme 0.25 <![CDATA[ddH2O]]> Up to 25

[0060] The A-tailing reaction procedure is: incubate in a metal bath at 72°C for 30 minutes. After the reaction is completed, the product is connected to the PMD-19T vector. The reaction system is shown in Table 7:

[0061] Table 7 Positive plasmid ligation reaction system

[0062]

[0063]

[0064] The ligation reaction procedure is: incubate at 16°C in a metal bath for 30 minutes. After the ligation is completed, the product is transformed into competent cells DH5α for transformation and amplification. The plasmid is extracted using the plasmid extraction kit of Quanshijin Company, and the constructed standard positive plasmid is named PMD-19T-PRRSV-1. The extracted plasmid is sequenced to ensure that the recombinant plasmid is successfully constructed for subsequent experiments.

[0065] 5. Design and synthesis of crRNA

[0066] The Cas13d protein does not need to recognize specific PAM / PFS sequences. The crRNA design can be completed only by relying on the inherent hairpin structure sequence of Cas13d and the target gene sequence. For the ORF6 gene of PRRSV-1, select its highly conserved target region, and ensure that its 3′ end is A, U or C, and select 28 nucleotides upstream from this point. Use NCBI's BLAST tool to align the selected sequences to verify their specificity. The designed crRNA was sent to Shanghai Sangon Biotechnology Co., Ltd. for synthesis. The synthesized crRNA was diluted with RNase-free H2O to the required experimental concentration, aliquoted and stored at -80°C for use. The designed crRNA sequences are detailed in Table 8:

[0067] Table 8 crRNA sequences

[0068]

[0069] 6. Design and synthesis of fluorescent RNA probes

[0070] The ssRNA fluorescent reporter molecule was synthesized by Sangon Biotech (Shanghai) Co., Ltd., and its sequence is 5′-FAM-UUUUU-BHQ1-3′. The probe has a FAM fluorophore and BHQ1 quenching modifications at both ends, and will only produce fluorescence under specific conditions. When Cas13d binds to crRNA to form a Cas13d / crRNA complex, it specifically binds to and cuts the complementary target RNA (RNA obtained by T7 transcription after PRRSV-1-specific RT-PCR amplification), thereby activating the side-cutting activity of the complex, non-selectively degrading the ssRNA probe, and then generating a fluorescent signal.

[0071] 7EsCas13d expression, purification and concentration

[0072] 7.1 Transformation of exogenous plasmid into cloning strain

[0073] The purchased plasmid (pET28a-MH6-EsCas13d) powder was operated according to the instructions, and Kana (kanamycin) was selected as the resistance of its culture medium. The protein expressed by the plasmid is about 112.4 kDa, the total length of the plasmid is 8242 bp, and the inserted target protein fragment is 2907 bp in size. This fragment carries a 6×His tag to facilitate the subsequent separation and purification of the protein.

[0074] The EsCas13d protein plasmid was transformed into Escherichia coli DH5α competent cells with a transformation ratio of 1:10 (plasmid: competent cells). After mixing, it was placed on ice for 25 minutes, then heat-shocked in a 42°C metal bath for 90 seconds, and then cooled in an ice water bath for 10 minutes. Then 500 μL of pre-warmed LB medium was added, incubated in a 37°C metal bath for 10 minutes, the shaking shaker was adjusted to 37°C, cultured at 70 rpm for 10 minutes, then adjusted to 100 rpm for 25 minutes, and finally cultured at 120 rpm until the bacterial solution became turbid. The mixed bacterial solution was evenly spread on the LB culture dish containing Kana resistance, placed in a 37°C incubator for forward culture for 1.5 hours, and then inverted for overnight culture. A single colony was picked as a DNA template, and ordinary PCR amplification verification was performed according to the primers provided by add gene. PCR reaction conditions were: 95°C for 4 minutes; 95°C for 30 seconds, 58°C for 30 seconds, 72°C for 30 seconds, for a total of 35 cycles; 72°C for 10 minutes. The amplified product was subjected to gel electrophoresis. Figure 3 The remaining part of the single colony was transferred to LB liquid medium containing Kana resistance (500 μL) and cultured on a shaker at 37°C for 12 hours. The positively amplified monoclonal strain was stored as glycerol bacteria (the ratio of bacterial liquid to 50% glycerol was 1:1) and frozen at -80°C for later use.

[0075] Table 9 EsCas13d plasmid verification primers

[0076] Primers Sequence 5'-3' Cas13d-F SEQ ID NO.12: ATAATCCGCTGCGTCGTT Cas13d-R SEQ ID NO.13: CGGCTGTTGGTGCTTTCT

[0077] Table 10 Colony common PCR reaction system

[0078] Components Volume (μL) Upstream and downstream primers 1 each 2xTaqenzymeMix 10 Colony (DNA template) About 1 μg <![CDATA[ddH2O]]> Up to 20

[0079] The experimental results showed that the single colony picked was plasmid positive, as shown in Figure 3 .

[0080] 7.2 Inducible expression of EsCas13d protein

[0081] Use a small plasmid extraction kit to extract the plasmid from the positive DH5α bacterial solution, and transfer the extracted plasmid into Rosetta competent cells. The transformation process is carried out according to the above steps. After overnight culture, the bacterial solution is inoculated into a new 300mL liquid LB medium at a ratio of 1:100, and kanamycin is added at a concentration ratio of 1000:1. Incubate at 37℃ constant temperature shaker for 2-3 hours until the OD of the bacterial solution reaches 600 The value reached about 0.6. IPTG was then added at a final concentration of 500 μM, the culture temperature was lowered to 16°C, and the culture was cultured at 180 rpm for 18 h. After the culture was completed, the bacterial solution was collected for further protein treatment.

[0082] 7.3 EsCas13d protein purification

[0083] Transfer the liquid LB inoculated with seed liquid and cultured overnight into a 50mL centrifuge tube and centrifuge at 4000rpm and 4℃ for 15min. After centrifugation, resuspend the precipitate with 10mL PBS, pour the resuspended liquid into a new 50mL centrifuge tube and repeat the centrifugation step until all the bacterial liquid is concentrated. Resuspend the concentrated bacteria with PBS to a final volume of 20mL. Add lysozyme and protease inhibitor (PMSF) at a ratio of 1:100, mix well and then perform ultrasonic disruption. The ultrasonic disruption program is: ф6, 30% power, ultrasonic 2s, interval 4s, ultrasonic time 40min. The disrupted bacterial liquid is placed in a centrifuge tube and centrifuged at 5000rpm at 4℃ for 40min. Filter the supernatant through a 0.22μm filter membrane for subsequent protein purification.

[0084] The supernatant was mixed with binding buffer in a 1:1 ratio. First, 2 times the volume of the nickel column was used to flow naturally through the nickel column (HisTrap FF crude column), and then the mixed protein sample was loaded. After the protein sample naturally flowed through the column, the loading was repeated twice to ensure that the nickel column fully adsorbed the protein. Then, 2 times the volume of the nickel column was used to elute the binding buffer to remove non-specific adsorbed impurities. The HisTrap pre-packed column was eluted with elution buffer of different gradient concentrations. The elution gradient was 10%, 25%, 50%, 75% and 100%, and 10 mL of elution buffer was prepared for each concentration. Each 1 mL of elution was collected in a 1.5 mL clean EP tube for subsequent SDS-PAGE and Western-blot protein identification. Finally, 3 volumes of binding buffer and 1 volume of 20% ethanol were used to equilibrate the chromatography medium and stored at 4°C to prevent bacterial contamination.

[0085] The results of SDS-PAGE experiments showed that the impurity proteins of Cas13d protein were basically removed in the 8th tube under the 50% concentration of elution buffer, and were basically eluted completely in the 7th tube under the 75% concentration of elution buffer. For specific experimental results, see Figure 4 .

[0086] The eluted protein solution in tube 7 under 50% concentration of elution buffer was selected, and the His tag on the protein was verified by Western-blot. The results of Western-blot experiment showed that the prokaryotic expression of Cas13a protein was successful. For specific experimental results, see Figure 5 .

[0087] 7.4 Concentration of EsCas13d protein

[0088] The protein sample that is basically free of protein impurities after SDS-PAGE identification is transferred to a 30000MWCO Millipore ultrafiltration column for ultrafiltration concentration. Place the ultrafiltration column in a centrifuge precooled to 4°C and centrifuge at 4000r / min for 15 minutes. Then add the remaining protein sample, and repeat the sample addition and centrifugation steps until the final protein solution volume reaches 500μL. Next, add PBS solution to a total of 1000μL, and repeat the addition of PBS three times until the liquid in the ultrafiltration tube is completely replaced by PBS, and the final protein solution volume is about 200μL. Transfer the protein solution to a clean EP tube and freeze it at -80°C. The concentrated and stored protein sample is identified by SDS-PAGE, and the specific results are shown in Figure 6 .

[0089] 8 Establishment of a detection method for porcine reproductive and respiratory syndrome virus type 1 based on CRISPR-Cas13d technology

[0090] 8.1 Activity verification of crRNA and Cas13d protein

[0091] The sequence of the fluorescent reporter molecule is 5′-FAM-UUUUU-BHQ1-3′, which was synthesized by Sangon Biotech (Shanghai) Co., Ltd. The probe has a FAM fluorophore and BHQ1 quenching modification at both ends, and will only produce fluorescence under specific conditions. When Cas13d binds to crRNA to form a Cas13d / crRNA complex, it specifically binds to and cuts the complementary target RNA (RNA transcribed by T7 after PRRSV-1-specific RT-PCR amplification), activates the side cutting activity of the complex, non-selectively cuts the fluorescent RNA probe, and then produces a fluorescent signal. In order to verify the activity of crRNA and Cas13d protein, the PRRSV-1 genome (provided by the Biotechnology Center of Sichuan Agricultural University) was used as a template, and the ssRNA obtained by in vitro transcription of double-stranded DNA after RAA amplification was used as a template. Cas13d protein, crRNA, fluorescent RNA probe, protein reaction buffer and RNase inhibitor were added to the reaction system and reacted at 37°C for 1 hour. The reaction system is shown in Table 10.

[0092] The experimental results showed that the first crRNA had good reactivity, so this crRNA was used to establish the subsequent detection method. The specific experimental results are shown in Figure 7 .

[0093] Table 10 crRNA and Cas13d protein activity verification system

[0094] Reaction components Dosage Cas13a protein 100nM Protein Reaction Buffer 5μL crRNA 100nM RNA fluorescent reporter probe 5μM RNase inhibitor 1μL ssRNA template 100ng RNase-free water Make up to 25 μL

[0095] 8.2 Establishment of a porcine reproductive and respiratory syndrome virus type 1 detection method combining RAA and CRISPR-Cas13d technology and clinical sample testing

[0096] The above-screened crRNA (crRNA 1) was selected to construct a CRISPR-Cas13d detection method based on RAA. The RAA reaction was combined with the Cas13d verification system, and the specific operation was as follows: the product after the first step RAA reaction was added to the CRISPR-Cas13d reaction system, and the system was shown in Table 11. The q-PCR instrument was set at 37°C, and the fluorescence value was read every 30 seconds for 60 cycles to verify the specificity and sensitivity.

[0097] use Viral DNA / RNAMini Kit (purchased from Nanjing Novozyme Biotechnology Co., Ltd.) was used to extract total nucleic acid from PRRSV-1 positive samples and the genomes of porcine reproductive and respiratory syndrome virus type 2 (PRRSV-2), porcine circovirus type 2 (PCV2), porcine circovirus type 3 (PCV3), porcine epidemic diarrhea virus (PEDV), classical swine fever virus (CSFV) and porcine Japanese encephalitis virus (JEV) (provided by the Biotechnology Center of Sichuan Agricultural University). The RAA-based EsCas13d system was used to amplify the DNA and cDNA genomes of the above 7 virus strains and the template-free control to evaluate the specificity of the RAA-based CRISPR-Cas13d technology for the detection of porcine circovirus type 2. The specific results are shown in Figure 8 .

[0098] To evaluate the sensitivity of this detection system, different dilution gradients of positive plasmid pMD-19T-PRRSV-1 were used as templates. The template concentrations were 10 8 , 10 7 , 10 6 , 10 5 , 10 4 , 10 3 , 10 2 , 10 1 , 10 0 The experimental results show that the method used in the present invention can detect a minimum template concentration of 100 copies / μL, and the results can still be visualized at this sensitivity level. Fig. 9. Subsequently, the system after the reaction was placed under blue light or ultraviolet light for observation to verify the consistency of the above detection system results with the visual observation results and the practicality of visualization. The experimental results show that the positive results obtained in the established detection system are consistent with the visual observation results under blue light / ultraviolet light conditions. For specific experimental results, see Fig. 9 and Fig.10 .

[0099] Table 11 PRRSV-1 detection system combining RAA and CRISPR-Cas13d technology

[0100]

[0101]

[0102] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for detecting crRNA of PRRSV-1 based on the CRISPR-Cas13d system, characterized in that: The sequence of the crRNA molecule is shown in SEQ ID NO.

9.

2. A product for detecting PRRSV-1 based on the CRISPR-Cas13d system, characterized in that: The product contains the crRNA molecule described in claim 1.

3. The product according to claim 2, characterized in that The product is a reagent or a kit.

4. The product according to claim 3, characterized in that The kit also contains Cas13d protein, ssRNA fluorescent reporter probe, and upstream primers and downstream primers for recombinase polymerase nucleic acid amplification.

5. The kit according to claim 4, characterized in that The ssRNA fluorescent reporter probe is 5′-FAM-UUUUU-BHQ1-3′.

6. Use of the crRNA molecule described in claim 1 in preparing a product for detecting PRRSV-1.

7. The use according to claim 6, characterized in that The product is a test kit.

8. The use according to claim 7, characterized in that The kit also contains Cas13d protein, ssRNA fluorescent reporter probe, and upstream primers and downstream primers for recombinase polymerase nucleic acid amplification.

9. The use according to claim 8, characterized in that The method of using the kit is as follows: (1) Using the cDNA of the sample to be tested as a template to amplify the product, and transcribe in vitro to obtain ssRNA; (2) reacting the ssRNA, Cas13d protein, ssRNA fluorescent reporter probe, and crRNA molecule obtained in step (1) in a CRISPR-Cas13d reaction system; (3) After the reaction in step (2) is completed, the reaction system is placed under blue light or ultraviolet light to observe the detection results.

10. The use according to claim 8, characterized in that The sequences of the upstream primer and downstream primer for recombinase polymerase nucleic acid amplification are shown in SEQ ID NO.3 and SEQ ID NO.6 respectively.

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