CrRNA for detecting porcine circovirus type 2 based on RAA-CRISPR-Cas13d technology, kit and application

Abstract

The invention discloses a crRNA for detecting porcine circovirus type 2 based on an RAA-CRISPR-Cas13d technology, a kit and application, and belongs to the technical field of molecular biology diagnos.A target sequence is efficiently amplified by adopting an isothermal amplification technology (RAA), and specific recognition and signal amplification are performed in combination with a CRISPR-Cas13d system, so that high-sensitivity and high-specificity detection is realized. Meanwhile, a simplified nucleic acid extraction method and a diversified result display technology are introduced, so that the detection process is optimized, result interpretation is simplified, and the detection efficiency is remarkably improved. By means of the improvement, the defects that an existing commercial nucleic acid extraction kit is tedious in step and complex in result interpretation are overcome, and greater possibility is provided for application of the kit in on-site instant detection.

Description

Technical Field

[0001] The present invention relates to the field of molecular biological diagnostic technology, and specifically to crRNA, a kit, and applications for detecting porcine circovirus type 2 based on RAA-CRISPR-Cas13d technology. Background Art

[0002] Porcine circovirus (PCV) is a single-stranded, closed circular, negative-sense DNA virus belonging to the Circoviridae family and the genus Circovirus. Its genome length is approximately 1.7–2.0 kb. Four genotypes have been identified: PCV 1, 2, 3, and 4. Clinical manifestations of PCV 2 include dermatitis and nephropathy syndrome (PDNS), post-weaning multisystemic wasting syndrome, congenital tremor, and reproductive failure. PCV 2 is characterized by diverse infection routes and rapid transmission.

[0003] Currently, the main methods for detecting PCV2 include PCR, qPCR, and ELISA. In addition, new technologies such as LAMP, nanoPCR, RAA, and dPCR have also been developed. However, these methods struggle with sensitivity, specificity, operational complexity, cost, and detection time. Therefore, a comprehensive approach is needed, encompassing effective surveillance, biosafety measures, vaccination, and accurate pathogen diagnostics.

[0004] CRISPR / Cas13d technology has recently garnered attention due to its high specificity, sensitivity, and reliability, becoming a hot research topic in molecular biology detection. The CRISPR / Cas system is an adaptive immune system of bacteria that defends against foreign DNA, plasmids, and bacteriophages. The system consists of the CRISPR locus and the Cas gene. The CRISPR locus contains a leader sequence, repeat sequences, and a spacer sequence. The Cas gene encodes various CRISPR-related proteins (such as Cas9, Cas12a, and Cas13a). The functions of different Cas proteins have been exploited to develop gene editing, gene therapy, and molecular detection technologies. CRISPR / Cas13d utilizes a designed crRNA sequence of approximately 58 base pairs to direct the Cas13d protein to cleave target RNA or ssRNA and triggers side-stepping, further cleaving other RNA in the environment. RNA detection is achieved by adding a reporter RNA that produces fluorescence during cleavage and observing the reaction using a fluorescence quantifier or blue light transilluminator. However, due to its limited sensitivity, the CRISPR / Cas system alone for pathogen detection often requires a nucleic acid pre-amplification step to enhance detection. Isothermal amplification (IAMP) has become a popular method for point-of-care (POCT) testing due to its ability to efficiently amplify target DNA at a constant temperature without the need for complex equipment. However, IAMP faces the challenge of nonspecific amplification, which can lead to inaccurate results. Therefore, IAMP can be combined with CRISPR / Cas technology to address these shortcomings. Commonly used IAMP techniques include recombinase polymerase amplification (RPA), recombinase-assisted amplification (RAA), and loop-mediated isothermal amplification (LAMP). These methods offer advantages such as high amplification efficiency and strong resistance to inhibitory components.

[0005] Currently, there are some technologies that combine RAA-CRISPR-Cas13d technology to detect porcine circovirus type 2, but there is still the problem of nucleic acid extraction relying on instruments. This experiment combines a nucleic acid extraction-free kit, which can adapt to more scenarios and achieve true instrument independence. It can play a greater role in some resource-scarce areas and rural areas.

[0006] Based on this, the present invention designs crRNA, kits and applications for detecting porcine circovirus type 2 based on RAA-CRISPR-Cas13d technology to solve the above problems. Summary of the Invention

[0007] In response to the above-mentioned shortcomings of the prior art, the present invention provides crRNA, kits and applications for detecting porcine circovirus type 2 based on RAA-CRISPR-Cas13d technology.

[0008] To achieve the above objectives, the present invention is implemented through the following technical solutions: A crRNA for detecting porcine circovirus type 2 based on RAA-CRISPR-Cas13d technology, wherein the nucleotide sequence of the crRNA is SEQ ID NO.7.

[0009] In order to better achieve the purpose of the present invention, the present invention also provides a reagent for detecting porcine circovirus type 2 based on RAA-CRISPR-Cas13d technology, wherein the reagent contains the crRNA.

[0010] In order to better achieve the purpose of the present invention, the present invention also provides a RAA-CRISPR-Cas13d detection system for detecting porcine circovirus type 2, wherein the CRISPR-Cas13d detection system includes the crRNA or the reagent.

[0011] Furthermore, it also includes Cas13d protein, ssRNA fluorescent reporter probe, and upstream primers and downstream primers for recombinase polymerase nucleic acid amplification.

[0012] Furthermore, the sequence of the ssRNA fluorescent reporter probe is: 5′‐FAM-UUUUU-BHQ1-3′.

[0013] Furthermore, the upstream primer sequence is: taatacgactcactatagggGATGATCTACTGAGACTGTGTGATCGATATCC; The downstream primer sequence is: CCTTCCTCCGTGGATTGTTCTGTAGCATTCTT.

[0014] In order to better achieve the purpose of the present invention, the present invention also provides a kit for detecting porcine circovirus type 2 based on RAA-CRISPR-Cas13d technology, wherein the kit includes the crRNA or the reagent or the CRISPR-Cas13d detection system.

[0015] In order to better achieve the purpose of the present invention, the present invention also provides an application of the crRNA for detecting porcine circovirus type 2 based on RAA-CRISPR-Cas13d technology in the preparation of a product for detecting porcine circovirus type 2.

[0016] Furthermore, the product for detecting porcine circovirus type 2 is a kit.

[0017] Furthermore, the kit also includes Cas13d protein, ssRNA fluorescent reporter probe, and upstream primers and downstream primers for recombinase polymerase nucleic acid amplification.

[0018] Compared to existing technologies, the present invention offers the following advantages: 1. By designing primers and crRNA, the present invention enables efficient detection of clinical PCV2-positive samples. Combined with the use of an RNA fluorescent reporter, it achieves real-time, visual detection of PCV2. The detection process is simple: simply illuminate the reaction system with a fluorescence quantifier or blue light transilluminator and observe the generation of a fluorescent signal to quickly determine the test result. Furthermore, this method also supports quantitative detection using qPCR instruments, and the entire detection process can be completed in just half an hour.

[0019] 2. The kit of this invention demonstrates high specificity and sensitivity, making it more convenient to operate and enabling rapid detection and visual results. By combining RAA and CRISPR-Cas13d technology, this invention provides a novel technical approach for on-site detection of PCV2, improving detection efficiency and ensuring the accuracy of results. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. Those skilled in the art can also derive other drawings based on these drawings without inventive effort.

[0021] Figure 1 Detection results of RAA amplification products using different RAA primer combinations.

[0022] Figure 2 This is a fluorescence amplification profile demonstrating Cas13d protein activity. Reaction 1 represents intact components, reaction 2 represents the absence of crRNA, reaction 3 represents the absence of target RNA, and reaction 4 represents the absence of protein.

[0023] Figure 3 Different crRNAs were added for different types of crRNA amplification profiles.

[0024] Figure 4 This is the fluorescence amplification map of the PCV2 detection system combined with RAA-CRISPR-Cas13d technology.

[0025] Figure 5 Visualization results of the PCV2 detection system combined with RAA-CRISPR-Cas13d technology.

[0026] Figure 6 This is a sensitivity test diagram of the PCV2 detection system combined with RAA-CRISPR-Cas13d technology.

[0027] Figure 7 This is a specificity test diagram of the PCV2 detection system combined with RAA-CRISPR-Cas13d technology. DETAILED DESCRIPTION

[0028] To make the purpose, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts shall fall within the scope of protection of the present invention.

[0029] Example 1. Design and synthesis of RAA primers We identified conserved regions within the PCV2 ORF1 gene and designed RAA primers based on RAA primer design principles. A T7 promoter sequence (in lowercase letters) was added to the upstream primer to facilitate in vitro transcription. The designed primer sequences are shown in Table 1. After design, the primers were sent to Sangon Biotech (Shanghai) Co., Ltd. for synthesis.

[0030] Table 1 PCV2 ORF1 gene T7-RAA primers The principles of primer design are as follows: When designing RAA primers, it is generally recommended that the primer length be between 30 and 35 base pairs. If the primer length is too short, it will affect its binding efficiency with the recombinase, resulting in a slow amplification process. If the primer length is too long, it is likely to form primer dimers or secondary structures during amplification, thus affecting the amplification effect. In the primer sequence, repeated G and C sequences should be avoided at the 5' end, while G or C bases are recommended at the 3' end. In addition, the GC content of the primer is ideally controlled between 40% and 50%, which helps improve primer stability and amplification efficiency. For optimal amplification, the product size is generally recommended to be between 100 and 300 base pairs, which helps improve amplification sensitivity and accuracy. The 5' end of the primer should avoid excessive GC content or the presence of single nucleotide repeats, especially repeats of no more than 5 bases. At the 3' end, the G to C ratio is even more important, effectively enhancing target binding stability and ensuring efficient amplification.

[0031] 2. RAA amplification and RAA primer screening Use the RAA nucleic acid amplification kit, select the primers listed in Table 1, and use the PCV2 genome as a template to perform RAA amplification. The reaction conditions are set to 37°C and the reaction time is 20 minutes. After amplification, take 5 μL of sample and perform agarose gel electrophoresis analysis. The electrophoresis results are as follows: Figure 1 According to the amplification effect, the most suitable RAA primer R3+F3 was selected for subsequent experiments.

[0032] 3. Construction of standard positive plasmid Using the PCV2 ORF1 genome as a template, PCR primers were selected and amplified using the high-fidelity Gflex enzyme, resulting in a 448-bp amplified fragment. After completion of the reaction, the amplified fragment was verified by 1% agarose gel electrophoresis. After confirming the correct size of the amplified fragment, the fragment was purified using a universal DNA purification kit. A small amount of the purified sample was sequenced, and the resulting assembly was compared using NCBI's BLAST database to verify the accuracy of the result. The fragment was then ligated with the PMD-19T vector. After ligation, it was transformed into competent DH5α cells for transformation and amplification. A plasmid extraction kit was used to extract the plasmid. The resulting standard positive plasmid, designated PMD-19T-PCV2, was sequenced in appropriate quantities to ensure successful recombinant plasmid construction for subsequent experiments.

[0033] 4. Design and Synthesis of crRNA The crRNA is designed for the highly conserved target region amplified by RAA, ensuring that its 3′ end is an A, U, or C. Then, 28 nucleotides are selected from this point onward. Sequence alignment is performed using the NCBI BLAST tool to verify sequence specificity. Based on the designed crRNA, gDNA is then designed for subsequent in vitro transcription to produce the crRNA.

[0034] Table 2 crRNA sequences 5. Synthesis of Fluorescent Probes The ssRNA fluorescent reporter molecule sequence is 5′-FAM-UUUUU-BHQ1-3′ and was synthesized by Sangon Biotech (Shanghai) Co., Ltd. The probe, which has a FAM fluorophore and BHQ1 quenchers at both ends, does not produce fluorescence under specific conditions. However, when the EsCas13d protein binds to the crRNA to form a complex, it specifically binds to the target RNA and activates the protein's cleavage activity, indiscriminately cleaving the ssRNA probe to produce fluorescence.

[0035] 6. Expression of EsCas13d protein The purchased plasmid (pET28a-MH6-RspCas13d) powder was diluted according to the manufacturer's instructions and then transformed into Rosetta competent cells. The mixed culture was evenly spread onto a Kana-resistant LB plate using a spreader rod and incubated in a 37°C incubator in the forward direction for 1.5 hours. Positive clones were then incubated inverted overnight to screen for positive clones. Positive clones were selected, expanded, and verified by PCR. The verified culture was then inoculated into a fresh 300 ml LB medium at a 1:100 ratio (bacteria: medium = 1:100, medium: Kana = 1000:1). Culture was continued at 37°C on a shaker until the OD600 nm value reached approximately 0.6. Protein expression was induced by adding IPTG to a final concentration of 500 μM and incubating at 180 rpm at 18°C for 18 hours. The culture was transferred to a 50 ml centrifuge tube and centrifuged at 4000 rpm for 15 minutes at 4°C to collect the cell pellet. Resuspend the pelleted cells in PBS and wash by centrifugation three times. Resuspend the concentrated cells in PBS to a volume of 20 ml. Add lysozyme and the protease inhibitor PMSF at a ratio of 1:100, mix well, and disrupt with sonication at a setting of φ6, 30% power, 2 seconds per sonication, 4 seconds per sonication interval, and 40 minutes. Place the thoroughly disrupted cells in a centrifuge tube and centrifuge at 5000 rpm at 4°C for 40 minutes. Filter the supernatant through a 0.22 μm filter membrane and purify and concentrate the protein using a His-tag protein purification kit. Measure the concentration of the purified protein sample.

[0036] 7. Protein activity verification and crRNA screening 7.1. Target RNA Preparation The RAA amplification product was purified using a universal DNA purification and recovery kit. The purified product was in vitro transcribed at 37°C for 4 h using a high-yield T7 RNA in vitro transcription kit according to the instructions. The excess DNA template was degraded using RNase-free DNase I, and then purified using a column-based RNA purification kit. After purification and concentration determination, the product was aliquoted and stored at -80°C.

[0037] 7.2. crRNA Preparation Each of the three gDNAs was mixed with an equal volume of the T7 promoter to a final concentration of 10 μM. The mixture was incubated at 95°C for 10 minutes in a metal bath and then at room temperature for 30 minutes. The product was then transcribed into crRNA in vitro and purified using a column-based RNA purification kit. After purification and concentration determination, the product was aliquoted and stored at -80°C.

[0038] 7.3 Protein Activity Verification and crRNA Screening To verify the activity of crRNA and Cas13d protein, the product obtained in step 7.1 was used as a template, and Cas13d protein, crRNA, fluorescent RNA probe, protein reaction buffer and RNase inhibitor were added, and the reaction was carried out at 37°C for 30 min.

[0039] The experimental results showed that the protein was active and 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 2 、 Figure 3 .

[0040] Table 3 CrRNA and Cas13d protein activity verification system 8. Rapid visual detection of PCV2 based on RAA-CRISPR-Cas13d 8.1. Establishment of detection method According to the instructions of the Rapid Nucleic Acid Releaser (DNA Type)-II, nucleic acids were extracted from three positive samples and three negative samples: 100 μL of liquid samples (serum, blood) were added with 16 μL, D-1 and 4 μL, spleen, lymph and other tissue samples were added with PBS and ground, centrifuged at 12000 rpm and 4°C for 10 min, then 100 μL of the supernatant was taken and added with 16 μL D-1 and 4 μL D-2, and lysed on a metal bath at 37°C for 5 min. 5 μL of the supernatant of the lysate was taken as a template and used directly in the RAA amplification reaction. After the reaction, 5 μL of the reaction product was taken for in vitro transduction. The obtained product was purified and concentrated using a column-type RNA rapid concentration and purification kit. The reaction was then carried out at 37°C for 30 min, and the fluorescence signal was collected. The test results are as follows. Figure 4 .

[0041] 8.2 Portability and visualization of detection methods The system was placed in a 37°C metal bath for 30 minutes, and then observed under UV light to see the results. The results showed that after the system reacted in the metal bath for 30 minutes, it was placed under UV light and fluorescence could be observed in the right eye, while no fluorescence was observed in the negative eye. Figure 5 .

[0042] 8.3. Sensitivity of the Detection Method To evaluate the sensitivity of this detection system, the positive plasmid pMD-19T-gE with different dilution gradients was used as the template, and the template concentration per uL was 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. Figure 6 .

[0043] 8.4 Specificity of the Detection Method Rapid nucleic acid release agent (DNA type)-II or rapid nucleic acid release agent (RNA type)-II D was used to release nucleic acid from PCV2-positive samples and genomes of porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 3 (PCV3), porcine epidemic diarrhea virus (PEDV), classical swine fever virus (CSFV), porcine Japanese encephalitis virus (JEV), and porcine parvovirus (SVV) (provided by the Biotechnology Center of Sichuan Agricultural University). The specificity of this method was then verified. The specific results are shown in the table below. Figure 7 The results showed that only PCV2 was positive while the others were negative, indicating that this method has good specificity.

[0044] 8.5 Clinical Sample Testing In addition to the positive samples used in step 8.2, 18 additional clinical samples were tested for PCV2. The qPCR method specified in the national standard was used as a control. The test results showed that the positive rate was consistent with the national standard qPCR test results.

[0045] This invention utilizes isothermal amplification (RAA) to efficiently amplify target sequences and combines it with the CRISPR-Cas13d system for specific recognition and signal amplification, achieving highly sensitive and specific detection. Furthermore, it incorporates simplified nucleic acid extraction methods and diverse result display technologies to optimize the detection process and simplify result interpretation, significantly improving detection efficiency. This improvement overcomes the shortcomings of existing commercial nucleic acid extraction kits, which often involve cumbersome procedures and complex result interpretation, and opens up greater potential for their application in point-of-care testing.

[0046] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements will not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A method for detecting crRNA of porcine circovirus type 2 based on RAA-CRISPR-Cas13d technology, characterized in that: The nucleotide sequence of the crRNA is SEQ ID NO.

7.

2. A reagent for detecting porcine circovirus type 2 based on RAA-CRISPR-Cas13d technology, characterized in that The reagent contains the crRNA described in claim 1.

3. A RAA-CRISPR-Cas13d detection system for detecting porcine circovirus type 2, characterized in that: The CRISPR-Cas13d detection system includes the crRNA according to claim 1 or the reagent according to claim 2.

4. The RAA-CRISPR-Cas13d detection system for detecting porcine circovirus type 2 according to claim 3, characterized in that It also includes Cas13d protein, ssRNA fluorescent reporter probe, and upstream primers and downstream primers for recombinase polymerase nucleic acid amplification.

5. The RAA-CRISPR-Cas13d detection system for detecting porcine circovirus type 2 according to claim 4, characterized in that The sequence of the ssRNA fluorescent reporter probe is: 5′-FAM-UUUUU-BHQ1-3′.

6. The RAA-CRISPR-Cas13d detection system for detecting porcine circovirus type 2 according to claim 4, characterized in that The upstream primer sequence is: taatacgactcactatagggGATGATCTACTGAGACTGTGTGATCGATATCC; The downstream primer sequence is: CCTTCCTCCGTGGATTGTTCTGTAGCATTCTT.

7. A kit for detecting porcine circovirus type 2 based on RAA-CRISPR-Cas13d technology, characterized in that: The kit comprises the crRNA according to claim 1 or the reagent according to claim 2 or the CRISPR-Cas13d detection system according to any one of claims 3 to 6.

8. An application of crRNA for detecting porcine circovirus type 2 based on RAA-CRISPR-Cas13d technology according to claim 1 in the preparation of a product for detecting porcine circovirus type 2.

9. The use according to claim 8, characterized in that The product for detecting porcine circovirus type 2 is a kit.

10. The use according to claim 9, characterized in that The kit also includes Cas13d protein, ssRNA fluorescent reporter probe, and upstream primers and downstream primers for recombinase polymerase nucleic acid amplification.

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