RAA-CRISPRCas13a method for detecting iridovirus fluorescence of micropterus salmoides
By integrating RAA-CRISPRCas13a into a single-tube reaction system, the complexity and false positive problems of LMBV detection in existing technologies are solved, enabling rapid and simplified LMBV detection suitable for aquaculture sites.
Patent Information
- Application Number
- CN202511926199.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-19
- Publication Date
- 2026-03-27
AI Technical Summary
In existing technologies, LMBV detection relies on laboratory molecular methods such as real-time quantitative PCR, which is complex and time-consuming, making it difficult to promote in farms. Furthermore, existing RAA amplification and CRISPR detection involve two steps, which are cumbersome and carry the risk of false positives, making it impossible to construct a simple and rapid on-site detection method.
By integrating recombinase-mediated isothermal amplification with a CRISPR/Cas13a detection system into a single-tube reaction system, the entire process of nucleic acid amplification and signal detection is achieved at a single constant temperature, simplifying the operation process, and generating a detection signal by Cas13a protein cleaving fluorescent reporter molecules.
It enables rapid and simplified LMBV detection, with high specificity and sensitivity, suitable for aquaculture sites, reducing reliance on precision instruments and improving detection speed and result reliability.
Smart Images
Figure CN121737347A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biological detection technology, and more specifically, to a RAA-CRISPRCas13a method for detecting fluorescence of largemouth bass iridovirus. Background Technology
[0002] Largemouth bass (Micropterus salmoides) is an important freshwater aquaculture species with a massive aquaculture industry. However, with increasing stocking densities, disease problems are becoming increasingly prominent, among which diseases caused by Largemouth bass iridovirus (LMBV) are one of the main threats to the healthy development of the industry. LMBV infection can cause farmed largemouth bass to exhibit symptoms such as darkening of body color, lethargy, spiral swimming, and necrosis of internal organs. The disease has a rapid onset and high mortality rate, often causing huge economic losses to fish farmers. This virus is highly contagious and, once an outbreak occurs, spreads rapidly within the farmed population. Therefore, establishing a rapid, sensitive, and specific early detection method for LMBV is crucial for timely early warning, isolation, control, and scientific treatment of the disease, and is a key link in ensuring the sustainable development of largemouth bass aquaculture.
[0003] Currently, LMBV detection mainly relies on laboratory molecular methods, such as real-time quantitative PCR (qPCR). While this method is sensitive and accurate, it depends on sophisticated instruments and professional operators, and the process is complex and time-consuming, making it difficult to promote and apply in grassroots fields such as farms. Isothermal amplification technologies (such as RAA) and CRISPR gene editing technologies offer new directions for rapid detection. RAA technology does not require complex temperature changes and can complete nucleic acid amplification in a short time; the CRISPR / Cas13a system can activate its cleavage activity after recognizing specific viral RNA, generating a fluorescent signal by cleaving the reporter probe, thereby achieving highly specific detection. However, existing technologies often separate RAA amplification and CRISPR detection into two independent steps. This "two-step" process still suffers from problems such as cumbersome operation steps, easy contamination of amplification products leading to false positives, and overall detection time not being ideal. In particular, how to efficiently couple and optimize these two technologies to construct a simple, closed-tube, rapid, and field-suitable LMBV detection method remains a technical challenge that urgently needs to be solved in this field. Summary of the Invention
[0004] To address the aforementioned technical problems, this invention proposes a RAA-CRISPRCas13a method for detecting fluorescence of largemouth bass iridovirus. This method integrates recombinase-mediated isothermal amplification with a CRISPR / Cas13a detection system into a single-tube reaction system, enabling the entire process from nucleic acid amplification to signal detection to be completed at a single constant temperature, greatly simplifying the operation procedure.
[0005] This invention provides a RAA-CRISPRCas13a method for detecting fluorescence of largemouth bass iridovirus, comprising the following steps: S1: Based on the specific gene sequence of largemouth bass iridovirus, prepare specific RAA primer pairs and guide crRNA; S2: Construct a single-tube reaction system, wherein the single-tube reaction system contains RAA amplification components, Cas13a protein, guide crRNA, and a single-stranded RNA reporter molecule with fluorescent and quenching groups; S3: Extract the nucleic acid from the sample to be tested as a template, and add the template to the single-tube reaction system; S4: Incubate the single-tube reaction system under a single isothermal condition of 37-42℃ for 15-30 minutes. When the guide crRNA recognizes the specific target sequence in the RAA amplification product, it activates the trans-cleavage activity of the Cas13a protein, cuts the fluorescent reporter group of the single-stranded RNA, separates the fluorescent group and the quencher group, and generates a detectable fluorescent signal. S5: Read the fluorescence signal. When the fluorescence signal intensity exceeds the preset judgment threshold, the sample to be tested is identified as positive for largemouth bass iridovirus.
[0006] In this scheme, in step S1, the specific gene sequence of the largemouth bass iridovirus is the major capsid protein gene sequence.
[0007] In this scheme, in step S2, the RAA amplification component, Cas13a protein, guide crRNA and reporter molecule are premixed lyophilized powders and exist in the reaction tube.
[0008] In this scheme, in step S2, the Cas13a protein is LwCas13a protein, the sequence of the single-stranded RNA reporter molecule is polyuridine nucleotide, the 5' end is labeled with a FAM fluorescent group, and the 3' end is labeled with a BHQ1 quencher group.
[0009] In this scheme, in step S2, the guide crRNA includes at least two types, which target different conserved regions of the LMBV genome respectively, and the single-stranded RNA reporter molecule includes reporter molecules that correspond to different guide crRNAs and are labeled with different fluorescence spectra.
[0010] In this scheme, the different conserved regions are selected from the major capsid protein gene, the DNA polymerase gene, and the ribonuclease reductase large subunit gene.
[0011] In this scheme, a nucleic acid enrichment step is included before step S3: the inactivated suspected virus sample is mixed with the pretreatment system and incubated. The pretreatment system contains Cas13a protein, guide crRNA and biotin-labeled RNA reporter molecule. Add streptavidin-tagged magnetic beads to capture biotin-tagged reporter molecule fragments generated after cleavage of activated Cas13a protein; After washing, the magnetic beads containing the Cas13a protein complex were used as nucleic acid templates for the test sample.
[0012] In this scheme, after step S4, a signal amplification step is also included: when the detection result is positive or a critical value, a portion of the reaction solution of the single tube reaction system is taken and added to the secondary amplification reaction system containing only single-stranded RNA reporter molecules; Incubation at a constant temperature allows the activated Cas13a protein in the reaction solution to cleave the reporter molecule in a secondary amplification reaction system, generating an enhanced and amplified fluorescence signal.
[0013] In this scheme, in step S5, the fluorescence detection device is a portable fluorescence detector or a microplate fluorescence detector.
[0014] A second aspect of the present invention provides a kit for detecting fluorescence of largemouth bass iridovirus using the RAA-CRISPR / Cas13a method, the kit comprising at least one reaction tube pre-loaded with the components of the single-tube reaction system of claim 1, wherein the components are, as described in claim 3, premixed lyophilized powder.
[0015] This invention addresses the shortcomings of the prior art and has the following beneficial effects: (1) This invention integrates recombinase-mediated isothermal amplification with CRISPR / Cas13a detection system into a single-tube reaction system, realizing the entire process from nucleic acid amplification to signal detection at a single constant temperature, simplifying the operation process and reducing the dependence on precision instruments.
[0016] (2) This invention can effectively identify infections with low viral loads and has high specificity for detecting largemouth bass iridovirus, effectively avoiding cross-reaction with other common aquatic viruses. In addition, the method of this invention has a fast detection speed and can obtain reliable results in a short time. Furthermore, the reagent form of premixed freeze-dried powder further enhances the stability and portability of the reagent, providing strong technical support for rapid screening in aquaculture sites and its promotion and application in grassroots laboratories. Attached Figure Description
[0017] Figure 1This is a schematic diagram illustrating the detection principle and process of the RAA-CRISPR / Cas13a method for detecting fluorescence of largemouth bass iridovirus in an embodiment of the present invention. Figure 2 This is a diagram showing the specificity verification results in an embodiment of the present invention; Figure 3 This is a graph showing the sensitivity verification results in an embodiment of the present invention; Figure 4 This is a diagram showing the clinical sample validation results in an embodiment of the present invention. Detailed Implementation
[0018] To better understand the above-mentioned objectives, features, and advantages of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. It should be noted that, unless otherwise specified, the embodiments and features described in these embodiments can be combined with each other.
[0019] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and therefore the scope of protection of the invention is not limited to the specific embodiments disclosed below. Example
[0020] The RAA-CRISPR / Cas13a method for detecting fluorescence of largemouth bass iridovirus is as follows: S1: Spleen and kidney tissue samples were collected from suspected diseased fish in a largemouth bass farm. Based on the conserved region of the major capsid protein gene of largemouth bass iridovirus, specific RAA primer pairs (SEQ ID NO: 1 and SEQ ID NO: 2) and guide crRNA (SEQ ID NO: 3) were prepared. The forward primer (SEQ ID NO: 1) was 5'-ACTGGCCACCACCTCTACTC-3'; the reverse primer (SEQ ID NO: 2) was 5'-AGACACGTTGATGCTTGCGT-3'; and the guide crRNA (SEQ ID NO: 3) was 5'-UAAUUUCUACUAAGUGUAGAUCGGAACACAUCCGACACUCUUUU-3'.
[0021] S2: Construct a single-tube reaction system. Prepare a 20 μL total reaction volume in a 0.2 mL clear reaction tube. The single-tube reaction system contains 10 μL of 2×RAA reaction buffer, which is necessary for RAA amplification. This buffer includes: recombinase, single-stranded DNA binding protein, and strand displacement DNA polymerase; 0.5 μL of LwCas13a protein, 0.4 μL of guide crRNA, and 0.2 μL of single-stranded RNA reporter molecule with fluorescent and quenching groups, which are necessary for CRISPR / Cas13a detection; and make up to 20 μL with nuclease-free water.
[0022] S3: Total DNA was extracted from 100 mg of tissue sample using a magnetic bead-based nucleic acid extraction kit. DNA concentration and purity were determined using a NanoDrop2000 micro-spectrophotometer. All samples were uniformly diluted to 50 ng / μL as templates. 2.0 μL of template solution was added to a single-tube reaction system, gently mixed, and then briefly centrifuged.
[0023] S4: Place the reaction tube in a preheated constant-temperature metal bath and incubate at 39°C for 20 minutes. The RAA amplification reaction and the CRISPR / Cas13a detection reaction are performed sequentially. The RAA amplification system rapidly amplifies the target DNA under constant-temperature conditions, generating RAA amplification products. The RAA amplification products are specifically bound to guide crRNA to form a Cas13a-crRNA-DNA ternary complex. The activated Cas13a protein non-specifically cleaves the single-stranded RNA reporter molecule. The cleavage separates the fluorescent group from the quencher group, generating a detectable fluorescent signal.
[0024] S5: Use a portable fluorescence detector to read the fluorescence signal. The preset judgment threshold is three times the standard deviation of the average fluorescence value of the negative control. When the fluorescence signal intensity exceeds the preset judgment threshold, the sample to be tested is considered positive for largemouth bass iridovirus. Positive and negative controls are also set up for quality control.
[0025] The RAA-CRISPR / Cas13a method for detecting fluorescence of largemouth bass iridovirus was validated for specificity, sensitivity, and clinical samples.
[0026] In the specificity validation, common aquatic viruses such as LMBV-positive samples, infectious spleen and kidney necrosis virus (ISKNV), nerve necrosis virus (NNV), carp spring viremia virus (SVCV), and grass carp reovirus (GCRV) were detected. Figure 2 As shown, the results indicate that only LMBV-positive samples produced specific fluorescent signals, while other pathogens were detected negatively, demonstrating the high specificity of the method.
[0027] In sensitivity validation, the LMBV positive plasmid standard (pMD-LMBV-MCP) was serially diluted 10-fold (4.6 × 10⁻⁶). 8 -4.6×10 0 (copies / μL), detected using the method described in this embodiment. Figure 3 As shown in the figure, the results indicate that this method can detect down to a minimum of 4.6 × 10¹ copies / μL, and its sensitivity is significantly improved compared to conventional PCR methods.
[0028] In clinical sample validation, 30 clinical samples were tested, such as... Figure 4 As shown, the positive detection rate of this method is 40% (12 / 30), and the concordance rate with the qPCR method is 100%. All operations are completed within 40 minutes, simplifying the operation process and outperforming traditional detection methods. Example
[0029] This embodiment is basically the same as the steps in Embodiment 1, except that a nucleic acid enrichment step is added before step S2: the inactivated suspected virus sample is mixed and incubated with the pretreatment system, which contains Cas13a protein, guide crRNA, and biotin-labeled RNA reporter molecules; streptavidin-labeled magnetic beads are added to capture biotin-labeled reporter molecule fragments generated after the activated Cas13a protein is cleaved; after washing, the magnetic beads with Cas13a protein complex are used as nucleic acid templates for the test sample in a single-tube reaction system.
[0030] The detection method prepared in this embodiment is applicable to the detection of samples with low viral load, verifying the feasibility and reliability of the method of the present invention in improving detection sensitivity. Example
[0031] This embodiment is basically the same as the steps in Embodiment 1. The difference is that after step S4, when the detection result is positive or at the critical value, a signal amplification step is added. A portion of the reaction solution of the single tube reaction system is taken and added to a secondary amplification reaction system containing only a high concentration of single-stranded RNA reporter molecules. The system is incubated at a constant temperature for 10 minutes. The reporter molecules are cleaved by the activated Cas13a protein in the reaction solution in the secondary amplification reaction system, generating an enhanced and amplified fluorescence signal for detection.
[0032] The detection method prepared in this embodiment can amplify weak positive signals, improve the reliability and accuracy of detection results, and is suitable for confirmatory detection of critical value samples.
[0033] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.
Claims
1. A RAA-CRISPR / Cas13a method for detecting fluorescence of largemouth bass iridovirus, characterized in that, Includes the following steps: S1: Based on the specific gene sequence of largemouth bass iridovirus, prepare specific RAA primer pairs and guide crRNA; S2: Construct a single-tube reaction system, wherein the single-tube reaction system contains RAA amplification components, Cas13a protein, guide crRNA, and a single-stranded RNA reporter molecule with fluorescent and quenching groups; S3: Extract the nucleic acid from the sample to be tested as a template, and add the template to the single-tube reaction system; S4: Incubate the single-tube reaction system under a single isothermal condition of 37-42℃ for 15-30 minutes. When the guide crRNA recognizes the specific target sequence in the RAA amplification product, it activates the trans-cleavage activity of the Cas13a protein, cuts the fluorescent reporter group of the single-stranded RNA, separates the fluorescent group and the quencher group, and generates a detectable fluorescent signal. S5: Read the fluorescence signal. When the fluorescence signal intensity exceeds the preset judgment threshold, the sample to be tested is identified as positive for largemouth bass iridovirus.
2. The RAA-CRISPR / Cas13a method for detecting largemouth bass iridovirus fluorescence according to claim 1, characterized in that, In step S1, the specific gene sequence of the largemouth bass iridovirus is the major capsid protein gene sequence.
3. The RAA-CRISPR / Cas13a method for detecting largemouth bass iridovirus fluorescence according to claim 1, characterized in that, In step S2, the RAA amplification components, Cas13a protein, guide crRNA, and reporter molecule are premixed lyophilized powders present in the reaction tube.
4. The RAA-CRISPR / Cas13a method for detecting largemouth bass iridovirus fluorescence according to claim 1, characterized in that, In step S2, the Cas13a protein is LwCas13a protein, and the sequence of the single-stranded RNA reporter molecule is polyuridine nucleotide, with a FAM fluorescent group labeled at the 5' end and a BHQ1 quencher group labeled at the 3' end.
5. The RAA-CRISPR / Cas13a method for detecting fluorescence of largemouth bass iridovirus according to claim 1, characterized in that, In step S2, the guide crRNA comprises at least two types, each targeting a different conserved region of the LMBV genome, and the single-stranded RNA reporter molecule comprises a reporter molecule corresponding to the different guide crRNAs and labeled with different fluorescence spectra.
6. The RAA-CRISPR / Cas13a method for detecting largemouth bass iridovirus fluorescence according to claim 5, characterized in that, The different conserved regions are selected from the major capsid protein gene, DNA polymerase gene, and ribonuclease reductase large subunit gene.
7. The RAA-CRISPR / Cas13a method for detecting fluorescence of largemouth bass iridovirus according to claim 1, characterized in that, Before step S3, a nucleic acid enrichment step is also included: the inactivated suspected virus sample is mixed with a pretreatment system and incubated, the pretreatment system containing Cas13a protein, guide crRNA and biotin-labeled RNA reporter molecule; Add streptavidin-tagged magnetic beads to capture biotin-tagged reporter molecule fragments generated after cleavage of activated Cas13a protein; After washing, the magnetic beads containing the Cas13a protein complex were used as nucleic acid templates for the test sample.
8. The RAA-CRISPR / Cas13a method for detecting fluorescence of largemouth bass iridovirus according to claim 1, characterized in that, The process after step S4 also includes a signal amplification step: when the detection result is positive or a critical value, a portion of the reaction solution of the single-tube reaction system is taken and added to a secondary amplification reaction system containing only single-stranded RNA reporter molecules. Incubation at a constant temperature allows the activated Cas13a protein in the reaction solution to cleave the reporter molecule in a secondary amplification reaction system, generating an enhanced and amplified fluorescence signal.
9. The RAA-CRISPR / Cas13a method for detecting fluorescence of largemouth bass iridovirus according to claim 1, characterized in that, In step S5, the fluorescence detection device is a portable fluorescence detector or a microplate fluorescence detector.
10. A kit for storing and performing the RAA-CRISPR / Cas13a method for detecting largemouth bass iridovirus fluorescence as described in any of claims 1-9, characterized in that, The kit comprises at least one reaction tube pre-loaded with the components of the single-tube reaction system of claim 1, wherein the components are, as described in claim 3, premixed lyophilized powder.