Method for detecting ricefield eel rhabdovirus based on CRISPR-CAS13a and transverse chromatographic test strip visualization

Abstract

The invention discloses a ricefield eel rhabdovirus detection method based on CRISPR-CAS13a and transverse chromatography test strip visualization, and belongs to the technical field of aquatic virus molecule detection. Comprising an RPA primer, Cas13a protein and crRNA, the RPA primer comprises primers as shown in SEQ ID NO.4 and SEQ ID NO.5, and the RPA primer comprises primers as shown in SEQ ID NO.4 and SEQ ID NO.5; and the sequence of the crRNA is as shown in SEQ ID NO. 6. The invention provides a method for detecting monopterus albus rhabdovirus based on CRISPR-CAS13a and transverse chromatographic test strip visualization. The method is good in detection specificity and high in sensitivity, the detection limit value can reach 1.14 * 10 < 1 > copies, better specificity and compatibility can be achieved, the detection cost is low, and operation is convenient and rapid.

Description

Technical Field

[0001] This invention relates to the field of molecular detection technology for aquatic viruses, and in particular to a method for detecting loach rhabdovirus based on CRISPR-CAS13a and visualization using transverse chromatography test strips. Background Technology

[0002] The swamp eel (Monopterus albus), also known as the yellow eel, is a freshwater burrowing, bottom-dwelling fish belonging to the order Gilfishes and family Synbranchidae. It is relatively resilient and commonly found in lakes, streams, rice paddies, and ditches. Its distribution is mainly in some Asian countries, and in my country, it is found throughout most regions except for the northwest and northeast. Swamp eel farming is also widespread in my country, occurring in the lower reaches of the Yellow River and the middle and lower reaches of the Yangtze River. Swamp eels are delicious, nutritious, and rich in various trace elements and minerals essential for human metabolism, giving them high nutritional and economic value. Their fat is rich in DHA and EPA, which have medicinal value, such as delaying aging and lowering blood lipids. According to the 2024 Fisheries Yearbook, swamp eel farming production reached 350,000 tons in 2023, a 6.28% increase compared to 2022. Wild eels have strong resistance to disease in natural waters and rarely suffer from illness. However, in recent years, due to factors such as high farming density and deterioration of aquaculture water resources, the eels' resistance to disease has decreased, and the incidence of disease has become increasingly serious, which has restricted the healthy and green development of the eel farming industry.

[0003] Rhabdoviruses are a class of negative-sense RNA viruses that resemble bullets or rods in shape. The viral RNA is encapsulated by nucleoproteins, and the nucleocapsid is surrounded by a lipoprotein envelope and glycoprotein spikes (Fields virology 6th). Because they appear bullet-shaped under ultrastructural observation, Melnick et al. suggested naming these viral particles rhabdoviruses. In 1970, the International Committee on Virus Nomenclature adopted this suggestion and officially named them the Rhabdoviridae family. Currently, the Rhabdoviridae family has many members and a wide host range, including mammals, plants, insects, and fish, and can cause mild, acute, or even fatal diseases. The morphology of rhabdoviruses infecting animals, plants, and insects generally differs; the former are usually bullet-shaped, while the latter are generally rod-shaped.

[0004] Due to the unique morphological characteristics of viral particles, rhabdoviruses distinguish themselves from the other seven members of the single-stranded negative-sense RNA virus family: Sunviridae, Bornaviridae, Mymonaviridae, Paramyxoviridae, Filoviridae, Nyamiviridae, and Pneumoviridae.

[0005] Currently, based on the clinical symptoms of diseases affecting swamp eels, nearly 20 bacterial diseases are mainly reported, including whitehead disease and hemorrhagic disease. These pathogens are generally conditionally induced, causing swamp eel diseases to exhibit a clear seasonality and conditionality, with outbreaks concentrated in spring, summer, and autumn. The water temperatures during these three seasons are more suitable for the growth of most pathogens, such as whitehead disease and skin rot. Currently, more than 15 parasites have been reported infecting swamp eels in China, including *Polyoncobothrium magnum*, *Neosentis celatus*, *Trypanosomamonopteri*, *Eel tail fluke*, *Diplostomum hubeiensis*, and *Diplostomum niedashui*. In addition, there are reports of diseases caused by fungi and some non-pathogenic biological factors leading to death. One pathogen, rhabdovirus, has been reported as a cause of viral diseases. Because artificial breeding technology for eels started relatively late, existing research mainly focuses on identifying pathogenic agents, with limited in-depth studies on the pathogenesis of diseases. Furthermore, it's possible that the same disease can be caused by multiple pathogens, or that different pathogens can infect eels and cause the same clinical symptoms. Therefore, the identification of pathogens causing eel diseases is complex and diverse. Eel diseases have become one of the bottlenecks restricting the green and healthy development of the industry. Currently, among all eel diseases, bacterial pathogens are the most frequently reported. In addition, there are reports of eels being infected with various parasites. Only one viral pathogen has been found: eel rhabdovirus. This virus, identified as belonging to the genus Perhabdovirus in the family Rhabdoviridae, is a highly pathogenic agent that has caused serious harm to eel farming.

[0006] In recent years, in addition to the reported parasitic and bacterial diseases, viral diseases have also hindered the healthy and green development of my country's eel farming industry. In 2013, Ou et al. identified a rhabdovirus in farmed eels in Hubei Province. The clinical symptoms of this disease include bleeding on the body surface, and internal organ dissection also revealed bleeding. Furthermore, in 2017, Sun et al. discovered that snakehead rhabdovirus could also infect farmed eels, with clinical symptoms similar to those observed by Ou et al., also causing bleeding in the fish. Therefore, this virus may be capable of cross-species transmission. In 2019, Liu et al. isolated and identified a strain of Chineserice-field eel rhabdovirus from eel larvae at an artificial breeding base in Qianjiang City, Hubei Province, tentatively named Chineserice-field eel rhabdovirus (CrERV). This virus can cause explosive mortality in artificially bred larvae, with a mortality rate as high as 90%. The main clinical symptoms are swollen head and bleeding on the body surface of the affected fish. The bleeding is similar to that reported by Ou et al. In 2023, Liu Wenzhi et al. isolated a strain of smelt rhabdovirus from diseased smelt tissue. Whole-genome sequencing analysis revealed that CrERV (CrERV) had the highest homology (94.4%) with mandarin fish rhabdovirus (SCRV). It also showed high homology (92.3%) with some rhabdovirus gene sequences sequenced by Ou et al. Epidemiological surveys showed that CrERV developed at water temperatures of 17-22℃. Therefore, these findings indicate that smelt rhabdovirus is a major reported viral disease causing explosive mortality in smelt. Current research on smelt rhabdovirus is mainly based on preliminary isolation and identification of the pathogen. Further in-depth research on rapid detection methods for the virus is needed. Therefore, in-depth research on rapid detection methods for smelt rhabdovirus is crucial to provide technical support for effective prevention and control of smelt viral diseases and healthy smelt farming.

[0007] Real-time quantitative PCR (RT-qPCR or qPCR) originated from a concept proposed by Higuchi in 1992: ethidium bromide (EB) is a fluorescent dye that can be embedded between the two bases of double-stranded nucleic acids. Under ultraviolet light excitation, the fluorescence intensity of EB incorporated during the annealing or extension phases of the PCR reaction is measured, thereby enabling real-time monitoring of the entire PCR cycle. Subsequently, based on this concept, PerkinElmer (PE) developed TaqMan fluorescent probe quantification technology in 1995, making quantitative PCR truly "real-time" and significantly improving the specificity and sensitivity of detection. Compared with traditional quantitative techniques (such as semi-quantitative PCR and competitive quantitative PCR), real-time quantitative PCR has advantages such as good reproducibility, specificity and sensitivity, accurate quantification, simple operation, less sample contamination, and high automation. This technology is now widely used in basic scientific research, genetically modified products and food safety testing, medical diagnostics, drug development, customs inspection and quarantine, and other scientific and practical fields.

[0008] Recombinase polymerase amplification (RPA) is one of the most advanced isothermal amplification technologies, first introduced, applied, and developed by Niall Armes in 2006. RPA uses a recombinase to pair with nucleic acid primers on a DNA template, and the polymerase synthesizes a new nucleic acid chain, achieving exponential amplification in a very short time (approximately 15 minutes). RPA amplification products can be detected in real-time by fluorescence or by endpoint detection methods such as electrophoresis and lateral flow dipstick (LFD). After several years of development, it has been successfully applied to the detection of aquatic animal diseases. This technology can rapidly detect target objects at temperatures of 37-42 °C, and the amplified products are visualized by combining them with colloidal gold test strips for result interpretation. This method requires a lower amplification temperature than other detection methods, making it more suitable for clinical on-site diagnosis. It is simple to operate, does not require repeated temperature adjustments, has a relatively short amplification time, and has low dependence on specialized equipment. It only requires a specific temperature environment to achieve exponential nucleic acid amplification, making it a relatively ideal rapid nucleic acid detection method.

[0009] CRISPR-Cas (clustered regularly spaced short palindromic repeats-CRISPR-related proteins) is an adaptive immune system encoded by genetic elements in most archaea and many bacteria, which defend against invading viruses and plasmids. Currently, the most widely used CRISPR systems are CRISPR Cas9, CRISPR Cas12, CRISPR Cas13, and CRISPR Cas14, while tam2 belongs to type II CRISPR systems. Its effector template is a single protein, which is easily editable and can be used for nucleic acid detection.

[0010] Cas13 is an RNA-guided RNA endonuclease that specifically cleaves ssRNA under the guidance of crRNA, retaining non-specific endonuclease activity after cleavage to cleave other non-target ssRNAs. East Seletsky first used LbuCas13a to detect phage λRNA and endogenous β-actin mRNA in 2016. This method activates the trans-cleavage activity of Cas13a by targeting ssRNA, degrading fluorescently labeled ssRNA (ssRNA-FQ), and ultimately releasing a fluorescent signal, thus achieving rapid detection with detection limits reaching 1-10 pM. Most existing CRISPR / Cas13-based detection technologies are based on Sherlock: the target sequence is pre-amplified by RPA or RT-RPA, then transcribed into ssRNA by T7 RNA polymerase, causing Cas13 to cleave the fluorescent probe of single-stranded RNA and release a fluorescent group. This allows Cas13 to cleave the fluorescent probe of ssRNA and release the fluorescent group. The entire reaction process can rapidly detect RNA and DNA at 37 °C and amol concentration. SHERLOCK V2 is an improvement on this research, adding four advantages: (1) it achieves quadruple detection in a single tube; (2) by adjusting the RPA primers to control the exponential amplification process, quantitative detection at the amol concentration level is achieved; (3) the cascade activation of Csm6 nuclease activity triggered by Cas13a side chain cleavage improves the sensitivity of the detection system; (4) target sequences as low as 2 am can be visualized within 90 min. The signal amplification capability of CRISPR-based trans-cleavage activity is determined by enzyme digestion kinetic parameters, which are essential for determining the detection limits of various Cas nucleases. Although CRISPR / Cas13a can directly recognize ssRNA and cleave probes at room temperature to generate signals, the enzyme digestion kinetic parameters of Cas13a make it difficult to detect low copy RNA. Fozouni et al. constructed multiple CRISPR / Cas13a systems using CRISPR / Cas13a technology. In their preliminary study, they achieved specific recognition of SARS-CoV-2 RNA sequences and successfully improved signal amplification efficiency through a strategy of multiple activation of CRISPR / Cas13a enzyme digestion activity. Simultaneously, a highly sensitive fluorescence detector was used to detect weak fluorescence signals. Shinoda et al. proposed an RNA detection strategy based on the CRISPR / Cas13a cleavage enzyme—the CRISPR-based microdroplet digital RNA detection method (SATORI). This method disperses Cas13a, target RNA, and chromosomal substrate strands into 120,000 microdroplets containing 3 fL each, promoting CRISPR / Cas13a activation by increasing the average droplet RNA concentration. Furthermore, by collecting the fluorescent groups generated by the enzyme digestion products in the microdroplets, concentrated detection of the fluorescence signal can be achieved.Furthermore, by improving the signal amplification process and detection equipment, the SATORI method has the potential to enhance the detection capability of target RNA. Cas13 is a nucleic acid detection platform with aM sensitivity and single-base specificity, suitable for developing multifunctional, rapid, convenient, and cost-effective nucleic acid detection technologies. This is crucial for point-of-care testing, epidemiological surveillance, and pathogen detection in environments with limited infrastructure. Summary of the Invention

[0011] The purpose of this invention is to provide a detection method for cristatitis cerevisiae (CrERV) based on CRISPR-Cas13a and transverse chromatography test strip visualization, thereby addressing the problems existing in the prior art. This invention combines RPA-CRISPR Cas13a to develop a detection system that proposes a rapid method suitable for clinical cristatitis cerevisiae detection at the grassroots level. This method utilizes three approaches to read the results: acquiring fluorescence signals, ultraviolet excitation for visible fluorescence, and test strip visualization. It also provides an empirical reference for the diagnostic research of other bacterial and viral diseases.

[0012] To achieve the above objectives, the present invention provides the following solution: One of the technical solutions of this invention is an RPA-CRISPR / Cas13a-LFA system for visually detecting loach rhabdovirus, comprising RPA primers, Cas13a protein and crRNA; The RPA primers include primers as shown in SEQ ID NO.4 and SEQ ID NO.5; The sequence of the crRNA is shown in SEQ ID NO.6.

[0013] The second technical solution of the present invention is a method for detecting loach bullet virus for non-disease detection or treatment purposes, which uses the RPA-CRISPR / Cas13a-LFA system to detect the sample to be tested and determine whether it contains loach bullet virus.

[0014] The third technical solution of the present invention is a reagent kit for visually detecting loach rhabdovirus, comprising the RPA-CRISPR / Cas13a-LFA system.

[0015] Based on the above technical solution, the present invention has the following technical effects: This invention provides a method for detecting loach rhabdovirus based on CRISPR-CAS13a and lateral chromatography test strip visualization. The method exhibits good specificity, high sensitivity, and a detection limit of 1.14 × 10⁻⁶. 1 Copies offer better specificity and compatibility, lower detection costs, and are convenient and quick to operate. Attached Figure Description

[0016] Figure 1 This is a flowchart of the experiment.

[0017] Figure 2 For RPA screening. Where M represents Marker, 1 represents a negative result for the first RPA primer pair, 2 represents a negative result for the first RPA primer pair, 3 represents a negative result for the second RPA primer pair, 4 represents a negative result for the second RPA primer pair, 5 represents a negative result for the third RPA primer pair, 6 represents a negative result for the third RPA primer pair, 7 represents a negative result for the fourth RPA primer pair, 8 represents a negative result for the fourth RPA primer pair, 9 represents a negative result for the fifth RPA primer pair, and 10 represents a negative result for the fifth RPA primer pair.

[0018] Figure 3 The system was established. Here, A represents the temperature-controlled fluorescence value, B represents the results of the temperature-controlled test strip and UV spectrophotometer, and C represents the relationship between T7 and mg2. ＋ Proportional fluorescence value, D represents T7 to mg 2＋ The results of the ratio test strip and UV spectrophotometer are shown. E represents the fluorescence value of the ratio of LwaCas13a Nuclease to CrRNA to probe, and F represents the results of the ratio test strip and UV spectrophotometer for LwaCas13a Nuclease to CrRNA to probe.

[0019] Figure 4 These refer to the various forms that the test strips may appear in. Among them, A: fluorescence value measured by RPA-CRISPR / Cas13a-Flu fluorescence quantitative instrument; B: detection by RPA-CRISPR / Cas13a-LFA test strip; C: final fluorescence value of RPA-CRISPR / Cas13a-Flu; D: fluorescence color development under ultraviolet spectrophotometry.

[0020] Figure 5 The results are from sensitivity experiments. A: Fluorescence value measured using an RPA-CRISPR / Cas13a-Flu fluorescence quantitative analyzer; B: Final fluorescence value of RPA-CRISPR / Cas13a-Flu; C: Detection using an RPA-CRISPR / Cas13a-LFA test strip; D: Fluorescence color development under a UV spectrophotometer.

[0021] Figure 6 The results are from sensitivity experiments. A: Fluorescence value measured using an RPA-CRISPR / Cas13a-Flu fluorescence quantitative analyzer; B: Final fluorescence value; C: Detection using an RPA-CRISPR / Cas13a-LFA test strip; D: Fluorescence color development under a UV spectrophotometer.

[0022] Figure 7These are specific experimental results. A: Fluorescence value measured using an RPA-CRISPR / Cas13a-Flu fluorescence quantitative analyzer; B: Final fluorescence value; C: Detection using an RPA-CRISPR / Cas13a-LFA test strip; D: Fluorescence color development under ultraviolet spectrophotometry.

[0023] Figure 8 These are the results of clinical testing experiments. A: RPA-CRISPR / Cas13a-LFA test strip detection; B: Fluorescence color development under UV spectrophotometry; C: qRCR method detection; D: Fluorescence value measured by RPA-CRISPR / Cas13a-Flu fluorescence quantitative instrument; E: Comparison of the three detection methods; F: Comparison of tissue samples (liver, spleen, kidney, intestine) detected by the three detection methods.

[0024] Figure 9 In the above, A: fluorescence value measured by RPA-CRISPR / Cas13a-Flu fluorescence quantitative instrument; B: detection by qRCR method; C: detection by RPA-CRISPR / Cas13a-LFA test strip; D: fluorescence color development under ultraviolet spectrophotometer. Detailed Implementation

[0025] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0026] This invention provides an RPA-CRISPR / Cas13a-LFA system for visually detecting loach rhabdovirus, comprising RPA primers, Cas13a protein, and crRNA; The RPA primers include primers as shown in SEQ ID NO.4 and SEQ ID NO.5; The sequence of the crRNA is shown in SEQ ID NO.6.

[0027] In some specific implementations, the RPA-CRISPR / Cas13a-LFA system comprises: one RT-RMA basal type lyophilized microsphere (DNA / RNA polymerase, dNTP, Mg... 2+ (etc.), 2 µl RPA primers, 1 µl activator (magnesium ions), 4 µl sample to be tested, 2 µl LwaCas13a Nuclease (10 µm), 4 µl CrRNA (10 µm), 2 µl HOLMES-LFA ssRNA reporter 1 (FAM / Biotin) (20 µm), 2 µl 10×HOLMES Buffer for Cas13a, 1M Mg 2+1.5 μl, TranscriptMax Enzyme Mix 1 μl, NTP mix 4 μl, ddH2O to make up to 30 μl.

[0028] This invention also provides a method for detecting loach rhabdovirus for non-disease detection or treatment purposes, which uses the RPA-CRISPR / Cas13a-LFA system to detect the sample to be tested and determine whether it contains loach rhabdovirus.

[0029] In some specific implementations, the conditions for testing the sample are as follows: after incubation at 37°C for 1.5 minutes, the sample is tested using a visual test strip (LFS).

[0030] In some specific implementation schemes, the method for determining whether the eel contains a rhabdovirus is as follows: 1. Operating Steps a) Take the required number of test strips (CRISPR single-system lateral chromatography test strips, with two bands: a lower control line (C line) and an upper detection line (T line). The C line is coated with avidin (SA), and the T line is coated with goat anti-mouse secondary antibody. The colloidal gold is labeled with anti-FITC / FAM monoclonal antibody. A complete CRISPR system probe (labeled with biotin at one end and FAM or FITC at the other) allows the colloidal gold to be completely captured at the C line. When a probe is cleaved by Cas enzyme, the colloidal gold bound to the cleaved fragment cannot be captured by the C line, forming the T line. The presence or absence of the T line indicates whether Cas enzyme has been activated), and label them accordingly.

[0031] b) Add 50 µL of the CRISPR reaction product or 50 µL of the diluted product to the PCR tube.

[0032] c) Insert the test strip with the sample pad end facing down, and leave it at room temperature for 5-10 minutes before interpreting the results.

[0033] d) After use, put the amplification product and test strip into a sealed bag and dispose of them properly.

[0034] 2. Result Interpretation a) Positive (+): A positive result is indicated by the C line showing color and the T line being visible to the naked eye, meaning that some of the nucleic acid probe has been cleaved by the Cas enzyme. If the C line is not colored and the T line is visible to the naked eye, it is considered positive, indicating that the nucleic acid probe has been almost completely cleaved by the Cas enzyme.

[0035] b) Negative (-): The C line shows color, while the T line does not, indicating that the nucleic acid probe has not been cleaved by the Cas enzyme.

[0036] c) Invalid: If neither the C nor T line shows color, it indicates an operational error or that the test strip has deteriorated and become ineffective. In this case, you should carefully read the instructions again and retest.

[0037] This invention also provides a kit for visually detecting loach rhabdovirus, comprising the RPA-CRISPR / Cas13a-LFA system. Example 1 1. Extraction of virus, clinical samples, and RNA, and preparation of cDNA The cristatin (CrERV) isolate from the swamp eel (GenBank accession number: PV185937, named strain XY0907) obtained in our laboratory was stored in the Pathogenic Microbiology Laboratory of the College of Animal Science, Yangtze University. Five liver, five kidney, five spleen, five intestine, and 24 mixed samples were collected from the aquatic product market. Both the virus and the samples were stored at -80℃ for subsequent experiments.

[0038] DNA / RNA Nucleic Acid Extraction Kit (Qingdao Lijian Biotechnology Co., Ltd., Shandong, China) extracts RNA from clinical samples (clinical samples obtained from Hubei Geshuo Biotechnology Co., Ltd.) and stores the obtained RNA at -80℃.

[0039] 2. Preparation of standard plasmids RNA was reverse transcribed into cDNA using the HiScript II 1st Strand cDNA Synthesis Kit (Vazyme, China). The obtained cDNA was then amplified by PCR using CrERV-specific primers (CrERV-F, CrERV-R). The amplified product was purified and ligated with the pMD-18T vector to construct a recombinant plasmid. Using the CrERV plasmid as the detection target, the purified plasmid was transformed into competent cells, cultured in solid medium containing ampicillin, and sequenced.

[0040] 3. RPA primer and crRNA design The CrERV N gene sequence (GenBank accession number: PV185937) obtained from NCBI was used as a reference to design 5 pairs of RPA primers (Table 1). The N gene is a conserved gene of CrERV, which can reduce the possibility of gene sequence mutations during experiments. The RPA primers should follow the design principles of the TranscriptMax T7 High Yield RNA Synthesis Kit (Tulu Harbor), with a T7 promoter added to the 5' end of the forward primer. Primer sets with high specificity were selected for experiments, and the coverage regions were screened. The regional sequences were provided to Boles Biotechnology Co., Ltd. for CrRNA design. Based on the different requirements of fluorescence and test strips, two types of probes were designed in this experiment: a reporter group for fluorescence (FAM-UUUUUU-BHQ1) and a reporter group for test strips (FAM-AAUUUUUUAAUU-Biotin).

[0041] HOLMES-Fluo ssRNA reporter 1 (FAM) is a single-stranded RNA oligonucleotide probe, labeled with a FAM fluorescent reporter group at the 5' end and a fluorescent quencher group at the 3' end. When the HOLMES ssRNA reporter probe is intact, the fluorescent signal emitted by the reporter group is quenched by the quencher group. When the HOLMES ssRNA reporter probe is cleaved, the FAM fluorescent reporter group and the quencher group at the 5' end separate, thereby emitting a detectable fluorescent signal. The common Cas13 enzyme, after forming a ternary complex with guide RNA and target RNA, is excited to exhibit trans-cleavage activity (or bypass cleavage activity) against ssRNA, cleaving the HOLMES ssRNA reporter probe and emitting a fluorescent signal.

[0042] Table 1 .

[0043] 4. RPA Response and Screening RPA reactions were performed strictly according to the Tolo Basic RT-RMA Kit (Lyophilized Microbeads) requirements. Primer length was 30-35 bp. The system consisted of one RT-RMA basic lyophilized microbead (DNA / RNA polymerase, dNTPs, Mg... 2+ Add 1 µl of RPA-F, 1 µl of RPA-R, 4 µl of template, 1 µl of activator (magnesium ions), and H2O to a final volume of 25 µl. Incubate at 37 °C for 25 minutes.

[0044] Five primer pairs were tested individually, and the best primers were selected. The RPA primer screening results showed that the second RPA primer pair was the optimal one.

[0045] 5. One-tube RPA-CRISPR / Cas13a-Flu RPA amplification, T7 transcription, and the CRISPR / Cas13a system were combined in the experiment, and LFS was detected by fluorescence and visualization strip test. A single-tube method was used for detection; the reaction system consisted of one RT-RMA basal type lyophilized microsphere (DNA / RNA polymerase, dNTPs, Mg2+). 2+ (etc.), 1 µl RPA-F, 1 µl RPA-R, 1 µl activator (magnesium ions), 4 µl template, 2 µl LwaCas13a Nuclease (10 μm), 4 µl CrRNA (10 μm), 2 µl HOLMES-LFA ssRNA reporter 1 (FAM / Biotin) (20 μm), 2 µl 10×HOLMES Buffer for Cas13a, 1M Mg 2+ Add 1.5 μl of TranscriptMax Enzyme Mix, 1 μl of NTP mix, and ddH2O to a final volume of 30 μl. Incubate the mixture at 37°C for 1 h, and then detect fluorescence every 30 s using a quantitative fluorescence analyzer. When the HOLMES ssRNA reporter probe is intact, the fluorescence signal emitted by the reporter group is quenched by the quenching group. When the HOLMES ssRNA reporter probe is cleaved, the 5' end FAM fluorescent reporter group and the quenching group separate, thus emitting a detectable fluorescence signal. Common Cas13 enzymes, after forming a ternary complex with guide RNA and target RNA, are excited to exhibit trans-cleavage activity (or bypass cleavage activity) against ssRNA, cleaving the HOLMES ssRNA reporter probe and emitting a fluorescence signal.

[0046] First, set the "positive signal threshold": use the highest fluorescence signal of the negative control as a benchmark, multiply it by 2-3 times to obtain the judgment threshold.

[0047] Key judgment criteria: If the sample fluorescence signal exceeds the set threshold and the curve shows a typical specific pattern of "rapid rise - stable plateau" → virus is present (positive).

[0048] If the sample fluorescence signal is consistently below the threshold, or the curve shows no significant increase and only approaches the baseline, then there is no virus (negative).

[0049] If the signal is close to the threshold (within ±10%), or the curve rises slowly without a clear plateau period, it is suspicious (re-examination is required).

[0050] 6. One-tube RPA-CRISPR / Cas13a-LFA RPA amplification, T7 transcription, and the CRISPR / Cas13a system were tested together, and the results were detected using fluorescence and visual test strips (LFS). The CRISPR single-system lateral chromatography test strip has two bands: a lower control line (C line) and an upper detection line (T line). The C line is coated with avidin (SA), and the T line is coated with goat anti-mouse secondary antibody. Anti-FITC / FAM monoclonal antibody is labeled on the colloidal gold. A complete CRISPR system probe (labeled with biotin at one end and FAM or FITC at the other) allows the colloidal gold to be completely captured at the C line. When a probe is cleaved by the Cas enzyme, the colloidal gold bound to the cleaved fragment cannot be captured by the C line, forming the T line. The presence or absence of the T line indicates whether the Cas enzyme has been activated.

[0051] The single-tube method was used for detection. The reaction system consisted of one RT-RMA basic type lyophilized microsphere (DNA / RNA polymerase, dNTPs, Mg). 2+ The following ingredients were purchased from Tolo Biotechnology Co., Ltd.: 1 µl RPA-F, 1 µl RPA-R, 1 µl activator (magnesium ions), 4 µl template, 2 µl LwaCas13a Nuclease (10 µm), 4 µl CrRNA (10 µm), 2 µl HOLMES-LFA ssRNAreporter 1 (FAM / Biotin) (20 µm), 2 µl 10×HOLMES Buffer for Cas13a, and 1M Mg 2+ Add 1.5 μl of TranscriptMax Enzyme Mix, 1 μl of NTP mix, and 4 μl of ddH2O to a final volume of 30 μl. Incubate the mixture at 37°C for 1.5 minutes, then detect using a visual test strip (LFS).

[0052] Judgment criteria: a) Positive (+): A positive result is indicated by the C line showing color and the T line being visible to the naked eye, meaning that some of the nucleic acid probe has been cleaved by the Cas enzyme. If the C line is not colored and the T line is visible to the naked eye, it is considered positive, indicating that the nucleic acid probe has been almost completely cleaved by the Cas enzyme.

[0053] b) Negative (-): The C line shows color, while the T line does not, indicating that the nucleic acid probe has not been cleaved by the Cas enzyme.

[0054] c) Invalid: If neither the C nor T line shows color, it indicates an operational error or that the test strip has deteriorated and become ineffective. In this case, you should carefully read the instructions again and retest.

[0055] 7. Sensitivity and Specificity 7.1 Sensitivity test of the RPA-CRISPR / Cas13a detection method The CrERV plasmid was serially diluted 1.14 × 10⁻⁶. 9 -1.14×10 0 The limit of detection (LoD) was determined by measuring copies / μL. Using plasmids of different dilutions as templates, the products were amplified via RPA reaction, and the sensitivity of the RPA-CRISPR / Cas13a method was analyzed. The established two-step RPA-CRISPR / Cas13a method (using RPA to amplify CrERV DNA, combining with T7 transcription to transcribe the amplified DNA into RNA, and then using Cas13a to recognize the target, activating its associated cleavage activity to cleave the RNA reporter molecule, thereby amplifying the signal) was compared with a quantitative fluorescence method.

[0056] 7.2 Specificity test of RPA-CRISPR / Cas13a detection method To further determine the specificity of the established RPA-CRISPR / Cas13a detection method, seven common aquatic pathogens were tested. These pathogens included Aeromonas hydrophila, Aeromonas sobria, Aeromonas veronii, swamp eel rhabdovirus (CrERV), mandarin fish rhabdovirus (SCRV), largemouth bass rhabdovirus (MSRV), and swamp eel infectious hemorrhagic syndrome virus (IHSV).

[0057] 8. Clinical testing Fish samples showing obvious signs of disease were collected (24 samples were obtained from Hubei Geshuo Biotechnology Co., Ltd.) and pretreated. The CRISPR / Cas13 detection system established in this invention was compared with the conventional CrERV detection method (qPCR) used in the laboratory. Samples already confirmed to be virus-free were selected as simulants (6 positive and 4 negative), and the CRISPR / Cas13 detection system established in this invention was compared with the conventional CrERV detection method (qPCR) used in the laboratory.

[0058] 9. Experimental Results 9.1 Adjustments were performed at 36, 27, 38, and 39℃. Results showed that 37℃ yielded the highest amplification efficiency, indicating it was the optimal temperature. Adjustments were also made to T7 and Mg...2+ The ratio was set with three gradients (1:1, 1:1.5, 1:2). The results showed that 1:1.5 was optimal, so the amount of T7 added was determined to be 1 μl, and Mg... 2+ The initial addition volume was 1.5 μl. The optimal addition ratios of Cas enzyme, CrRNA, and fluorescent reporter group were adjusted (1:1:1, 1:2:1, 1:2:2, 2:1:1, 2:2:1). The results showed that 1:2:2 was optimal, and the final addition volume was determined to be 2 μl of Cas enzyme (10 μm), 4 μl of CrRNA (10 μm), and 2 μl of FQ (20 μm). The final system consisted of one RT-RMA basic lyophilized microsphere (DNA / RNA polymerase, dNTPs, Mg...). 2+ The following ingredients were added: 1 μl each of primers (upper and lower), 1 μl of activator (magnesium ions), 4 μl of template, 2 μl of Cas enzyme (10 μm), 4 μl of CrRNA (10 μm), 2 μl of FQ (20 μm), 2 μl of 10× buffer, and Mg2+. ＋ Add 1.5 μl of T7, 1 μl of NTP, and ddH2O to a final volume of 30 μl.

[0059] 9.2 The final sensitivity of the two-step method is: 1.14 × 10⁻⁶ 0 copies, such as Figure 5 As shown. One-step sensitivity: 1.14 × 10⁻⁶ 1 copies, such as Figure 6 As shown.

[0060] 9.3 Specificity test results show that this detection method is only positive when detecting loach rhabdovirus, indicating good specificity.

[0061] 9.4 Clinical testing results showed that the positive detection rates of RPA-CRISPR / Cas13a-Flu and RPA-CRISPR / Cas13a-LFA were the same, slightly lower than those of the qPCR method. (See [link to relevant documentation]). Figure 8 .

[0062] 9.5 Simulated sample test results show that the positive detection rates of the three detection methods are the same.

[0063] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. An RPA-CRISPR / Cas13a-LFA system for visually detecting loach rhabdovirus, characterized in that, Including RPA primers, Cas13a protein, and crRNA; The RPA primers include primers as shown in SEQ ID NO.4 and SEQ ID NO.5; The sequence of the crRNA is shown in SEQ ID NO.

6.

2. The RPA-CRISPR / Cas13a-LFA system according to claim 1, characterized in that, The RPA-CRISPR / Cas13a-LFA system comprises: 1 RT-RMA basic lyophilized microsphere, 2 µl RPA primers, 1 µl magnesium ions as activator, 4 µl of the sample to be tested, 2 µl of LwaCas13a Nuclease (10 µm), 4 µl of CrRNA (10 µm), 2 µl of HOLMES-LFA ssRNA reporter1 (FAM / Biotin) (20 µm), 2 µl of 10×HOLMES Buffer for Cas13a, and 1 M Mg 2+ 1.5 μl, 1 μl of TranscriptMax Enzyme Mix and 4 μl of NTP mix, and ddH2O to make up to 30 μl.

3. A method for detecting loach rhabdovirus for non-disease detection or treatment purposes, characterized in that, The RPA-CRISPR / Cas13a-LFA system described in claim 1 or 2 is used to detect the sample to be tested to determine whether it contains the loach bullet virus.

4. The detection method according to claim 3, characterized in that, The conditions for testing the sample are as follows: after incubation at 37°C for 1.5 hours, the sample is tested using the Visualized Test Strip (LFS).

5. A reagent kit for visually detecting loach rhabdovirus, characterized in that, Includes the RPA-CRISPR / Cas13a-LFA system as described in claim 1 or 2.

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