A kit for detecting mandarin frog iridovirus and infectious spleen and kidney necrosis virus, as well as a method and application thereof

Abstract

The present invention belongs to the field of biotechnology, and in particular to a kit for detecting mandarin frog iridescent virus and infectious spleen and kidney necrosis virus, as well as its method and application. The present invention combines RT-RPA technology with the CRISPR / Cas system, designs RPA primers and crRNA for the MCP genes of MRV and ISKNV viruses, and successfully establishes a rapid detection method for MRV and ISKNV based on RPA-CRISPR / Cas12a, which can detect templates as low as 1 copy per μL (MRV) and 0.1 copies per μL (ISKNV), with good specificity and repeatability, and can react at a constant temperature of 37-39°C without the need for complex instruments and equipment. The RT-RPA reaction system designed by the present invention can reverse transcribe RNA, so the mRNA transcribed from the MCP genes of MRV and ISKNV viruses in the sample will also be detected, further improving the detection rate.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a kit for detecting mandarin frog iridovirus and infectious spleen and kidney necrosis virus, as well as a method and application thereof. Background Art

[0002] Iridoviridae are large, icosahedral, cytoplasmic, double-stranded DNA viruses that are among the most prevalent and pathogenic viral pathogens in farmed fish. According to the latest report of the International Committee on Taxonomy of Viruses (https: / / ictv.global / taxonomy), Iridoviridae are primarily divided into two subfamilies, α and β, encompassing 22 species in seven genera. Among these, two species that pose a significant threat to mandarin fish (Siniperca chuatsi) aquaculture are Mandarin fishranavirus (MRV) and infectious spleen and kidney necrosisvirus (ISKNV).

[0003] MRV belongs to the genus Ranavirus, within the subfamily Alpha. Ranaviruses have a wide host range, causing systemic infection targeting the kidneys, liver, and gastrointestinal tract. The virus is transmitted through ingestion, parenteral injection, direct contact, or environmental exposure. Acute infection with Ranavirus can result in morbidity and mortality rates of up to 100%, significantly impacting fish, reptiles, and amphibians, particularly farmed fish, resulting in significant economic losses. MRV, first discovered and classified in 2013, is an emerging pathogen in mandarin fish aquaculture, causing mass mortalities in natural populations of adult mandarin fish. Studies have also shown a 100% mortality rate in hybrid mandarin fish juveniles within five days of infection.

[0004] ISKNV belongs to the genus Isovirus of the α subfamily. In 1997, an outbreak of virus-infected mandarin fish diseases occurred in various breeding grounds in Guangdong. A spherical virus was first discovered in the spleen of the diseased mandarin fish. It was subsequently confirmed that ISKNV was the pathogen causing the outbreak of infectious diseases in mandarin fish, and that the spleen and kidneys were the main target organs, causing swelling and necrosis of the spleen and kidney cells of mandarin fish. Therefore, the virus was named infectious spleen and kidney necrosis virus.

[0005] Currently, the detection and identification of these two viruses are commonly performed using methods such as nested PCR, fluorescent quantitative PCR, and microscopy. Nested PCR requires two rounds of PCR cycles and a final electrophoresis test, which is time-consuming. Fluorescent quantitative PCR, on the other hand, requires an expensive fluorescent quantitative PCR instrument. Conventional optical microscopy can only provide a preliminary assessment of viral infection; definitive identification of the virus requires purified virus and an electron microscope. Therefore, there is a need for a simpler, less demanding detection method that is convenient for on-site operation and allows for visual interpretation. Summary of the Invention

[0006] The first aspect of the present invention aims to provide a primer set.

[0007] The second aspect of the present invention aims to provide a reagent.

[0008] The third aspect of the present invention is to provide a kit.

[0009] The fourth aspect of the present invention aims to provide applications of the primer set of the first aspect of the present invention, and / or the reagent of the second aspect of the present invention, and / or the kit of the third aspect of the present invention.

[0010] The fifth aspect of the present invention aims to provide a method for detecting Siniperca chuatsi iridovirus and / or infectious spleen and kidney necrosis virus for non-diagnostic purposes.

[0011] In order to achieve the above-mentioned purpose of the present invention, the technical solution adopted by the present invention is:

[0012] A first aspect of the present invention provides a primer set comprising a primer pair for detecting Rana chuatsi iridovirus and / or a primer pair for detecting infectious spleen and kidney necrosis virus.

[0013] Preferably, the primer pair for detecting the mandarin frog iridescent virus comprises a forward primer for amplifying the mandarin frog iridescent virus and a reverse primer for amplifying the mandarin frog iridescent virus.

[0014] Preferably, the forward primer for amplifying the Rana chuatsi iridovirus comprises a nucleic acid sequence comprising at least one of SEQ ID NOs: 5 to 7.

[0015] Preferably, the reverse primer for amplifying the Rana chuatsi iridovirus comprises a nucleic acid sequence comprising at least one of SEQ ID NOs: 8 to 10.

[0016] Preferably, the primer pair for amplifying infectious spleen and kidney necrosis virus comprises a forward primer for amplifying infectious spleen and kidney necrosis virus and a reverse primer for amplifying infectious spleen and kidney necrosis virus.

[0017] Preferably, the forward primer for amplifying infectious spleen and kidney necrosis virus comprises at least one of SEQ ID NOs: 11-13.

[0018] Preferably, the reverse primer for amplifying infectious spleen and kidney necrosis virus comprises at least one of SEQ ID NOs: 14-16.

[0019] Preferably, the nucleic acid sequences of the primer set for amplifying Rana chuatsi iridovirus are shown in SEQ ID NOs: 5 and 8.

[0020] Preferably, the nucleic acid sequences of the primer set for amplifying infectious spleen and kidney necrosis virus are shown in SEQ ID NOs: 13 and 14.

[0021] The second aspect of the present invention provides a reagent comprising the primer set of the first aspect of the present invention, and crRNA for detecting Rana chuatsi iridovirus and / or crRNA for detecting infectious spleen and kidney necrosis virus.

[0022] Preferably, the crRNA sequence for detecting Rana chuatsi iridovirus comprises at least one of SEQ ID NOs: 17 to 21;

[0023] Preferably, the crRNA sequence for detecting infectious spleen and kidney necrosis virus includes at least one of SEQ ID NOs: 22 to 25.

[0024] Preferably, the crRNA sequence for detecting Rana chuatsi iridovirus includes SEQ ID NO: 18.

[0025] Preferably, the crRNA sequence for detecting infectious spleen and kidney necrosis virus includes at least one of SEQ ID NOs: 23 to 25; further preferably, it is SEQ ID NO: 23.

[0026] The third aspect of the present invention provides a kit comprising the reagent of the second aspect of the present invention.

[0027] Preferably, the kit further comprises a fluorescent probe.

[0028] Preferably, the fluorescent probe comprises a nucleic acid sequence, the 5' end of the nucleic acid sequence is labeled with a fluorescent reporter group, and the 3' end of the nucleic acid sequence is labeled with a quencher group;

[0029] Preferably, the fluorescent group includes at least one of FAM, ROX, HEX, CY3, and CY5;

[0030] Preferably, the quenching group includes at least one of BHQ1, BHQ2, and BHQ3.

[0031] Preferably, the kit further comprises a Cas protein.

[0032] Preferably, the Cas protein is Cas12a.

[0033] Preferably, the kit further comprises an RPA enzyme preparation.

[0034] Preferably, the RPA enzyme preparation comprises recombinase, recombinase loading factor, single-strand binding protein, and strand-displacing DNA polymerase.

[0035] Preferably, the RPA enzyme preparation may further include reverse transcriptase.

[0036] Preferably, the kit further comprises RPA buffer, Mg 2+ , CRISPR reaction buffer and DEPC water.

[0037] In some embodiments of the present invention, the fluorescent probe comprises:

[0038] Probe 1: 5′-FAM-TTATT-BHQ1-3′;

[0039] Probe 2: 5′-HEX-TTATT-BHQ1-3′;

[0040] Probe 3: 5′-CY3-TTATT-BHQ2-3′;

[0041] Probe 4: 5′-ROX-TTATT-BHQ2-3′;

[0042] Probe 5: 5′-CY5-TTATT-BHQ3-3′.

[0043] Preferably, the fluorescent probe is at least one of probe 1 and probe 4.

[0044] The fourth aspect of the present invention provides the use of the primer set of the first aspect of the present invention, and / or the reagent of the second aspect of the present invention, and / or the kit of the third aspect of the present invention in any one of a1) to a3):

[0045] a1) preparing products for detecting Rana chuatsi iridovirus;

[0046] a2) application in the preparation of a product for screening subjects infected with Rana chuatsi iridovirus;

[0047] a3) Detection of Rana chuatsi iridovirus for non-diagnostic purposes;

[0048] a4) preparing products for detecting infectious spleen and kidney necrosis virus;

[0049] a5) application in the preparation of a product for screening subjects infected with infectious spleen and kidney necrosis virus;

[0050] a6) Detection of infectious splenorenal necrosis virus for non-diagnostic purposes.

[0051] Preferably, the infected object includes at least one of fish, frogs, shrimps, crabs, shellfish and turtles.

[0052] A fifth aspect of the present invention provides a method for detecting Siniperca chuatsi iridovirus and / or infectious spleen and kidney necrosis virus for non-diagnostic purposes.

[0053] The detection method comprises the following steps:

[0054] b1) Sample extraction: Take the sample to be tested and extract nucleic acid;

[0055] b2) RPA amplification: The primer set described in the third aspect of the present invention, RT-RPA enzyme preparation, RPA buffer, Mg 2+ , DEPC water is mixed with the nucleic acid extracted in step b1) and subjected to RPA amplification;

[0056] b3) CRISPR detection: The crRNA, Cas protein, fluorescent probe, CRISPR reaction buffer, and DEPC water described in the third aspect of the present invention are mixed with the amplified product obtained in step b2) to perform CRISPR reaction detection and determine the fluorescence change result.

[0057] In some embodiments of the present invention, the method for extracting nucleic acid comprises the following steps:

[0058] Take a pea-sized sample and place it in a 2 mL centrifuge tube. Add 1 mL of PBS or normal saline and homogenize it with a homogenizer until there are no obvious tissue pieces. Centrifuge briefly and collect the supernatant.

[0059] Subsequent processing methods include precipitation, magnetic bead method, column extraction method and automatic nucleic acid purification instrument method to extract DNA / RNA; simplified nucleic acid release schemes can also be used on site where equipment is lacking, including the use of rapid nucleic acid release agent treatment, high-temperature lysis method and proteinase K treatment.

[0060] In some embodiments of the present invention, the RPA reaction system is: RT-RPA buffer, forward primer, reverse primer, DEPC water, RT-RPA enzyme lyophilized powder, magnesium acetate initiator and DNA / RNA of the sample to be tested; more preferably, the reaction system of the RT-RPA reaction is a 50 μL system: 25 μL RT-RPA buffer, 2 μL 10 μM forward primer, 2 μL 10 μM reverse primer, 1 portion of RT-RPA enzyme lyophilized powder, DNA / RNA template of the sample to be tested plus DEPC water for a total of 18 μL (the dilution ratio is adjusted according to the template concentration) and 3 μL of magnesium acetate initiator; the RT-RPA buffer, RT-RPA enzyme lyophilized powder and magnesium acetate initiator were purchased from Le Shang Biotechnology (Wuxi) Co., Ltd.

[0061] In some embodiments of the present invention, magnesium acetate is added to the system last as a reaction initiator to control the start of the reaction; the reaction temperature of the RPA is 39°C, and the reaction time is 20-40 minutes; more preferably, the reaction time is 30 minutes; 5 minutes after the start of the reaction, the reaction unit is removed, gently flicked to mix, and briefly centrifuged, and then returned to the constant temperature instrument to continue the reaction.

[0062] In some embodiments of the present invention, the reaction system of the CRISPR / Cas12a reaction is: CRISPR reaction buffer, Cas12a protein, crRNA, fluorescent probe, RPA reaction product and DEPC water; more preferably, the reaction system of the CRISPR / Cas12a reaction is a 20 μL system: 2 μL of CRISPR reaction buffer, 0.5 μL of 10 μM Cas12a protein, 1 μL of 10 μM crRNA, 1 μL of 10 μM fluorescent probe, 2-10 μL of RPA reaction product, and 20 μL of DEPC water; wherein the final concentration of the Cas protein is 0.05-0.25 μM, preferably 0.25 μM; the final concentration of the crRNA is 0.05-0.5 μM, preferably 0.5 μM; the final concentration of the fluorescent probe is 0.125-10 μM.

[0063] In some embodiments of the present invention, the CRISPR / Cas12a reaction temperature is 37° C., and the reaction time is 15-60 min; more preferably, the reaction time is 15 min.

[0064] In some embodiments of the present invention, the method for determining the fluorescence change result is:

[0065] (1) When using probe 1, 2, 3, or 4, observe through an orange filter under blue light or directly observe under ultraviolet light. Prepare a negative control at the same time. If the fluorescence intensity is greater than the negative control, it is positive; otherwise, it is negative. Preferably, a blue light gel cutting instrument and ultraviolet imaging system can be used; or

[0066] (2) When using probe 1, 2, 3, 4, or 5, use a fluorescence quantitative instrument to detect and prepare a negative control at the same time. Compare the fluorescence intensity of the sample to be tested and the negative control at the same time. If the fluorescence intensity of the sample to be tested is greater than that of the negative control, it is positive, otherwise it is negative; or

[0067] (3) When high concentration (final concentration 10 μM) of probe 1 or probe 4 was used, the color change was observed directly with the naked eye.

[0068] The negative control treatment method is to replace the sample to be tested with DEPC water, and completely synchronize the treatment method of the sample to be tested from the start of the RT-RPA reaction.

[0069] The beneficial effects of the present invention are:

[0070] The present invention combines RT-RPA technology with the CRISPR / Cas system, designs RPA primers and crRNA for the MCP genes of MRV and ISKNV viruses, and successfully establishes a rapid detection method for mandarin frog iridescent virus and infectious spleen and kidney necrosis virus based on RPA-CRISPR / Cas12a. This method can detect templates as low as 1 copy per μL (MRV) and 0.1 copies per μL (ISKNV), with good specificity and repeatability. Moreover, the method can react at a constant temperature of 37-39°C without the use of complex instruments and equipment. The RT-RPA reaction system designed by the present invention can reverse transcribe RNA, so the mRNA transcribed from the MCP genes of MRV and ISKNV viruses in the sample will also be detected, further improving the detection rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] Figure 1is the experimental result of RT-RPA primer screening, where A is the result of MRV amplification experiment, 1 represents MRV-RPA-F1 / MRV-RPA-R1, 2 represents MRV-RPA-F1 / MRV-RPA-R2, 3 represents MRV-RPA-F1 / MRV-RPA-R3, 4 represents MRV-RPA-F2 / MRV-RPA-R1, 5 represents MRV-RPA-F2 / MRV-RPA-R2, 6 represents MRV-RPA-F2 / MRV-RPA-R3, 7 represents MRV-RPA-F3 / MRV-RPA-R1, 8 represents MRV-RPA-F3 / MRV-RPA-R2, and 9 represents MRV-RPA-F3 / MRV-RPA-R3, + represents positive, and - represents negative; B represents the result of ISKNV amplification experiment Test results, where 1 represents ISKNV-RPA-F1 / ISKNV-RPA-R1, 2 represents ISKNV-RPA-F1 / ISKNV-RPA-R2, 3 represents ISKNV-RPA-F1 / ISKNV-RPA-R3, 4 represents ISKNV-RPA-F2 / ISKNV-RPA-R1, 5 represents ISKNV-RPA-F2 / ISKNV-RPA-R2, 6 represents ISKNV-RPA-F2 / ISKNV-RPA-R3, 7 represents ISKNV-RPA-F3 / ISKNV-RPA-R1, 8 represents ISKNV-RPA-F3 / ISKNV-RPA-R2, and 9 represents ISKNV-RPA-F3 / ISKNV-RPA-R3, + represents positive, and - represents negative.

[0072] Figure 2 Figure 1 is the experimental result of crRNA screening, where A is the crRNA screening experimental result of MRV; B is the crRNA screening experimental result of ISKNV.

[0073] Figure 3 These are the results of the CRISPR / Cas reaction system Cas12a protein and crRNA dosage optimization experiment, where A is the MRV reaction system optimization experiment result; B is the ISKNV reaction system optimization experiment result.

[0074] Figure 4 Figure 1 is a graph showing the results of an experiment to optimize the amount of fluorescent probes used in the CRISPR / Cas reaction system. Figure 1 shows ...

[0075] Figure 5 Figure 1 is the experimental result of the system detecting RNA targets, where A is the result of the MRV detection experiment; B is the result of the ISKNV detection experiment.

[0076] Figure 6Figure 1 is the result of the threshold experiment, where A is the result of the MRV detection threshold experiment; B is the result of the ISKNV detection threshold experiment.

[0077] Figure 7 Figure 1 is the result of the sensitivity test, where A and C are the results of the MRV sensitivity test; B and D are the results of the ISKNV sensitivity test.

[0078] Figure 8 It is the result diagram of the specificity experiment, among which A is the result of the MRV specificity test experiment; B is the result of the ISKNV specificity test experiment.

[0079] Figure 9 It is the result diagram of the repeatability experiment, where A is the result of the MRV repeatability test experiment; B is the result of the ISKNV repeatability test experiment.

[0080] Figure 10 This is the result of a probe optimization experiment without the need for additional excitation light. DETAILED DESCRIPTION

[0081] The following will clearly and completely describe the concept and technical effects of the present invention in conjunction with the embodiments to fully understand the purpose, features and effects of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0082] Example 1 Analysis and Screening of MRV and ISKNV Specific Gene Targets

[0083] According to the latest report of the International Committee on Taxonomy of Viruses (https: / / ictv.global / taxonomy), the Iridoviridae family is mainly divided into two subfamilies, with seven genera and a total of 22 species. MRV and ISKNV belong to the genera Ranavirus and Cytomegalovirus, respectively. Using the BLAST function of the NCBI website (https: / / www.ncbi.nlm.nih.gov / ), it was found that the MCP genes of different strains of MRV or ISKNV viruses were highly similar, but differed greatly from the genes of other viruses, as shown in Tables 1 and 2. Therefore, the MCP gene was selected as the detection target.

[0084] Table 1 Conservative analysis of MRV virus MCP gene

[0085]

[0086]

[0087] Table 2 Conservative analysis of ISKNV virus MCP gene

[0088]

[0089]

[0090] Example 2 Preparation of MRV and ISKNV Major Capsid Protein Gene (MCP) Standard Plasmids

[0091] 1. Nucleic Acid Extraction

[0092] The MRV disease material (GenBank: MG941005.3) and ISKNV disease material (GenBank: AF370008.1) deposited by our group were used to infect the mandarin fish larvae cell line MFF-1 (PMID: 18485510). Samples were collected after 72 hours, and nucleic acids were extracted according to the instructions of the FastPure Viral DNA / RNA Mini Kit V2 (RC313) of Nanjing Novozymes Biotechnology Co., Ltd. The templates were recorded as MRV-DNA1 and ISKNV-DNA1.

[0093] 2. PCR amplification of target gene fragments

[0094] The target gene fragment was synthesized using TOYOBO's high-success-rate PCR enzyme KOD FX (KFX-101). The reaction system is shown in Table 3, and the templates were the nucleic acids MRV-DNA1 and ISKNV-DNA1 extracted in Step 1. PCR primers are shown in Table 4. Reaction conditions are shown in Table 5.

[0095] Table 3 PCR system

[0096]

[0097] Table 4 PCR primers

[0098]

[0099] Table 5 PCR cycling conditions

[0100]

[0101] 3. Preparation of linear vector by enzyme digestion

[0102] TaKaRa's restriction enzyme QuickCut was used TM Sal I and restriction enzyme QuickCut TM The pCMV-HA-C empty plasmid (stored in this laboratory) was digested with Kpn I, and the product was recovered using the Cycle Pure Kit (D6492) from OMEGA.

[0103] 4. Connection and conversion

[0104] Use Shanghai Tolo Port Biotechnology Co., Ltd. Use the Universal CloneMix (#24305) kit and prepare the reaction system on ice according to Table 6. Incubate at 37°C for 15 min. Then, transform the recombinant product into competent cells and plate them with ampicillin-resistant culture medium.

[0105] Table 6 Connection system

[0106]

[0107] 5. Extraction of Recombinant Plasmid

[0108] The monoclonal colonies on the plate in step 4 were picked and cultured in ampicillin-resistant liquid LB medium for 4 h to obtain a turbid bacterial solution, which was sent to Guangzhou Tianyi Huiyuan Gene Technology Co., Ltd. for plasmid sequencing. The sequencing results were compared with the target plasmid sequence, and the strains with consistent alignment were selected for expansion culture; the plasmid was extracted using the FastPureEndoFree Plasmid Maxi Kit (DC202-01) of Nanjing Novozymes Biotechnology Co., Ltd.; the concentration of the recombinant plasmid was determined using a Nanodrop2000 ultramicro UV spectrophotometer, and the copy number was converted according to the plasmid concentration, and the recombinant plasmid was diluted 10 times to 10 - 1 copies / μL, stored at -20℃ for future use, and the plasmid stock solution stored at -80℃ for future use.

[0109] Example 3 Design and screening of RPA primers

[0110] The reference sequences of the MCP genes of MRV and ISKNV (MRV: MG941005.3, ISKNV: AF370008.1) were downloaded from NCBI. Primers were designed using Snapgene software. Three forward primers and three reverse primers were designed, and they could be combined in pairs for a total of nine pairs of primers. The specific sequences are shown in Table 7. After design, they were synthesized by Guangzhou Tianyi Huiyuan Gene Technology Co., Ltd.

[0111] Table 7 RPA primer sequences

[0112]

[0113] The RPA reaction was performed using the RT basic nucleic acid amplification kit of Leshan Biotechnology (Wuxi) Co., Ltd. The reaction system and operation were referred to the kit instructions. The specific steps are as follows:

[0114] 1. Prepare the reaction premix (single sample / reaction) according to Table 8, mix thoroughly and centrifuge briefly.

[0115] Table 8 RPA reaction system

[0116]

[0117] 2. Transfer the above 47 μL reaction premix into the reaction unit, flick the tube to fully dissolve the RT-RPA enzyme lyophilized powder, and centrifuge briefly.

[0118] 3. Add 3 μL of magnesium acetate starter to the reaction tube cap, carefully close the tube cap, and briefly centrifuge to allow the starter to enter the premixed solution. Mix by hand and briefly centrifuge.

[0119] 4. After adding the initiator, quickly place the reaction tube in the instrument with pre-set parameters for reaction at 39°C for 30 minutes. After 5-7 minutes, remove the reaction unit, flick it to mix thoroughly, centrifuge briefly, and return it to the instrument to continue the reaction.

[0120] The RPA reaction was performed according to the above steps using the primer pairs in Table 7. The templates were MRV-DNA1 and ISKNV-DNA1 in Example 2, and the amount used was 5 μL. DEPC water was used as a blank. After the reaction, 1:1 phenol / chloroform was added to extract DNA, and DNA agarose gel electrophoresis was performed.

[0121] The results of the MRV amplification experiment were as follows: Figure 1 As shown in Figure A, numbers 1-9 represent the amplification results of each primer pair, respectively. 1 represents MRV-RPA-F1 / MRV-RPA-R1, 2 represents MRV-RPA-F1 / MRV-RPA-R2, 3 represents MRV-RPA-F1 / MRV-RPA-R3, 4 represents MRV-RPA-F2 / MRV-RPA-R1, 5 represents MRV-RPA-F2 / MRV-RPA-R2, 6 represents MRV-RPA-F2 / MRV-RPA-R3, 7 represents MRV-RPA-F3 / MRV-RPA-R1, 8 represents MRV-RPA-F3 / MRV-RPA-R2, and 9 represents MRV-RPA-F3 / MRV-RPA-R3. + represents positive, and - represents negative. It can be seen that under the same conditions, all nine primer pairs except MRV-RPA-F2 / MRV-RPA-R1 amplified specific bands, and no bands were produced in the negative test. Based on the levels of specific products and nonspecific by-products, the primer pair MRV-RPA-F1 / MRV-RPA-R1 was selected as the optimal primer pair for subsequent experiments.

[0122] The results of the ISKNV amplification experiment are as follows Figure 1As shown in Figure B, numbers 1-9 represent the amplification results of each primer pair, respectively. 1 represents ISKNV-RPA-F1 / ISKNV-RPA-R1, 2 represents ISKNV-RPA-F1 / ISKNV-RPA-R2, 3 represents ISKNV-RPA-F1 / ISKNV-RPA-R3, 4 represents ISKNV-RPA-F2 / ISKNV-RPA-R1, 5 represents ISKNV-RPA-F2 / ISKNV-RPA-R2, 6 represents ISKNV-RPA-F2 / ISKNV-RPA-R3, 7 represents ISKNV-RPA-F3 / ISKNV-RPA-R1, 8 represents ISKNV-RPA-F3 / ISKNV-RPA-R2, and 9 represents ISKNV-RPA-F3 / ISKNV-RPA-R3. + represents positive, and - represents negative. It can be seen that under the same conditions, all nine primer pairs amplified specific bands, and no bands were produced in the negative test. The amplification abilities of each primer pair were different. Finally, the primer pair ISKNV-RPA-F3 / ISKNV-RPA-R1 was selected as the optimal primer pair based on the specific product yield and non-specific by-product level for subsequent experiments.

[0123] Example 4 Design and Synthesis of crRNA

[0124] According to the activation principle of CRISPR / Cas12a protein, the region with the PAM sequence in the RPA product was selected as the target for designing crRNA. The specific sequence is shown in Table 9.

[0125] Table 9

[0126]

[0127] According to the requirements of the Cas12a High Yield crRNA Synthesis and Purification Kit of Shanghai Tolo Port Biotechnology Co., Ltd., the reverse complementary DNA sequence of the RNA sequence in Table 9 was taken, and the reverse complementary DNA sequence of the T7 promoter was added to the 3' end and sent to Guangzhou Tianyi Huiyuan Gene Technology Co., Ltd. for synthesis. It was annealed with the Cas12a Sense Oligo in the kit as Target Antisense Oligo, and the annealed product was used as a transcription template. The reagents in the kit were used to perform in vitro transcription reaction and crRNA purification according to the instructions.

[0128] The concentration of the purified crRNA was determined using a Nanodrop2000 ultra-micro UV spectrophotometer, and the copy number was calculated using Snapgene software. The product was diluted to 100 μM with DEPC water as a storage solution and stored at -80°C. When used, it was diluted to 10 μM as a working solution and stored at -20°C.

[0129] Example 5 Screening of crRNA

[0130] The crRNA purified in Example 4 was used for CRISPR / Cas12a reaction, and the reaction system is shown in Table 10.

[0131] The CRISPR reaction buffer was 10× HOLMES Buffer 1, a product of Shanghai Tolo Biotechnology Co., Ltd.; the Cas12a protein was the enhanced version of LbaCas12a (Cpf1) protein, a product of Shenzhen Yizhi Biotechnology Co., Ltd.; and the fluorescent probe was 5′-FAM-TTATT-BHQ1-3′, synthesized by Guangzhou Tianyi Huiyuan Gene Technology Co., Ltd.

[0132] Table 10 CRISPR / Cas12a reaction system

[0133]

[0134]

[0135] The MRV-DNA1 nucleic acid and ISKNV-DNA1 nucleic acid in Example 2 were used as templates in an amount of 5 μL, DEPC water was used as a negative template, and the primer pairs MRV-RPA-F1 / MRV-RPA-R1 and ISKNV-RPA-F3 / ISKNV-RPA-R1 were used for RPA reaction. After 20 minutes of reaction, the product was directly used as the target DNA for the CRISPR / Cas12a reaction without purification.

[0136] A 96-well real-time fluorescence quantitative PCR instrument was used, and the reaction temperature was set to 37°C. The fluorescence signal was collected every 30 seconds. The results were as follows: Figure 2 As shown, Figure 2 A in the middle is the result of MRV experiment. Figure 2 Figure B shows the ISKNV experiment results. As can be seen from the figure, the crRNAs with the highest fluorescence values ​​at 15 minutes were MRV-crRNA2 and ISKNV-crRNA2, respectively. Therefore, MRV-crRNA2 and ISKNV-crRNA2 were selected as the optimal crRNAs for subsequent experiments.

[0137] Example 6 Optimization of CRISPR / Cas12a Reaction System

[0138] The concentration of Cas12a protein and crRNA in the system of Table 10 is regulated, and the remaining conditions are exactly the same as in Example 5, and the best Cas12a protein and crRNA dosage are screened. The specific reaction system is shown in Table 11.

[0139] Table 11 Screening of optimal usage of Cas12a protein and crRNA

[0140]

[0141] A 96-well real-time fluorescence quantitative PCR instrument was used, and the reaction temperature was set to 37°C. The fluorescence signal was collected every 30 seconds. The results were as follows: Figure 3 As shown, Figure 3 A in the middle is the result of MRV experiment. Figure 3 Figure B shows the results of the ISKNV experiment. As can be seen from the figure, the fluorescence value increases with the increase in the addition amount after 15 minutes of reaction. The fluorescence value of the group with an addition amount of 10μM Cas12a protein 0.5μL and 10μM crRNA 1μL is significantly higher than that of the other groups. Therefore, this addition amount was selected as the optimal addition amount and was used in subsequent experiments.

[0142] After confirming the optimal Cas12a protein and crRNA concentrations, the concentration of the fluorescent probe in the system was changed. The remaining conditions were the optimal conditions described above. The reaction system is specifically shown in Table 12.

[0143] Table 12 Optimal usage of ssDNA reporter (FAM) for screening

[0144]

[0145] A 96-well real-time fluorescence quantitative PCR instrument was used, and the reaction temperature was set to 37°C. The fluorescence signal was collected every 30 seconds. The results were as follows: Figure 4 As shown, Figure 4 A in the middle is the result of MRV experiment. Figure 4 Figure B shows the results of the ISKNV experiment. As can be seen from the figure, at 15 minutes of reaction, the greater the amount of fluorescent probe added, the stronger the fluorescence value. Combined with the results observed under blue light, and considering both observation quality and cost, 1 μL of 10 μM fluorescent probe was selected as the optimal addition amount for subsequent experiments. In fact, even the lowest dosage of 0.25 μL achieved good observation results.

[0146] Example 7 Detection of RNA Targets by the System

[0147] 1. RNA Extraction

[0148] The MRV disease material (GenBank: MG941005.3) and ISKNV disease material (GenBank: AF370008.1) deposited by our group were used to infect the mandarin fish larvae cell line MFF-1 (PMID: 18485510). Samples were collected after 72 hours and the cells were cultured according to the method of Promega. The Super Total RNA Extraction Kit instructions were used to extract RNA, and the templates were designated as MRV-RNA1 and ISKNV-RNA1;

[0149] 2. Detection of RNA targets

[0150] MRV-RNA1 and ISKNV-RNA1 were used as templates to be tested, with a usage of 5 μL. MRV-DNA1 and ISKNV-DNA1 were used as positive controls, with a usage of 5 μL. DEPC water was used as negative. RT-RPA reaction was performed under the aforementioned optimal conditions. After 30 minutes of reaction, 5 μL of the product was taken directly as the target DNA for CRISPR / Cas12a reaction without purification. The remaining conditions were the aforementioned optimal conditions. The product was placed in a preheated 37°C real-time fluorescence quantitative PCR instrument and the fluorescence value was detected every 30 seconds. After 15 minutes of reaction, the experimental results were as follows. Figure 5 As shown, it can be seen that the system successfully detected the RNA nucleic acid targets of MRV and ISKNV.

[0151] Example 8 Threshold Experiment

[0152] Fifty mandarin fish were obtained from a farm and dissected after an ice bath. A small amount of brain, spleen, liver, and kidney tissues were mixed into one tube. The first 20 fish were in a single tube, and three tissue samples of each fish were mixed into one tube for the last 30 fish, for a total of 30 samples, numbered 1-30. 1 mL of PBS buffer was added to each homogenate and centrifuged using a handheld centrifuge. 300 μL of supernatant was used for viral nucleic acid extraction (the extraction method was the same as step 1 in Example 2). Nested PCR was used to detect the presence of MRV and ISKNV. The results showed that all 30 samples were free of MRV, and samples 1-29 were free of ISKNV.

[0153] These samples were subjected to RT-RPA-CRISPR / Cas12a reactions using the previously optimized conditions. 15 μL of template was added to the RPA reaction. After 30 minutes of reaction, 5 μL of the product was directly used as target DNA for the CRISPR / Cas12a reaction without purification. The reaction was continued for 60 minutes. Three replicates were set up for each sample and fluorescence was measured every 30 seconds in a preheated 37°C real-time fluorescence quantitative PCR instrument. MRV-DNA1 and ISKNV-DNA1 were used as positive controls.

[0154] Calculate the average and standard deviation of the fluorescence value of the sample at a specific time, and take the threshold = average + 3 × standard deviation as the detection threshold. If the fluorescence value at the same time is greater than this threshold, it is judged as positive, otherwise it is negative. Figure 6 shown.

[0155] When detecting MRV, the mean value of 30 samples in a 15-minute CRISPR / Cas reaction was 635.143, with a standard deviation of 44.628, a threshold of 769.028, and a positive score of 14285.8. Furthermore, to account for differences in sample loading time, this example provides thresholds for both 30-minute and 60-minute CRISPR / Cas reactions: 1034.753 for 30 minutes and 1610.207 for 60 minutes.

[0156] When detecting ISKNV, the mean value of 29 samples at a 15-minute CRISPR / Cas reaction was 533.304, with a standard deviation of 51.997, a threshold of 689.0295, and a positive score of 27472.3. Furthermore, to account for differences in sample loading time, this example provides thresholds for both 30-minute and 60-minute CRISPR / Cas reactions: 835.5907 for the 30-minute reaction and 1137.328 for the 60-minute reaction.

[0157] This example also demonstrates that this method has good specificity and does not react with host nucleic acids to produce false positives. This example is provided for reference only. Specific fluorescence values ​​may vary due to factors such as operating techniques, reagent batches, and manufacturers. Therefore, it is necessary to prepare a negative sample and compare it with the negative sample to determine positive / negative results.

[0158] Example 9 Sensitivity Test

[0159] The recombinant plasmid containing MRV-MCP and ISKNV-MCP gene DNA fragments at different concentrations prepared in Example 2 was used as a template, and the aforementioned optimal conditions were used to carry out RT-RPA-CRISPR / Cas12a reactions, wherein 15 μL of template was added to the RT-RPA reaction, and 5 μL of the product was taken directly as Target DNA without purification for CRISPR / Cas12a reaction after 30 min of reaction. Three replicates were set for each group, and the fluorescence value was detected every 30 s in a preheated 37 ° C real-time fluorescence quantitative PCR instrument. Three replicates were also set at the same time, and the cells were placed in a 37 ° C metal bath. They were taken out and placed in a blue light gel cutting instrument for observation at 15 min and 60 min.

[0160] The results are as follows Figure 7As shown in the figure, when detecting MRV, this method can detect a template as low as 1 copy / μL; when detecting ISKNV, this method can detect a template as low as 0.1 copy / μL.

[0161] Example 10 Specificity Test

[0162] The SCRV virus (reference GenBank: DQ399789.1) preserved in this laboratory was used to infect the MFF-1 cell line (PMID: 18485510), and the cell samples after the pathological changes were collected and RNA was extracted using the protocol described in Example 7. The RT-RPA-CRISPR / Cas12a reaction was detected using the aforementioned optimal conditions, wherein 15 μL of template was added to the RPA reaction, and after 30 minutes of reaction, 5 μL of the product was taken directly as the target DNA for CRISPR / Cas12a reaction without purification, and MRV-DNA1 and ISKNV-DNA1 were set as positive, and DEPC water was set as negative.

[0163] The fluorescence value was detected using a real-time fluorescence quantitative PCR instrument. After 15 minutes, the gel was taken out and observed under a blue light gel cutting instrument. The experimental results are as follows: Figure 8 At 15 minutes, only the fluorescence value of the positive sample was much higher than that of the negative sample, while no positive signal was detected in the other samples and the negative control. No cross-reaction between different viruses was observed, indicating that this method can specifically identify the target gene fragment and specifically detect MRV and ISKNV viruses.

[0164] Example 11 Repeatability Test

[0165] Using MRV-DNA1 and ISKNV-DNA1 prepared in Example 2 as templates, the RT-RPA-CRISPR / Cas12a reaction was performed using the aforementioned optimal conditions, wherein 15 μL of template was added to the RPA reaction, the reaction was carried out for 30 minutes, and DEPC water was negative. 6 RT-RPA amplification reactions were performed, and the 6 reaction products were added to the CRISPR / Cas system as target DNA. Each group had 3 replicate wells, 5 μL per well, and a real-time fluorescence quantitative PCR instrument was used to control the temperature and detect the fluorescence intensity. After 15 minutes, the gel was taken out and observed using a blue light gel cutting instrument. The experimental results are shown in Figure 2. Figure 9 As shown, the fluorescence values ​​detected six times for the same sample differed very little. The calculated coefficient of variation (CV) for MRV detection was 2.74%, and the coefficient of variation (CV) for ISKNV detection was 5.12%, indicating that the method established in the present invention has good repeatability.

[0166] Example 12 Optimization of probes without additional excitation light

[0167] We replaced different fluorescent and quenching groups and handed them over to Guangzhou Tianyi Huiyuan Gene Technology Co., Ltd. to synthesize a variety of reporter probes, namely:

[0168] Probe 1: 5′-FAM-TTATT-BHQ1-3′;

[0169] Probe 2: 5′-HEX-TTATT-BHQ1-3′;

[0170] Probe 3: 5′-CY3-TTATT-BHQ2-3′;

[0171] Probe 4: 5′-ROX-TTATT-BHQ2-3′;

[0172] Probe 5: 5′-CY5-TTATT-BHQ3-3′;

[0173] The ISKNV-DNA1 nucleic acid in Example 2 was used as a positive template in an amount of 5 μL, DEPC water was used as a negative template, and the primer pair ISKNV-RPA-F3 / ISKNV-RPA-R1 was used for RT-RPA reaction. After 30 minutes of reaction, the product was directly used as the target DNA for CRISPR / Cas12a reaction without purification. The fluorescent probes used were probe 1, probe 2, probe 3, probe 4, and probe 5, with a final concentration of 10 μM. The other conditions were the aforementioned optimal conditions. After 15 minutes of reaction, the color change was observed with the naked eye. The experimental results are shown in FIG. Figure 10 As shown, the color change of probe 1 and probe 4 is clearly visible to the naked eye without the need for ultraviolet light or blue light.

Claims

1. A reagent, characterized in that: Comprising a primer pair for detecting mandarin frog iridovirus and a primer pair for detecting infectious spleen and kidney necrosis virus, as well as crRNA for detecting mandarin frog iridovirus and crRNA for detecting infectious spleen and kidney necrosis virus; The nucleic acid sequences of the primer pair used to amplify the Rana chuatsi iridovirus are shown in SEQ ID NOs: 5 and 8; The nucleic acid sequences of the primer pair used to amplify infectious spleen and kidney necrosis virus are shown in SEQ ID NOs: 13 and 14; The crRNA sequence for detecting Rana chuatsi iridovirus is shown in SEQ ID NO: 18; The crRNA sequence for detecting infectious spleen and kidney necrosis virus is shown in SEQ ID NO:

23.

2. A kit, characterized in that: Comprising the reagent according to claim 1.

3. The kit according to claim 2, wherein: The kit further comprises a fluorescent probe; The fluorescent probe comprises a nucleic acid sequence, wherein the 5' end of the nucleic acid sequence is labeled with a fluorescent reporter group, and the 3' end of the nucleic acid sequence is labeled with a quencher group; The fluorescent reporter group includes at least one of FAM, ROX, HEX, CY3, and CY5; The quenching group includes at least one of BHQ1, BHQ2, and BHQ3.

4. The kit according to claim 2, wherein: The kit further comprises a Cas protein; The Cas protein is Cas12a; The kit also includes an RPA enzyme preparation; The RPA enzyme preparation includes recombinase, recombinase loading factor, single-strand binding protein, and strand displacement DNA polymerase; The RPA enzyme preparation also includes reverse transcriptase; The kit also includes RPA buffer, Mg 2+ , CRISPR reaction buffer and DEPC water.

5. Use of the reagent according to claim 1 and / or the kit according to any one of claims 2 to 4 in any one of a1) to a6): a1) Preparation of products for detecting Rana chuatsi iridovirus; a2) Application in the preparation of a product for screening subjects infected with Rana chuatsi iridovirus; a3) Detection of Rana chuatsi iridovirus for non-diagnostic purposes; a4) Preparation of products for detecting infectious spleen and kidney necrosis virus; a5) Application in the preparation of a product for screening subjects infected with infectious spleen and kidney necrosis virus; a6) Detection of infectious spleen and kidney necrosis virus for non-diagnostic purposes; The infected object includes at least one of fish, frogs, shrimps, crabs, shellfish and turtles.

6. A method for detecting Rana chuatsi iridovirus and / or infectious spleen and kidney necrosis virus for non-diagnostic purposes, comprising the following steps: b1) Sample extraction: Take the sample to be tested and extract nucleic acid; b2) RPA amplification: The primer set, RPA enzyme preparation, RPA buffer, MgCl2, 2+ , DEPC water, mixed with the nucleic acid extracted in step b1) and subjected to RPA amplification; b3) CRISPR detection: The crRNA, Cas12a protein, fluorescent probe, CRISPR reaction buffer, and DEPC water described in claim 4 are mixed with the amplified product obtained in step b2), and a CRISPR reaction detection is performed to determine the fluorescence change result.

7. The detection method according to claim 6, wherein: The final concentration of the Cas protein is 0.05-0.25 μM, and / or The final concentration of the fluorescent probe is 0.125-10 μM; and / or The final concentration of the crRNA is 0.05~0.5 μM.

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