A RAA detection primer probe combination for detecting multiple members of the genus Ranavirus and its application

Abstract

The present invention discloses a RAA detection primer-probe combination for detecting multiple members of the genus Ranavirus and its application, belonging to the field of biotechnology. The RAA detection primer-probe combination comprises an upstream primer RARAAF3, a downstream primer RARAAR3, and a probe RARAAP. By optimizing reaction conditions, the present invention ultimately establishes a method for detecting Ranaviruses based on the RAA method. This method can simultaneously detect multiple Ranaviruses, has the characteristics of rapid detection and simple operation, and can complete the reaction within 20 minutes. This provides a convenient and simple method for on-site screening of Ranaviruses and non-laboratory environment detection, and has high scientific research and practical value.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an RAA detection primer-probe combination for detecting multiple members of the genus Ranavirus and applications thereof. Background Art

[0002] Ranavirus is a large, linear, double-stranded DNA virus with a wide host range, infecting at least 175 species of cold-blooded vertebrates from 52 families, including reptiles, fish, and amphibians. Ranavirus can cause severe illness and varying degrees of mortality, resulting in significant economic losses to the aquaculture industry. Annual losses from aquatic diseases caused by ranavirus in my country amount to tens of billions of yuan. The World Organization for Animal Health (WOAH) lists ranavirus infection as a reportable aquatic animal disease. Ranavirus has a wide geographical distribution and has been found on all six continents except Antarctica.

[0003] There are many members in the genus Ranavirus. In June 2023, the International Committee on Taxonomy of Viruses (ICTV) renamed the seven species of Ranaviruses that have been included, namely Ranavirus ambystoma 1, Ranavirus alytes 1, Ranavirus perca 1, Ranavirus gadus 1, Ranavirus rana 1, Ranavirus micropterus 1 and Ranavirus epinephelus 1. The original name of Ranavirus type 1 was Frogvirus 3 (FV3), Ranavirus perca1 was originally named Epizootic haematopoietic necrosis virus (EHNV), and Ranavirus micropterus1 was originally named Santee-Cooper ranavirus (SCRV). The World Organisation for Animal Health (WOAH) Diagnostic Manual of Aquatic Animal Diseases uses a method for detecting ranaviruses based on restriction endonuclease analysis (REA) and sequence analysis of a portion of the major capsid protein (MCP) gene using PCR amplification. An alternative method recommended is PCR followed by sequence analysis (ADHyatt). Marsh et al. used PCR amplification of MCP sequences to detect ranaviruses from various sources in Australia, Europe, and the United States. Matthew et al. established a general detection method using the MCP gene as the target gene to detect FV3 virus from the American box turtle. Zhou Yong et al., Li Huifang et al., and Groocock GH et al. established a TaqMan real-time PCR detection method for frog viruses. The above detection method can detect FV3, Bohleiridovirus (BIV), European catfish virus (ECV), European sheatfish virus (ESV), and EHNV. None of these methods are described for Ranavirus micropterus 1.Pallister, Goldberg, Getchell, Gong Jinpeng, and others designed specific primers based on conserved sequences in MCP and amplified Ranavirus micropterus 1 using fluorescent quantitative PCR. Li Jiangyu et al. established a triple fluorescent PCR method for detecting Ranaviruses based on the MCP gene and TaqMan probes. Although these conventional PCR and quantitative PCR methods offer the advantage of high sensitivity, they have limitations. Their reliance on instrumentation restricts their application in field testing. Therefore, the development of rapid, sensitive, accurate, and field-suitable detection reagents and methods is of great importance.

[0004] Recombinase-mediated isothermal amplification (RAA) is a novel isothermal amplification technique with numerous advantages, including ease of use, high sensitivity, and specificity. It is considered a potential alternative to PCR for nucleic acid amplification. Unlike PCR, this technique does not require prior denaturation. Instead, it utilizes specific enzymes to open the template strands and amplify nucleic acids under constant temperature conditions, typically between 37°C and 42°C. Therefore, this technique is not limited by instrumentation or testing sites. Another significant advantage of this technique is its short detection time: the entire amplification reaction can be completed in 10–20 minutes, and the amplified product is detectable. The amplified product can be combined with a variety of detection methods for visual analysis. Therefore, real-time fluorescence RAA is a method that can be used for on-site portable diagnostics. Summary of the Invention

[0005] The present invention aims to provide a RAA detection primer-probe combination for detecting multiple members of the Ranavirus genus and its application, thereby addressing the aforementioned problems of the prior art. The present invention can simultaneously detect multiple Ranaviruses, features rapid detection, and is easy to operate. The reaction can be completed within 20 minutes, providing a convenient and simple method for on-site screening of Ranaviruses and non-laboratory detection, with high scientific and practical value.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides an RAA detection primer-probe combination for detecting multiple members of the genus Ranavirus, comprising an upstream primer RARAAF3, a downstream primer RARAAR3 and a probe RARAAP;

[0008] The nucleotide sequence of the upstream primer RARAAF3 is shown in SEQ ID NO.5;

[0009] The nucleotide sequence of the downstream primer RARAAR3 is shown in SEQ ID NO.6;

[0010] The probe RARAAP is composed of the sequence shown in SEQ ID NO. 11, tetrahydrofuran and the sequence shown in SEQ ID NO. 12 connected in sequence.

[0011] Furthermore, the 30th base of the sequence shown in SEQ ID NO. 11 is modified with a fluorescent reporter group; the 2nd base of the sequence shown in SEQ ID NO. 12 is modified with a fluorescent quencher group, and the 3' end is blocked.

[0012] Furthermore, the fluorescent reporter group includes FAM, and the fluorescent quencher group includes BHQ1.

[0013] Furthermore, the blocking treatment includes labeling C3 Spacer at the 3' end of the sequence shown in SEQ ID NO.12.

[0014] The present invention also provides an RAA detection kit for detecting multiple members of the genus Ranavirus, comprising the RAA detection primer-probe combination.

[0015] Furthermore, it also includes RAA reaction solution, Buffer B, negative quality control and positive quality control; the RAA reaction solution is a buffer solution containing recombinase, single-strand binding protein and strand displacement DNA polymerase; the Buffer B is magnesium acetate solution.

[0016] The present invention also provides the use of the RAA detection primer-probe combination or the RAA detection kit in the detection of RAA of multiple Ranavirus members.

[0017] The present invention also provides a method for detecting RAA of various members of the genus Ranavirus, comprising the steps of using the DNA of a sample to be tested as a template and performing an RAA reaction using the RAA detection primer-probe combination.

[0018] Furthermore, the RAA reaction system includes 29.4 μL of RAA reaction solution, 2.5 μL of Buffer B, 1 μL each of upstream and downstream primers, 0.45 μL of probe, 2 μL of DNA template and 15.65 μL of distilled water.

[0019] Furthermore, the RAA reaction conditions are 40° C., 40 cycles, and 30 seconds per cycle.

[0020] The present invention discloses the following technical effects:

[0021] The present invention designs a pair of primers and a probe to detect multiple ranaviruses based on the MCP gene sequence of members of the genus Ranavirus. By optimizing the reaction conditions, a method for detecting ranaviruses based on the RAA method is finally established. The method is simple to operate, does not require complicated procedures, and is suitable for outdoor and on-site operations. It has high sensitivity, with a minimum detection limit of up to 10 copies / μL; and has strong specificity, with no cross-reaction with KHV, TiLV, VNNV, CCV, CEV, IHNV, ISKNV, GcRV-2, and CyHV-2.

[0022] Compared with existing technologies, the present invention can detect multiple frog viruses at the same time, has the characteristics of rapid detection and simple operation, and can complete the reaction within 20 minutes. It provides a convenient and simple method for on-site screening and non-laboratory environment detection of frog viruses, and has high scientific research and practical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0024] Figure 1 is the primer pair screening result, where M is DL 2000Marker, 1 is primer RARAAF1 / RARAAR1, 2 is primer RARAAF1 / RARAAR2, 3 is primer RARAAF1 / RARAAR3, 4 is primer RARAAF1 / RARAAR4, 5 is primer RARAAF3 / RARAAR1, 6 is primer RARAAF3 / RARAAR2, 7 is primer RARAAF3 / RARAAR3, and 8 is primer RARAAF3 / RARAAR4;

[0025] Figure 2 The test results of different reaction temperatures of the present invention are as follows;

[0026] Figure 3 The results of the different primer concentration tests of the present invention are shown in FIG.

[0027] Figure 4 These are the test results of different probe concentrations of the present invention;

[0028] Figure 5 is the specific detection result of the RAA detection method of the present invention;

[0029] Figure 6 The sensitivity test results of the RAA detection method of the present invention using different template concentrations are shown. DETAILED DESCRIPTION

[0030] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0031] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0032] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0033] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0034] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0035] Example

[0036] 1. Design of real-time fluorescent RAA primers and probes

[0037] The MCP genes of FV3 (AY548484.1), EHNV (FJ433873.1), Bohlevirus (BIV) (NC_038507.1), Tiger frog virus (TFV) (MT512504.1), and Largemouth bass rana virus (LMBV) (FR682503.1) from GeneBank were used as detection targets. Following the principles of primer and probe design for recombinase isothermal amplification, Ranavirus RAA primers and probes were designed, as shown in Table 1. FV3, BIV, and TFV belong to Ranavirus rana1, EHNV belongs to Ranavirus perca1, and LMBV belongs to Ranavirus micropterus1. Primers and probes were synthesized by Liuhe BGI Genomics Co., Ltd.

[0038] Table 1 RAA primer and probe names and sequences

[0039]

[0040]

[0041] In Table 1, S, K, R, and Y are degenerate bases, with S referring to G or C, K to G or T, R to A or G, and Y to T or C. In the sequence of the probe RARAAP, the last thymine nucleotide from the 5' end of the sequence shown in SEQ ID NO. 11 is a thymine nucleotide modified with a FAM fluorescent reporter group (6-FAM-dT), and the first thymine nucleotide from the 5' end of the sequence shown in SEQ ID NO. 12 is a thymine nucleotide modified with a BHQ1 fluorescent quencher group (BHQ1-dT); THF (tetrahydrofuran) is present between SEQ ID NO. 11 and SEQ ID NO. 12; and the 3' end of SEQ ID NO. 12 is blocked to prevent sequence extension.

[0042] 2. Screening of real-time fluorescent RAA primers and probes

[0043] Standard plasmid construction: Using LMBV genomic DNA as a template, amplify the fragment using RARAAF4 (SEQ ID NO. 7) and RARAAR3 (SEQ ID NO. 6) to ensure that the amplified fragments cover the fragments amplified by all primers in Table 1. Purify the PCR product and ligate it into the pMD19-T vector to construct the recombinant plasmid, designated pMD19-T-LM. After verification by PCR and sequencing, determine the concentration of the recombinant plasmid using a full-wavelength reader and convert it to copy number. Dilute pMD19-T-LM to 1000 copies / μL as a template for the PCR reaction. PCR amplification was performed using the crossover combination of upstream and downstream primers listed in Table 1. The reaction system consisted of 12.5 μL of 2× PCR buffer, 1 μL of upstream primer, 1 μL of downstream primer, 2 μL of template, and HO to 25 μL. The reaction conditions were as follows: pre-denaturation at 94°C for 5 min, 30 cycles of denaturation at 94°C for 30 s, annealing at 53°C for 30 s, and extension at 72°C for 30 s; and post-extension at 72°C for 5 min.

[0044] The electrophoresis results of some primers are as follows Figure 1 As shown, the primers RARAAF3 / RARAAR3 with the highest sensitivity and amplification efficiency were finally selected from these primers.

[0045] 3. Real-time fluorescence RAA detection method for members of the genus Ranavirus

[0046] Using the genomic DNA of the sample to be tested as a template, primers RARAAF3 / RARAAR3 and probe RARAAP were used to perform the RAA reaction and detect the fluorescence signal. The specific steps are as follows:

[0047] The RAA detection system consists of a 50 μL volume containing 29.4 μL of RAA reaction solution, 1 μL of upstream and downstream primers (10 μM each), 0.45 μL of probe (10 μM), 2 μL of DNA template, and 13.65 μL of distilled water. The reaction mixture is vortexed to thoroughly dissolve the lyophilized powder in the reaction tube. Finally, 2.5 μL of Buffer B (280 mM) is added to each reaction tube. After mixing and centrifugation, the reaction tubes are quickly placed in a fluorescence quantitative amplification instrument and subjected to 40 cycles of 30 seconds per cycle at a constant temperature of 40°C. Fluorescence signals are collected once per cycle. The RAA reaction solution is a buffer containing the recombinase, single-strand binding protein, and strand-displacing DNA polymerase, and Buffer B is a magnesium acetate solution.

[0048] 4. Optimization of RAA reaction conditions

[0049] Using pMD19-T-LM as template, the optimal reaction conditions for RAA detection were established by optimizing the primer and probe concentrations.

[0050] The RAA detection system is 50 μL, including 29.4 μL of RAA reaction solution, 1 μL of upstream and downstream primers, 0.45 μL of probe, 2 μL of DNA template and 13.65 μL of distilled water. The reaction mixture is shaken to fully dissolve the lyophilized powder in the reaction tube. Finally, 2.5 μL of Buffer B (280 mM) is added to each reaction tube. After mixing and centrifugation, the reaction tube is quickly placed in a fluorescence quantitative amplification detector to start 40 cycles of constant temperature reaction, each cycle is 30 seconds, and the fluorescence signal is collected once in each cycle.

[0051] The final concentration of the fixed probe was 0.09 μmol / μL, and the final concentration of the primer was 0.4 μmol / μL. The RAA reaction was performed at 37°C, 38°C, 39°C, and 40°C to screen the optimal reaction temperature. The results showed that the RAA reaction could be performed at 37°C, 38°C, 39°C, and 40°C. Figure 2 .

[0052] The RAA reaction was performed at a reaction temperature of 40°C, with a final concentration of the fixed probe of 0.09 μmol / μL and primers at final concentrations of 0.2 μmol / μL, 0.4 μmol / μL, 0.6 μmol / μL, and 0.8 μmol / μL, respectively, to screen for the optimal primer concentration. The results showed that in the total system, RAA reactions could be performed at primer final concentrations of 0.2 μmol / μL to 0.6 μmol / μL. Figure 3 .

[0053] The RAA reaction was performed at a reaction temperature of 40°C, with a fixed primer final concentration of 0.4 μmol / μL and probe final concentrations of 0.09 μmol / μL, 0.12 μmol / μL, 0.15 μmol / μL, and 0.18 μmol / μL, respectively, to screen for the optimal probe concentration. The results showed that in the overall system, RAA reactions could be performed at probe final concentrations of 0.09 μmol / μL to 0.18 μmol / μL. Figure 4 .

[0054] 5. Specificity analysis of RAA detection methods

[0055] Based on the results of RAA reaction condition optimization, primers RARAAF3 / RARAAR3 and probe RARAAP were used to perform RAA detection on 9 common fish viruses, including KHV, TiLV, VNNV, CCV, CEV, IHNV, ISKNV, GcRV-2, and CyHV-2, as well as 5 Ranavirus members, including BIV, FV3, LMBV, TFV, and EHNV. The results showed that only Ranavirus members could produce amplification curves, see Figure 5 .

[0056] 6. Sensitivity analysis of RAA detection methods

[0057] Dilute pMD19-T-LM 10-fold to adjust the concentration to 10 6 The reaction was performed with a primer concentration of 0.4 μmol / μL, a probe concentration of 0.09 μmol / μL, a reaction temperature of 40°C, and a reaction time of 20 min. The results showed that the minimum detection limit of this method was 10 copies / μL. Figure 6 .

[0058] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A RAA detection primer-probe combination for detecting multiple members of the genus Ranavirus, characterized in that: Includes upstream primer RARAAF3, downstream primer RARAAR3 and probe RARAAP; The nucleotide sequence of the upstream primer RARAAF3 is shown in SEQ ID NO.5; The nucleotide sequence of the downstream primer RARAAR3 is shown in SEQ ID NO.6; The probe RARAAP is composed of the sequence shown in SEQ ID NO. 11, tetrahydrofuran, and the sequence shown in SEQ ID NO. 12 connected in sequence; The various Ranavirus genera include FV3, EHNV, BIV, TFV, and LMBV; The 30th base of the sequence shown in SEQ ID NO.11 is modified with a fluorescent reporter group; the 2nd base of the sequence shown in SEQ ID NO.12 is modified with a fluorescent quencher group, and the 3' end is blocked; The blocking treatment includes labeling C3 Spacer at the 3' end of the sequence shown in SEQ ID NO.

12.

2. The RAA detection primer-probe combination according to claim 1, characterized in that: The fluorescent reporter group includes FAM, and the fluorescent quencher group includes BHQ1.

3. A RAA detection kit for detecting multiple members of the genus Ranavirus, characterized in that: The invention comprises the RAA detection primer-probe combination according to claim 1 or 2.

4. The RAA detection kit according to claim 3, characterized in that It also includes RAA reaction solution, Buffer B, negative quality control and positive quality control; the RAA reaction solution is a buffer solution containing recombinase, single-strand binding protein and strand displacement DNA polymerase; the Buffer B is a magnesium acetate solution.

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