Primer probe group and kit for simultaneously detecting multiple frog viruses and application of primer probe group and kit

Abstract

The invention discloses a primer probe group and a kit for simultaneously detecting multiple frog viruses and application of the primer probe group and the kit, and belongs to the technical field of animal virus detection. The primer probe group for detecting the frog virus comprises a primer group of which the sequence is shown as SEQ ID NO.1-2 and a probe of which the sequence is shown as SEQ ID NO.3. The primer probe group for detecting the frog virus comprises a primer group of which the sequence is shown as SEQ ID NO.3. The primer and the probe are designed according to the D5family NTPase gene sequence of a frog virus member, the method for detecting the frog virus based on the qPCR method is finally established by optimizing reaction conditions, and the method has the characteristics of strong specificity, good repeatability, high sensitivity, simplicity in operation and reliability, and has good market application prospects and popularization and application values.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of animal virus detection, in particular to a primer probe set for simultaneously detecting multiple frog viruses, a kit and application thereof. BACKGROUND

[0002] Frog virus is a new pathogen that can infect reptiles, fish and amphibians, and the host range includes 175 species of poikilothermic vertebrates in 52 families, causing the population of various fish and amphibians to decrease, and causing significant economic losses to the aquaculture industry. The annual loss of aquatic diseases caused by frog virus in China is as high as tens of billions of yuan.

[0003] Currently, there are 7 virus species in the genus Ranavirus recognized by the International Committee on Taxonomy of Virus (ICTV), which are Ranavirus ambystoma 1, Ranavirus alytes 1, Ranavirus perca 1, Ranavirus gadus 1, Ranavirus rana 1, Ranavirus micropterus 1 and Ranavirus epinephelus 1, respectively. Their representative strains are Ambystoma tigrinum virus (ATV), common midwife toad virus (CMTV), epizootic haematopoietic necrosis virus (EHNV), cod iridovirus (CoIV), Frog virus 3 (FV3), largemouth bass virus (LMBV) and Singapore grouper iridovirus (SGIV), respectively. The current methods for identifying members of the genus Ranavirus include Restriction Endonuclease Analysis (REA), virus protein mapping, DNA sequence analysis, etc. The genome of the genus Ranavirus contains 26 relatively conserved core genes, which are involved in viral transcription, replication and structural functions. Through the analysis of the 26 core genes, it is found that the similarity of D5 family NTPase gene and Major Capsid Protein (MCP) gene is higher. MCP is a structural protein with a length of about 1300-1500 bp, and the similarity of MCP between the genus Ranavirus is more than 69%, which is often used for the diagnosis and identification of frog virus. While D5 family NTPase gene is involved in viral replication and repair, with a length of about 2700-2900 bp, and the similarity is more than 66%.

[0004] The similarity of D5 family NTPase gene to MCP gene is close, but the length is much larger than MCP gene, which is a very suitable gene for diagnosis and identification of members of Ranaviridae. According to the sequence of D5 family NTPase gene, members of Ranaviridae can be divided into three categories, the first category is FV3 type frog virus represented by FV3, including epizootic haematopoietic necrosis virus (EHNV), Frog virus 3 (FV3), Andrias davidianus iridovirus disease (ADIV), Soft-shelled turtle iridovirus (STIV), Bohle virus (BIV), Tiger frog Virus (TFV), Rana nigromaculata ranavirus (RNRV) and the like, the second category is SCRV type represented by Santee-Cooper ranavirus (SCRV), including LMBV and Siniperca chuatsi ranavirus (SCRaV), and the third category is SGIV type represented by SGIV, including SGIV and GIV.

[0005] The recommended method for diagnosing frog virus by the World Organization for Animal Health (WOAH) Manual of Diagnostic Tests and Vaccines for Terrestrial Animals is to perform PCR amplification of the MCP gene and then perform restriction enzyme analysis for FV3, BIV, European catfish virus (ECV), European sheatfish virus (ESV), and EHNV. The recommended method does not analyze SCRV virus. The fluorescent quantitative PCR method established by Li Hui-fang can detect members of the Ranavirus genus except for Singapore grouper iridovirus, but does not analyze whether SCRV can be detected. Zhang Li-feng designed three pairs of primers and three probes to identify members of the Ranavirus genus. Chinese patent document CN104099428A designs universal primers for three types of frog viruses based on the MCP sequence, discloses three pairs of primers and three probes, and can detect FV3, BIV, EHNV, ADIV, STIV, LMBV, and SGIV. Chinese patent documents CN112301168A, CN118703703A, and CN113293235A disclose nucleic acid combinations, kits, and detection methods for detecting individual members of the Ranavirus genus. Specifically, CN112301168A provides a LMBV fluorescent quantitative PCR detection method, CN118703703A provides a SCRaV fluorescent quantitative PCR detection method, and CN113293235 provides a PCR detection method for FV3. SUMMARY

[0006] The purpose of the present application is to provide a primer probe set for simultaneously detecting multiple frog viruses, a kit and its application, to solve the problems of complex identification methods, large workload, and single methods in the prior art. A pair of primers and a probe are designed for D5 family NTPase gene for multiple frog virus detection and quantitative analysis, which can detect multiple frog viruses, including FV3, BIV, EHNV, ADIV, STIV, LMBV, SGIV, SCRaV, TFV, and RNRV. The method has high sensitivity, with a minimum detection limit of 10 copies / μL. The use of a pair of specific primers and a probe can achieve the purpose of identifying members of the Ranavirus genus, with the advantages of high specificity, high sensitivity, simple operation, high detection efficiency, good market application prospect, and application value.

[0007] To achieve the above purpose, the present application provides the following solutions:

[0008] The first aspect of the present application provides a primer probe set for simultaneously detecting multiple frog viruses, comprising an upstream primer, a downstream primer and a probe.

[0009] The nucleotide sequence of the upstream primer is shown as SEQ ID NO. 1.

[0010] The nucleotide sequence of the downstream primer is shown as SEQ ID NO. 2.

[0011] The nucleotide sequence of the probe is shown as SEQ ID NO. 3.

[0012] Preferably, the 5' end of the probe sequence is modified with a fluorescent reporter group, and the 3' end of the probe sequence is modified with a fluorescent quencher group.

[0013] Preferably, the fluorescent reporter group comprises FAM, and the fluorescent quencher group comprises BHQ1.

[0014] The second aspect of the present application further provides a detection kit for simultaneously detecting multiple frog viruses, comprising the primer probe set.

[0015] Preferably, the detection kit further comprises PCR buffer, positive control and negative control.

[0016] The third aspect of the present application further provides the use of the primer probe set or the detection kit in the preparation of a detection reagent for detecting frog viruses.

[0017] The fourth aspect of the present application further provides a method for detecting multiple frog viruses for non-disease diagnosis purposes using the primer probe set or the detection kit, characterized in that the method comprises the step of using the primer probe set or the detection kit for qPCR reaction with the DNA of the sample to be tested as a template.

[0018] In the case that the positive control has an amplification curve and the negative control has no amplification curve, when the sample to be tested has an amplification curve and the Ct value is ≤ 35, it is determined that the frog virus nucleic acid in the sample to be tested is positive; when the sample to be tested has no amplification curve, it is determined that the frog virus nucleic acid in the sample to be tested is negative.

[0019] Preferably, the multiple frog viruses comprise FV3, BIV, EHNV, ADIV, STIV, LMBV, SGIV, SCRaV, TFV and RNRV.

[0020] Preferably, the qPCR reaction system is: PCR buffer 10 μL, upstream and downstream primers each 0.4 μL, probe 0.2 μL, DNA template 2 μL, and ddH2O supplemented to 20 μL.

[0021] Preferably, the qPCR reaction program is: 95℃, 15s, 62℃, 1min, 40 cycles.

[0022] The present application discloses the following technical effects:

[0023] The present application selects the conserved region of the D5 family NTPase gene of the 1-type Notophthalmus viridescens frog virus, 1-type Atelopus zeteki frog virus, 1-type Perca fluviatilis frog virus, 1-type Gadus morhua frog virus, 1-type Rana frog virus, 1-type Micropterus salmoides frog virus and 1-type Epinephelus frog virus to design primers and probes, which aims to realize accurate and rapid detection of frog viruses on site, and has the characteristics of simple, specific and efficient identification of multiple members of the frog virus genus.

[0024] The present application has a minimum detection limit of 10 copies / μL, can effectively detect FV3-type frog viruses, SCRV-type frog viruses and SGIV-type frog viruses in the frog virus genus in the sample, specifically including FV3, BIV, EHNV, ADIV, STIV, LMBV, SGIV, SCRaV, TFV and RNRV, and has strong specificity, and has no cross reaction with Koi herpesvirus (KHV), Tilapia Lake Virus (TiLV), viral nervous necrosis virus (VNNV), Channel catfish virus (CCV), Carp edema virus disease (CEV), Infectious hematopoietic necrosis virus (IHNV), Infection spleen and kidney necrosis virus (ISKNV), Grass carp reovirus II (GcRV-2) and Cyprinid herpesvirus II (CyHV-2). BRIEF DESCRIPTION OF DRAWINGS

[0025] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0026] Figure 1 The detection results of different reaction temperatures of the present application;

[0027] Figure 2 Results of detection of different primer concentrations of the present application;

[0028] Figure 3 Results of detection of different probe concentrations of the present application;

[0029] Figure 4 Results of specificity and sample detection of the present application;

[0030] Figure 5 Results of detection of SGIV template concentration sensitivity of the present application;

[0031] Figure 6 Results of detection of LMBV template concentration sensitivity of the present application;

[0032] Figure 7 Results of detection of FV3 template concentration sensitivity of the present application. DETAILED DESCRIPTION

[0033] Various exemplary embodiments of the present application will now be described in detail, which should be considered to be illustrative of certain aspects, features and embodiments of the present application, but not a limitation thereof.

[0034] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. Additionally, for a range of values of a parameter, unless otherwise stated, each intervening value of the parameter is also specifically included within the scope of the present application. The intervening values of the parameter are combined with a stated value of the parameter in range form. These are only examples of the various values that can be used, and equivalents thereof, which are within the scope of the present application. Other examples of the various values that can be used are within the scope of the present application. Still other examples of the various values that can be used are within the scope of the present application.

[0035] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice of the present application. All documents mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the documents are cited. In case of conflict between the present specification and any document incorporated by reference, the present specification will control.

[0036] Various modifications and changes can be made to the specific embodiments of the present application described herein without departing from the scope or spirit of the application. Other embodiments of the application will be apparent to those of ordinary skill in the art from the description and examples presented herein. The description and examples are illustrative of the application and are not intended to limit the scope of the application in any way.

[0037] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", "contains", "containing", or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises, includes, has, contains a list of elements does not include only those elements but can include other elements not expressly listed or inherent to such process, method, article, or apparatus.

[0038] SEQ ID NO. 1: 5'-CTGTTTGAGAARATGCTGGG-3';

[0039] SEQ ID NO. 2: 5'-CCGCGTTTATGGTYTCGTC-3';

[0040] SEQ ID NO. 3: 5'-TCCATCACKGCCCACCTCACTCC-3';

[0041] wherein R, Y and K are degenerate bases, R means G or A, Y means C or T, and K means G or T.

[0042] Design and synthesis of qPCR primers and probes

[0043] Primers and probes were designed according to the sequence characteristics of D5 family NTPase of members of Ranavirus in GeneBank.

[0044] The D5 family NTPase genes of FV3 (MH351268), BIV (NC_038507.1), EHNV (MT510734), TFV (MT512504.1), LMBV (FR682503.1), SCRaV (OQ267588), SGIV (AY521625), ADIV (KC865735.1), STIV (EU627010.1) and RNRV (MG791866.1) in GeneBank were used as detection targets, and the qPCR primers and probes were designed using software Oligo 7. The primers included forward primers with the sequence shown in SEQ ID NO. 1 and reverse primers with the sequence shown in SEQ ID NO. 2. The sequence of the specific probe was shown in SEQ ID NO. 3, and R, Y and K were degenerate bases, R meant G or A, Y meant C or T, and K meant G or T.

[0045] The sequence of the probe SEQ ID NO. 3 was modified with a FAM fluorescent reporter group at 5' and a BHQ1 fluorescent quencher group at 3'.

[0046] The primers and probes described above were synthesized by Shengong Huada Gene Technology Co., Ltd.

[0047] Example 2: qPCR detection method for members of Ranavirus

[0048] The genomic DNA of the sample to be tested was used as a template, and the primers and probes designed in Example 1 were used for qPCR reaction to detect the fluorescence signal. The specific steps are as follows:

[0049] The qPCR reaction system was as follows: 10 μL of fluorescence quantitative PCR buffer, 0.4 μL of each of the upstream and downstream primers, 0.2 μL of the probe, 2 μL of the DNA template, and ddH2O was used to make up to 20 μL of the reaction system. After mixing and centrifugation, the reaction tube was quickly placed in the fluorescence quantitative amplification detector to start the qPCR reaction, and the reaction conditions were as follows: 95℃, 15s, 62℃, 1min, 40 cycles; the corresponding fluorescence signal was collected at the 62℃ annealing and extension stage.

[0050] In the case that the positive quality control has an amplification curve and the negative quality control has no amplification curve, when the sample to be tested has an amplification curve and the Ct value is ≤35, it is determined that the frog virus nucleic acid in the sample to be tested is positive; when the sample to be tested has no amplification curve, it is determined that the frog virus nucleic acid in the sample to be tested is negative.

[0051] Example 3: Optimization of qPCR reaction conditions for members of the frog virus genus

[0052] The genomic DNA of SGIV was extracted, and SEQ ID NO. 1 and SEQ ID NO. 2 were used as primers for amplification. After purification of the PCR product, it was cloned into the pM19-T vector to construct a recombinant plasmid, named pMD19-SGIV. After PCR verification and sequencing identification, the concentration of the recombinant plasmid was determined by a full-wavelength reader. According to the formula: plasmid copy number (copies / μL) = 6.02×10 23 (copies / moL) × plasmid mass concentration (g / μL) / plasmid relative molecular mass (g / moL), the mass concentration of the recombinant plasmid standard was converted to copy number concentration.

[0053] The recombinant plasmid pMD19-SGIV was diluted to 10 6 copies / μL-10 3 copies / μL, which was used as a template to optimize the primer concentration, probe concentration and reaction temperature to establish the optimal reaction conditions for the qPCR detection method.

[0054] The qPCR reaction system was as follows: 10 μL of fluorescence quantitative PCR buffer, 0.4 μL of each of the upstream and downstream primers, 0.2 μL of the probe, 2 μL of the DNA template, and ddH2O was used to make up to 20 μL of the reaction system. After mixing and centrifugation, the reaction tube was quickly placed in the fluorescence quantitative amplification detector to start the qPCR reaction, and the reaction conditions were as follows: 95℃, 15s, 62℃, 1min, 40 cycles; the corresponding fluorescence signal was collected at the 62℃ annealing and extension stage.

[0055] The qPCR reaction was carried out under the conditions of a fixed probe final concentration of 0.2 μmol / μL, a primer final concentration of 0.4 μmol / μL, and a reaction temperature of 57°C, 58°C, 59°C, 60°C, 61°C, or 62°C, to screen the optimal reaction temperature. The results showed that qPCR reactions could be carried out at 57°C, 58°C, 59°C, 60°C, 61°C, or 62°C. See Figure 1 .

[0056] Under the condition of an annealing temperature of 62°C, the qPCR reaction was carried out under the conditions of a fixed probe final concentration of 0.2 μmol / μL and a primer final concentration of 0.1 μmol / μL, 0.2 μmol / μL, 0.3 μmol / μL, 0.4 μmol / μL, 0.5 μmol / μL, or 0.6 μmol / μL, to screen the optimal primer concentration. The results showed that qPCR reactions could be carried out under the conditions of a primer final concentration of 0.1 μmol / μL to 0.6 μmol / μL. See Figure 2 .

[0057] Under the condition of an annealing temperature of 62°C, the qPCR reaction was carried out under the conditions of a fixed primer final concentration of 0.4 μmol / μL and a probe final concentration of 0.05 μmol / μL, 0.1 μmol / μL, 0.15 μmol / μL, 0.2 μmol / μL, 0.25 μmol / μL, or 0.3 μmol / μL, to screen the optimal probe concentration. The results showed that qPCR reactions could be carried out under the conditions of a probe final concentration of 0.05 μmol / μL to 0.3 μmol / μL. See Figure 3 .

[0058] Example 4 Specificity analysis of the qPCR detection method for members of the Ranavirus genus

[0059] According to the optimization results of the above experiments, the primer and the probe were used for qPCR reactions of nine common fish viruses, KHV, TiLV, VNNV, CCV, CEV, IHNV, ISKNV, GcRV-2, and CyHV-2, and ten members of the Ranavirus genus, FV3, BIV, EHNV, ADIV, STIV, LMBV, SGIV, SCRaV, TFV, and RNRV. The results showed that only the members of the Ranavirus genus could produce amplification curves. See Figure 4 .

[0060] Example 5 Sensitivity analysis of the qPCR detection method for members of the Ranavirus genus

[0061] The viral genomic DNA of LMBV and FV3 is extracted, and standard plasmids are constructed according to the method of Example 3, and are named as pMD19-LMBV and pMD19-FV3, respectively. The plasmids pMD19-SGIV, pMD19-LMBV and pMD19-FV3 are diluted by 10 times, and the concentration is adjusted to 10 6 copies / μL-10 copies / μL, the primer concentration is 0.4 μmol / μL, the probe concentration is 0.2 μmol / μL, and the reaction conditions are: 95℃, 15s, 62℃, 1min (40 cycles). The results show that the minimum detection limit of the method is 10 copies, as shown in Figures 5-7 .

[0062] Example 6 Reproducibility analysis of qPCR detection method for members of Ranavirus

[0063] The plasmids pMD19-SGIV, pMD19-LMBV and pMD19-FV3 are diluted by 10 times, and the concentration is adjusted to 10 6 copies / μL-10 4 copies / μL. The above samples are tested by three different experimenters for three times according to the method of Example 2, and the standard deviation (SD) and coefficient of variation (CV) of Ct values between groups and within groups are used as the judgment standard. The analysis results are shown in Table 1.

[0064] Table 1 Reproducibility analysis of qPCR detection method

[0065]

[0066] As shown in the above table, the coefficient of variation within groups of the qPCR detection method established in the application is less than 2.76%, and the coefficient of variation between groups is less than 3.83%, which indicates that the real-time fluorescent quantitative PCR detection method established in the application has good reproducibility.

[0067] Example 7 Verification experiment

[0068] From 2024 to 2025, 42 samples of ornamental fish and 6 samples of frogs suspected to be positive were collected, and the conventional PCR method recommended by WOAH and the qPCR method of Example 2 were used for detection at the same time. The results show that 19 positive samples are detected by the conventional PCR method, and 42 positive samples are detected by the qPCR method. The positive coincidence rate of the two detection methods is 100%, and the detection rate of the qPCR method is higher than that of the conventional PCR detection method.

[0069] The above described embodiments are only to illustrate the preferred modes of the present application, and are not intended to limit the scope of the present application. Any modification and improvement made by those skilled in the art to the technical solutions of the present application without departing from the design spirit of the present application shall fall within the protection scope of the present application as defined by the claims.

Claims

1. A primer probe set for simultaneous detection of multiple frog viruses, characterized in that: including upstream primers, downstream primers and probes; The nucleotide sequence of the upstream primer is shown in SEQ ID NO.1; The nucleotide sequence of the downstream primer is shown in SEQ ID NO.2; The nucleotide sequence of the probe is shown in SEQ ID NO.

3.

2. The primer probe set according to claim 1, characterized in that The 5' end of the probe sequence is modified with a fluorescent reporter group, and the 3' end of the probe sequence is modified with a fluorescent quencher group.

3. The primer probe set according to claim 2, characterized in that The fluorescent reporter group includes FAM, and the fluorescent quencher group includes BHQ1.

4. A detection kit for simultaneously detecting multiple frog viruses, characterized in that: The method comprises the primer-probe set according to any one of claims 1 to 3.

5. The detection kit according to claim 4, characterized in that The detection kit also includes PCR buffer, positive quality control and negative quality control.

6. Use of the primer probe set according to any one of claims 1 to 3 or the detection kit according to any one of claims 4 to 5 in preparing a detection reagent for detecting ranavirus.

7. A method for detecting multiple ranaviruses for non-disease diagnosis purposes using the primer probe set according to any one of claims 1 to 3 or the detection kit according to any one of claims 4 to 5, characterized in that: The method comprises the steps of using the sample DNA to be tested as a template and performing a qPCR reaction using the primer probe set or the detection kit; When the positive quality control has an amplification curve and the negative quality control has no amplification curve, when the amplification curve of the test sample appears and the Ct value is ≤35, the ranavirus nucleic acid in the test sample is determined to be positive; when the test sample has no amplification curve, the ranavirus nucleic acid in the test sample is determined to be negative.

8. The method according to claim 7, characterized in that The multiple ranaviruses include FV3, BIV, EHNV, ADIV, STIV, LMBV, SGIV, SCRaV, TFV and RNRV.

9. The method according to claim 7, characterized in that The qPCR reaction system is as follows: 10 μL of PCR buffer, 0.4 μL of upstream and downstream primers, 0.2 μL of probe, 2 μL of DNA template, and ddH 2 O added to 20 μL.

10. The method according to claim 7, characterized in that The qPCR reaction program was: 95°C, 15s, 62°C, 1min, 40 cycles.

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