A detection primer and kit for the genotyping of a large yellow croaker ranavirus and application thereof

Abstract

The application relates to a detection primer, a kit and application of a large yellow croaker swollen cell virus, and relates to a large yellow croaker swollen cell virus detection primer, and based on the primer, a specific method amplification system and condition are used to detect the large yellow croaker swollen cell virus. The PCR detection primer is high in specificity and sensitivity, and can identify whether the large yellow croaker tissue contains the swollen cell virus. The application can be used for the detection of the large yellow croaker swollen cell virus, and can provide early diagnosis, gene typing for the large yellow croaker iridovirus disease, and can provide effective technical support for the molecular epidemiological investigation of the large yellow croaker iridovirus disease.

Description

Technical Field

[0001] This invention relates to the field of aquatic disease detection and identification technology, and in particular to a primer and reagent kit for typological detection of swollen cell virus and its application. Background Technology

[0002] Iridovirus disease is one of the major diseases affecting large yellow croaker fry, with mortality rates reaching 40%-80%, causing significant economic losses. The pathogen is an iridovirus belonging to the genus Megalovirus. Domestic and international scholars have conducted genetic diversity analysis of Megalovirus using mcp and ATPase sequences, identifying at least three genotypes. Through whole-genome and mcp sequence analysis of different Megalovirus isolates from large yellow croaker, two subtypes of Megalovirus have been identified: FD201807(LMIV) and SA. However, a rapid method for typing and detecting Megalovirus in large yellow croaker is currently lacking. Therefore, this invention proposes a primer and kit for Megalovirus typing to address the aforementioned problems. Summary of the Invention

[0003] (1) Technical problems to be solved

[0004] This invention overcomes the technical problems of existing technologies that cannot rapidly detect macula cell virus (MCV) typing in large yellow croaker, and cannot provide early diagnostic analysis and disease prevention. Therefore, it provides MCV typing detection primers, kits, and their applications.

[0005] To address the aforementioned technical problems, the present invention aims to provide a primer for detecting swollen cell virus in large yellow croaker.

[0006] Another object of the present invention is to provide a kit for detecting swollen cell virus typing in large yellow croaker;

[0007] Another objective of this invention is to provide a PCR detection method for pheocytic virus typing in large yellow croaker;

[0008] Another objective of this invention is to provide the application of the above primers in the preparation of a large yellow croaker edema cell virus typing detection reagent.

[0009] This invention can be used for highly sensitive and specific molecular detection of swollen cell virus in large yellow croaker, and can also be used for early diagnosis, virus typing, and molecular epidemiological investigation of iridovirus disease in large yellow croaker.

[0010] (2) Technical solution

[0011] To address the aforementioned technical problems, a first aspect of the present invention provides:

[0012] A primer for genotyping large yellow croaker edema cell virus, wherein the primers are 852F and 1272R, and their nucleotide sequences are as follows:

[0013] 852F: 5'-TATACTGGAGGACGACGAT-3',

[0014] 1272R: 5'-GCACTCTTCGCATGCGTTATC-3'.

[0015] A second aspect of the present invention provides:

[0016] A kit for detecting swollen cell virus typing in large yellow croaker, comprising the aforementioned primers, PCR reaction premix, and positive control.

[0017] The PCR reaction premix described above includes reagents commonly used in the art, such as Taq DNA polymerase, dNTPs, and Mg. 2+ Taq enzyme buffer.

[0018] Furthermore, the above kit also includes a positive control; the positive control is a recombinant plasmid containing the ORF026 nucleic acid sequence of the large yellow croaker edema cell virus strain FD201807.

[0019] A third aspect of the present invention provides:

[0020] A PCR detection method for pheocytosis of large yellow croaker includes the following steps:

[0021] 1) Extract DNA from large yellow croaker samples or virus fluid samples for later use;

[0022] 2) Using the DNA from the large yellow croaker sample or the viral fluid sample as a template, PCR amplification was performed using the primers 852F and 1272R described above or the kit described above to obtain amplification product A.

[0023] 3) Take the PCR amplification product A and perform 1% agarose gel electrophoresis to detect it. Compare it with the positive control and determine whether the sample has FD201807 (LMIV) type malformation virus. If a band of about 421 bp is amplified, the sample is positive.

[0024] The extraction of DNA from the sample to be tested in step 1) above includes extracting DNA from the sample using conventional DNA extraction techniques in the art or using existing DNA extraction kits.

[0025] Further, the PCR amplification system in step 2) is as follows: 10 μL of PCR reaction premix, 1 μL of template DNA, 1 μL each of primer 852F and primer 1272R with a concentration of 10 μmol / L, and 7 μL of sterile water.

[0026] Further, the PCR amplification program in step 2) is as follows: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, 53℃ annealing for 30 s, 72℃ extension for 35 s, for 30 cycles; 72℃ final extension for 5 min.

[0027] A fourth aspect of the present invention provides:

[0028] Application of the above primers in the preparation of detection reagents for maceration of large yellow croaker varicella virus.

[0029] A fifth aspect of the present invention provides:

[0030] The above-mentioned PCR detection method was applied in the prevention and epidemiological investigation of iridovirus disease in large yellow croaker farming, especially for iridoviruses of the genus *Mammillaria*.

[0031] (3) Beneficial effects

[0032] The beneficial effects of this invention are as follows: This invention designs a pair of primers targeting the ORF026 specific region of FD201807-LMIV type large yellow croaker edema cell virus, providing a PCR detection method for large yellow croaker edema cell virus typing. This solves the problem of the lack of a rapid detection method for large yellow croaker edema cell virus typing in the prior art. This detection method has a high detection rate, is simple and easy to operate, has a short time consumption, low detection cost, and high specificity. It is suitable for rapid diagnosis and can achieve specific and sensitive early diagnosis of FD201807-LMIV type large yellow croaker edema cell virus with higher reliability. Attached Figure Description

[0033] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0034] Figure 1 The image shows a gel electrophoresis result obtained by PCR primers or kits described in Example 1 of this invention: where M is the DNA molecular weight standard DL2000, lane 1 is the positive control, and lane 2 is the negative control.

[0035] Figure 2The image shows a gel electrophoresis result obtained by performing a specificity test on the PCR primers or kits described in Example 2 of this invention. In the image, M is the DNA molecular weight standard DL2000, lane 1 is the positive control, lane 2 is the tissue of a healthy large yellow croaker, lane 3 is red sea bream iridovirus (RSIV), lane 4 is Singapore grouper iridovirus (SGIV), lane 5 is yellowfin sea bream ascites iridovirus (SBV), lane 6 is mandarin fish frog virus (MRV), lane 7 is koi herpesvirus (KHV), lane 8 is nerve necrosis virus (NNV), lane 9 is Vibrio alginolyticus, and lane 10 is Pseudomonas proteus.

[0036] Figure 3 The gel electrophoresis image is obtained by performing a sensitivity test on the PCR primers or kit described in Example 3 of this invention, where M is the DNA molecular weight standard DL2000, and lanes 1-8 are 10... 7 10 6 10 5 10 4 10 3 10 2 10 1 10 0 copies / μL; lane 9 is the negative control.

[0037] Figure 4 The image is an electrophoresis diagram obtained by using the PCR primers or kits described in Example 4 of this invention to perform typological detection of large yellow croaker edema cell virus on the sample. In the image, M is the DNA molecular weight standard DL2000, lane 0 is the negative control, lane 20 is the positive control, and lanes 1-19 correspond to the large yellow croaker tissue samples 1-19 to be detected. Detailed Implementation

[0038] The present invention can be better understood from the following embodiments. However, those skilled in the art will readily understand that the specific material ratios, process conditions, and results described in the embodiments are for illustrative purposes only and should not, and will not, limit the invention as described in detail in the claims.

[0039] Unless otherwise specified, all experimental materials and reagents used are commercially available consumables and reagents.

[0040] Example 1: Preparation of the Large Yellow Croaker Swollen Cell Virus Typing Detection Kit:

[0041] 1. Primer design and synthesis

[0042] The culturing primers for large yellow croaker edema cell virus (ECV) described in this embodiment are based on the large yellow croaker ECV genome sequences (OQ475017.1, AY779031.1, OL774655.1, OL774654.1, OL774653.1) already registered in GenBank. By comprehensively applying various biological software for comparative analysis, the ORF026 specific region that can be used for genotyping identification was screened out. Based on this region, genotyping primers 852F and 1272R were cleverly designed and synthesized by a professional company. After synthesis, the primers are in lyophilized powder form and diluted with sterile water to a working concentration of 10 μM.

[0043] The nucleotide sequences of primer pairs 852F and 1272R are as follows:

[0044] 852F: 5'-TATACTGGAGGACGACGAT-3',

[0045] 1272R: 5'-GCACTCTTCGCATGCGTTATC-3'.

[0046] 2. PCR reaction premix

[0047] The PCR reaction premix used in this invention contains Taq enzyme, dNTPs, and Mg required for conventional PCR. 2+ And components such as buffer solution.

[0048] 3. Positive control

[0049] The 421bp positive PCR product of the large yellow croaker edema cell virus strain FD201807 was ligated into the 19-T vector, transformed into Escherichia coli DH5α under heat stress, and positive clones were screened out. After being cultured overnight in LB broth containing 100 μg / mL ampicillin, the positive recombinant plasmid was extracted and confirmed by PCR detection and sequencing. This plasmid DNA is the positive control used in the kit of this invention, and its sequence is shown in SEQ ID NO 3.

[0050] 4. Using the primers, PCR reaction premix, and positive control described above, the large yellow croaker edema cell virus typing PCR detection kit of this application was prepared.

[0051] Example 2 Specificity Detection

[0052] To determine the accuracy of the detection method, a specificity test was conducted. Positive plasmids, red sea bream iridovirus (RSIV), Singapore grouper iridovirus (SGIV), mandarin fish frog virus (MRV), yellowfin sea bream ascites disease iridovirus (SBV), koi herpesvirus (KHV), *Pseudomonas alginolyticus*, *Vibrio alginolyticus*, genomic DNA from the spleen tissue of healthy large yellow croaker, and cDNA libraries of neuronecrosis virus (NNV) were used as PCR amplification templates. Primer pairs 852F and 1272R were used for amplification.

[0053] The total volume of the PCR reaction is 20 μL. The optimal reaction system is as follows: 10 μL of PCR premix, 1 μL of template DNA, 1 μL each of primer 852F and primer 1272R at a concentration of 10 μmol / L, and 7 μL of sterile water.

[0054] The PCR amplification program was as follows: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, 53℃ annealing for 30 s, 72℃ extension for 35 s, for 30 cycles; and 72℃ final extension for 5 min.

[0055] After the reaction was completed, 6 μL of the PCR product was taken for gel electrophoresis to observe the specific PCR detection.

[0056] Using the primers and method of this invention, a band of approximately 421 bp was amplified with the positive control sample. However, no such band was observed in the PCR amplification products using cDNA libraries containing genomic DNA from red sea bream iridovirus (RSIV), Singapore grouper iridovirus (SGIV), mandarin fish frog virus (MRV), yellowfin sea bream ascites disease iridovirus (SBV), koi herpesvirus (KHV), *Pseudomonas alginolyticus*, *Vibrio alginolyticus*, healthy large yellow croaker spleen tissue, and neuronecrosis virus (NNV) genomes (see details). Figure 2 This indicates that the primers and method have good specificity.

[0057] Example 3 Sensitivity Detection

[0058] Genomic DNA was extracted from positive plasmids, and their concentration was determined using Nanodrop 2000. The plasmids were then diluted to 10⁻⁶. 6 10 5 10 4 10 3 10 2 10 1 10 0 Copies / μL were used as templates for PCR amplification according to the large yellow croaker maculavirus typing detection method. Sensitivity detection results are as follows: Figure 3 As shown, the template is 10. 6 10 5 10 4 103 10 2 Positive plasmids of copies / μL all showed positive bands of approximately 421bp in size. This indicates that this detection method can detect as few as 10 copies / μL. 2 Copies, with high sensitivity.

[0059] Example 4 uses the primers or kits designed in Example 1 to detect swollen cell virus typing in large yellow croaker.

[0060] 1. Preparation of Detection Samples and Template DNA

[0061] Samples 1-18 were large yellow croakers collected from the Ningde aquaculture area between 2018 and 2022. Samples 1-18 tested positive for PCR using the 1F / R primers in the national standard GB / T 36191—2018, while sample 19 tested negative using the same method. Cloning and sequencing analysis of the full-segment mcp gene of the samples that tested positive for macronasvir determined the viral genotype, as shown in Table 1. Five fish were taken from each batch, with 0.1–0.2 g taken from each fish. The samples were mixed and homogenized into a tissue homogenate. The DNA of the sample to be tested was prepared according to the steps of a commercially available animal tissue genomic DNA extraction kit, serving as the template DNA for macronasvir genotyping in large yellow croakers.

[0062] Table 1 Information on large yellow croaker samples used for typing detection

[0063]

[0064] 2. PCR amplification

[0065] Take 1 μL of the DNA from the sample prepared in step 1 as a template and perform the detection according to the PCR method for pheochromocytosis of large yellow croaker.

[0066] 3. Agarose gel electrophoresis

[0067] PCR amplification products were subjected to 1% agarose gel electrophoresis at a constant voltage of 120V for 40 minutes. Results were observed using a gel imaging system or a blue light spectrometer. Under normal conditions, a positive control should show a single 421bp band (attached to the image). Figure 4 The middle lane is 20), and the negative control showed no corresponding band (in the attached lane). Figure 4 The middle lane is lane 0). If the control result does not match the above, the test is invalid and needs to be repeated. If the control result is normal, and the test sample shows a band of approximately 421 bp, it is considered positive; if the test sample does not show a corresponding band, it is considered negative.

[0068] 4. Experimental Results

[0069] The test results for samples 1-17 are as follows: Figure 4As shown (and also in Table 1 above), the positive control showed a band of approximately 421 bp, the negative control showed no corresponding band, and samples 2, 4, 17, and 18 showed a band of approximately 421 bp. Samples 2, 4, 17, and 18 were determined to be FD type positive, and their typing results were consistent with the full-segment mcp gene sequence typing results of the virus in each sample. This indicates that the method or kit of the present invention can successfully perform typing detection of swollen cell virus in large yellow croaker, which can ensure the effectiveness and large-scale usability of the kit.

[0070] from Figure 4 As can be seen from the above, the PCR detection primer pair of this application has specificity for large yellow croaker maculavirus and can distinguish large yellow croaker maculavirus from other microorganisms. The primer pair of this application has high specificity and strong recognition. The detection primer pair of this invention can be used to detect maculavirus in the large yellow croaker farming environment, which plays an important guiding role in the prevention and control of diseases in large yellow croaker farming. Of course, it can also be used to detect whether maculavirus is present in the farming environment of large yellow croaker that has never been diseased, which can play a preventive role for large yellow croaker that has not been diseased. The detection primer pair of this application has strong practicality.

[0071] The above description is a detailed description of the preferred embodiments of the present invention. However, the embodiments are not intended to limit the scope of the patent application of the present invention. All equivalent changes or modifications made under the technical spirit of the present invention should fall within the patent scope covered by the present invention.

Claims

1. A detection primer for typing of a giant sea bass (Hypophthalmichthys nobilis) cell enlargement virus, characterized by, The primers are 852F and 1272R, and their nucleotide sequences are as follows: 852F: 5'-TATACTGGAGGACGACGAT-3', 1272R: 5'-GCACTCTTCGCATGCGTTATC-3'.

2. A detection kit for typing of the virus of the swollen cell disease of Larimichthys crocea, characterized in that, It includes the primer pair as described in claim 1, PCR reaction premix, and positive control.

3. The kit of claim 2, wherein The positive control was a recombinant plasmid containing the ORF026 nucleic acid sequence of the large yellow croaker edema cell virus strain FD201807.

4. The kit according to claim 2 or 3, characterized in that, The kit was used for PCR amplification to obtain amplification product A.

5. The kit of claim 4, wherein The PCR amplification system consisted of: 10 μL of PCR reaction premix, 1 μL of template DNA, 1 μL each of primer 852F and primer 1272R at a concentration of 10 μmol / L, and 7 μL of sterile water.

6. The kit of claim 4, wherein The PCR amplification program is as follows: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, 53℃ annealing for 30 s, 72℃ extension for 35 s, for 30 cycles; 72℃ final extension for 5 min.

7. The application of the large yellow croaker edema cell virus typing detection primers as described in claim 1 in the preparation of the large yellow croaker edema cell virus FD201807 detection reagent.

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