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Preparation method and application of VHSV (viral hemorrhagic septicemia virus) viral sample particles

A virus-like, particle-like technology, applied in biochemical equipment and methods, viruses, virus/bacteriophage, etc., can solve the problems that cannot reflect the influence of the nucleic acid extraction process, are easily degraded by ribonuclease, and hidden dangers in biological safety, and achieve great convenience The effects of large-scale promotion and application, avoiding the risk of false negatives, and solving stability problems

Inactive Publication Date: 2017-07-21
INSPECTION & QUARANTINE TECH CENT SHANDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Virus culture or cDNA obtained by reverse transcription of RNA is usually used as a standard, but the former has hidden dangers in biological safety and is easily degraded by ribonuclease, while the latter cannot reflect the influence of nucleic acid extraction process and reverse transcription on the test

Method used

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  • Preparation method and application of VHSV (viral hemorrhagic septicemia virus) viral sample particles
  • Preparation method and application of VHSV (viral hemorrhagic septicemia virus) viral sample particles
  • Preparation method and application of VHSV (viral hemorrhagic septicemia virus) viral sample particles

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment 1 prepares VHSV-N-VLPs

[0040] The preparation method of the virus-like particle standard substance containing VHSV-N gene, comprises the following steps:

[0041] (1) Construction of VLPs recombinant plasmid pET-NH-MS2his containing histidine tag

[0042] The pET32a plasmid was digested with Xba I and Nco I, and the sequence between the two restriction sites of Xba I and Nco I was removed, and the artificially synthesized 5'-PO4-CTAGATTACAAGGC-3' and 5'-PO4-CATGGCCTTGTAAT- The 3' two complementary oligonucleotides were annealed and inserted into it to construct the recombinant plasmid pET-NH, which was identified by sequencing.

[0043] A one-step amplification kit was used to amplify the MS2 target fragment with MS2Pf1 and MS2Pr1 as primers and MS2RNA as a template. The amplified product was detected by agarose gel electrophoresis, and the target fragment was recovered with an agarose gel electrophoresis recovery kit (TaKaRa), eluted with 50 μL ddH2O, an...

Embodiment 2V

[0052] The characteristic detection of embodiment 2VHSV-N-VLPs

[0053] To the characteristic detection of the virus-like particle standard substance containing VHSV N gene, the steps are as follows:

[0054] (1) RNA extraction and RT-PCR detection in VHSV-N-VLPs

[0055] Use 100 μL of the purified virus-like particle solution to extract RNA as a template, and perform RT-PCR detection according to the primer sequence of VHSV N in Power 4. The RT-PCR system is: 5×RT-PCR buffer 5 μL, dNTP mix 1 μL, Enzyme Mix 1 μL, Primers VHSV-F and VHSV-R each 1 μL, RNA template 5 μL, ddH2O make up 25 μL, the reaction conditions were reverse transcription at 50°C for 30 minutes, extinguishing and denaturing at 95°C for 10 minutes, then 1 minute at 95°C, 1.5 minutes at 59°C, and 1 minute at 72°C. 40 cycles, and finally extended at 72°C for 10 minutes; the amplified product was detected by 1.0% agarose gel electrophoresis to 1247bp, see figure 2 , send for sequencing.

[0056] (2) Detection ...

example 3

[0064] Example 3. Application of detecting VHSV infection in rainbow trout

[0065] VHSV-N-VLPs were used as quality control samples to detect VHSV RNA infection in rainbow trout, and the steps were as follows.

[0066] 1. Virus isolation

[0067] Take the brain, spleen and kidney of live rainbow trout. After the sample was homogenized according to conventional methods, the suspension was diluted 1:10 with culture medium containing 10 times antibiotics, and incubated overnight at 4°C or at room temperature for 1 hour to release the virus. Centrifuge at 12000rpm for 15min, and collect the supernatant. Dilute 10 times and 100 times, inoculate BF-2 cells. Incubate at 15°C (maximum not higher than 18°C) for 3 days. The suspension was collected and used for detection and identification after freezing and thawing once.

[0068] 2. Extraction of RNA

[0069] RNA was extracted using an RNA extraction kit.

[0070] 3. Primer probe

[0071] Forward primer VHSV P1: 5'-AAA-CTC-GCA...

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Abstract

The invention discloses a preparation method and application of VHSV (viral hemorrhagic septicemia virus) viral sample particles; the viral sample particles are RNA-protein complex of MS2 bacteriophage coded coat protein covered with VHSV N gene RNA; a coded histidine tagged sequence is inserted into a Beta hairpin loop structural sequence of the MS2 bacteriophage coded coat protein, so as to construct MS2 bacteriophage coat protein with recombinant histidine tag and Phn-MS2his recombinant plasmid for expressing maturase protein gene; VHSV N gene is amplified through RT-PCR (reverse transcriptase-polymerase chain reaction) and is cloned to Pnh-MS2his, so as to construct prokaryotic expression recombinant plasmid, named pNH-MS2his-VHSV-N; the obtained pNH-MS2his-VHSV-N plasmid is converted to expressed Escherichia coli BL21 to carry out induced expression. The VHSH-N-VLPs viral sample particles herein may act as standard material and quality control material for RT-PCR detection, are free of infectivity, good in safety and reliability and stability, and can tolerate ribonuclease.

Description

technical field [0001] The invention relates to a preparation method and application of virus-like particles containing viral hemorrhagic sepsis virus (VHSV) N gene. Background technique [0002] Viral hemorrhagic septicemia of salmonids (VHSV) is also known as trout ascites disease and new trout disease. The pathogen is a rhabdovirus, which can cause acute or chronic visceral diseases in fish, and the mortality rate is very high. [0003] Viral hemorrhagic sepsis has been prevalent in Europe for a long time, and it has been officially reported in France, Italy, Switzerland, Norway, Sweden, Poland and other European countries. In recent years, the disease has spread to North America, Oceania, Southeast Asia and other regions. [0004] VHSV mainly infects trout and other salmonids, but also affects other cultured non-salmonid fry. Usually when the water temperature drops to around 10°C, the disease will occur in fish schools. The main pathological changes of hemorrhagic s...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12N15/63C12N15/66C12N7/04C12Q1/70C12Q1/68
CPCC12N7/00C12N15/11C12N15/63C12N15/66C12N2760/20023C12Q1/701
Inventor 王群孙涛岳志芹姜帆梁成珠房保海
Owner INSPECTION & QUARANTINE TECH CENT SHANDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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