Red sea bream iridovirus molecular standard sample and its preparation method

A red sea bream iridescent virus and standard sample technology, applied in biochemical equipment and methods, methods based on microorganisms, microorganisms, etc., can solve the problems of aquatic animal disease, difficult to obtain viruses, and undetectable viruses, and achieve good uniformity, Guaranteed quality control, high stability effect

Active Publication Date: 2015-10-28
吴斌 +3
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Due to the deterioration of the breeding ecological environment, poor awareness of disease prevention, widespread drug abuse, poor quality of aquatic animal seedlings, and unsound epidemic prevention systems, etc., aquatic animals have disease
The fish samples sent for inspection in the system are usually relatively healthy visually, but the red sea bream iridescent virus usually only develops at a suitable temperature, and the current PCR method can only be detected from heavily infected fish showing clinical symptoms. Out of virus, unable to detect potential virus
With the increase of import and export trade, the time limit of the inspection process is shortened. Most fish diseases require cell proliferation first, and it is difficult for ordinary aquatic animal disease laboratories to obtain the virus. great challenge

Method used

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  • Red sea bream iridovirus molecular standard sample and its preparation method
  • Red sea bream iridovirus molecular standard sample and its preparation method
  • Red sea bream iridovirus molecular standard sample and its preparation method

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Experimental program
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Effect test

Embodiment 1

[0027] The preparation of embodiment 1 red sea bream iridovirus molecular standard sample

[0028] 1. Identification of red sea bream iridescent virus

[0029] According to the 2009 version of the OIE Aquatic Animal Diseases Diagnostic Manual Chapter2.3.7 in the fish red sea bream iridovirus polymerase chain reaction method.

[0030] (1) Treatment of tissue disease materials

[0031] Take 50 mg of tissue samples (including brain, liver, spleen and kidney) of diseased fish infected by red sea bream iridescent virus, add 150 μL of CTAB solution, cut into pieces with small scissors, grind and homogenize into a paste, and obtain the tissue homogenate of diseased fish;

[0032] (2) Extraction of DNA: In the diseased fish tissue homogenate obtained in step (1), add CTAB solution (by 2% CTAB, 1.4mol / L NaCl, 20mmol / L EDTA, 20mol / L Tris-HCl pH7.5 To prepare, add mercaptoethanol to a final concentration of 0.25% before use.) to 800 μL, place at 25°C for 2 hours, add 350 μL of extract ...

Embodiment 2

[0067] The homogeneity experiment of embodiment 2 red sea bream iridescent virus molecular standard sample

[0068] The homogeneity determination of red sea bream iridescent virus molecular standard sample is that the red sea bream iridescent virus molecular standard sample prepared by the method of embodiment 1 is randomly divided into two sample groups, i.e. sample group A and sample group B, each sample group has 15 standard samples, each sample was subjected to PCR amplification according to the following reaction conditions, and the concentration of PCR products (ng / μL) was measured. By comparing the concentration changes of PCR products, the uniformity of standard samples was evaluated according to statistical methods. The results are shown in Table 2 , 3 and 4.

[0069] PCR reaction system: Add 1 μL of red sea bream iridescent virus molecular standard sample (containing 1×10 -2 μg DNA), 10 μL of 10-fold buffer for Taq enzyme, 2.5 μL each of primers RSIV-F1 and RSIV-R1,...

Embodiment 3

[0078] Stability experiment of embodiment 3 red sea bream iridescent virus molecular standard sample

[0079] The red sea bream iridescent virus molecular standard sample prepared by the method of embodiment 1 is randomly grouped, and each sample is carried out PCR amplification according to the PCR reaction system described in embodiment 2 and the PCR reaction conditions, and measures the PCR product concentration (ng / μL) , by comparing the concentration changes of PCR products, evaluate the stability of standard samples according to statistical methods.

[0080] Two types of stability testing are employed:

[0081] One is the stability test at -20°C. 24 molecular standard samples of red sea bream iridescent virus were randomly selected, and 2 samples were tested every month for 12 consecutive months. The results are shown in Table 5.

[0082] The other is a stability test at a higher temperature of 20°C. 12 molecular standard samples of red sea bream iridescent virus were r...

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Abstract

The invention provides a standard sample of red sea bream iridobirus virus molecules and a preparation method for the standard sample. The preparation method specifically comprises the steps: selection of raw materials of RSIV (red sea bream iridobirus virus), preparation of the standard sample, checking of uniformity and stability, qualitative verification, value fixing and the like. The preparation method for the standard sample comprises the steps: extracting a DNA (deoxyribonucleic acid) of a virus from an RSIV solution, performing PCR (polymerase chain reaction) to multiply and purify a target gene fragment, connecting and converting the target fragment, and recycling plasmids. The standard sample of red sea bream iridobirus viruses is high in stability and high in uniformity, supplies requirements of the standard sample on detection researches, medicinal researches and application researches on the red sea bream iridobirus viruses, technically instructs a inspection and quarantine institution and technically supports service export enterprises. According to the standard sample disclosed by the invention, comparison of different laboratory results can be realized, so that the quality control in a laboratory is guaranteed.

Description

technical field [0001] The invention belongs to the technical field of virus culture standard products for detection and preparation methods thereof, and in particular relates to red sea bream iridescent virus standard samples and preparation methods thereof. Background technique [0002] my country is the world's largest producer and exporter of aquatic products. At present, the total amount of aquatic products in my country has accounted for 40% of the total global fishery output, ranking first in the world for 15 consecutive years, and the output of aquaculture aquatic products accounts for 70% of the world's total aquaculture output. Aquatic products have played an important role in ensuring national food security, increasing farmers' income and stabilizing the rural economy. The barriers faced by my country's seafood export are mainly green barriers, such as Japan's "Positive List System", the European Union's "EU Food and Feed Safety Management Regulations" and related...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12N15/70C12N15/66C12R1/93
CPCC12Q1/6806C12Q2531/113
Inventor 吴斌肇慧君林长军胡强
Owner 吴斌
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