Real-time fluorescence quantitative PCR detection method for fish iridovirus

A technology of iris virus and detection method, applied in the field of quantitative detection of large yellow croaker iris virus

Inactive Publication Date: 2006-09-06
THIRD INST OF OCEANOGRAPHY STATE OCEANIC ADMINISTATION
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  • Abstract
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  • Application Information

AI Technical Summary

Problems solved by technology

At present, fluorescent quantitative PCR technology has been widely used in the detection of various viruses, such as: respiratory syncytial virus, parainfluenza virus, hepatitis virus, HIV, adenovirus, etc., but there is no report on the application of fluorescent quantitative PCR to the detection of iridescent viruses

Method used

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  • Real-time fluorescence quantitative PCR detection method for fish iridovirus
  • Real-time fluorescence quantitative PCR detection method for fish iridovirus
  • Real-time fluorescence quantitative PCR detection method for fish iridovirus

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Experimental program
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Effect test

Embodiment Construction

[0012] 1. Preparation of PCR template:

[0013] 1.1 The spleen and kidney of healthy large yellow croaker naturally infected with the virus were taken respectively, added to phosphate buffer saline (PBS, pH 7.2) at a ratio of 1:10 (w / v), and homogenized in an ice bath.

[0014] 1. Centrifuge at 25000rpm at 4°C for 10min.

[0015] 1.3 Take 300ul of the centrifuged supernatant of the homogenate and mix it with an equal volume of DNA extraction buffer. At the same time, add proteinase K to a final concentration of 100ul / ml, and place it at 37°C for 15min.

[0016] 1.4 Add 400ul of balanced phenol, shake it by hand for 10min, then centrifuge at 12000rpm for 5min.

[0017] 1.5 Take the supernatant and extract it with an equal volume of phenol / chloroform / isopropanol (25:24:1) for 1-2 times.

[0018] 1.6 Extract again with chloroform / isopropanol (24:1) to completely remove the protein.

[0019] 1.7 Take the supernatant and add 2 times the volume of absolute ethanol, and place it a...

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Abstract

According to epidemic disease and electrical mirror morphological analysis to ill fish, the cause of disease is iris virus. Definite the iris virus ATPase gene conservative area sequence as augmentation target sequence, and design and synthesize double specificity primers and a molecule beacon probe to quantitative detect fish iris virus. According to the quantitative calibration curve made by different concentration gradient positive plasmid, the logarithm of different gradient quantitative mold number is correlation with Ct, and the correlation coefficient is 0.998; at the same time, the detection method has high sensibility, and it can detect at least 70 virus particles. Detecting other virus and LYCIV genes express that the method has good specificity.

Description

Technical field: [0001] The invention relates to a method for quantitative detection of fish iridescent virus, especially large yellow croaker iridescent virus. Background technique [0002] In recent years, with the rapid development of the fish farming industry, fish farming diseases caused by iridescent viruses are becoming more and more serious, which has caused a variety of economically valuable fish such as: large yellow croaker, grouper, mandarin fish, flounder, perch , American redfish and other large-scale deaths. It is estimated that the annual economic losses caused by iridescent virus-induced aquatic diseases amount to billions of dollars. For fish viral diseases, there is no specific treatment method at present, and early detection and diagnosis of viruses are still the most important means of prevention and control. Therefore, the establishment of a fast, sensitive and practical fish iridescent virus PCR detection technology can not only quickly diagnose the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 陈新华王小文
Owner THIRD INST OF OCEANOGRAPHY STATE OCEANIC ADMINISTATION
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