Reagent box for detecting prawn infectious subcutaneous and hemopoietic tissue necrosis virus and method thereof

A technology of hematopoietic tissue and necrotic virus, which is applied in the direction of biochemical equipment and methods, and microbial measurement/inspection, can solve the problems of low sensitivity, complicated operation, high cost, etc., and achieve high sensitivity and strong specificity

Inactive Publication Date: 2009-02-11
SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] In the past, there were mainly three methods for detecting IHHNV in prawns: 1. Electron microscope method, which is costly, time-consuming, low sensitivity, and not suitable for on-site detection; 2. Gene probe method, which is costly and complicated to operate; 3. Polymerase chain method Reaction method (PCR method), which is faster and more sensitive than the previous two methods, but requires an expensive PCR instrument

Method used

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Examples

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Embodiment Construction

[0051] The following examples are further illustrations of the present invention and should not be regarded as limiting the present invention.

[0052] Prepare the isothermal branch amplification detection kit for shrimp infectious subcutaneous and hematopoietic necrosis virus according to the following formula: tissue lysate A: 4MGTC, 25mM sodium citrate, 0.5% sodium lauryl sarcosine and 0.1M β-mercaptoethanol DNA adsorption solution B: 50% Golden Beads suspension

[0053] Hybridization solution C: 2M GTC, 0.5% BSA, 80mM EDTA, 400mM Tris-HCl pH7.5, 0.5% sodium lauryl sarcosine, 2.5nM IHHNV-C-probe and 25nM IHHNV-capture probe

[0054] Magnetic beads D: 4mg / ml SAV immunomagnetic beads

[0055] Probe capture solution E: 10mM Tris-HCl pH7.5, 1mM EDTA and 2M NaCl

[0056] Wash solution F: 10 mM Tris-HCl pH 8.0 and 1 mM EDTA

[0057] Ligation solution G: 20ul 1×ligase buffer and 0.25ul Taq DNA ligase (40U / ul)

[0058] RAM reaction solution H: 6ul 2.5mM dNTP, 5ul 10×Thermopol r...

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Abstract

This invention provides a reagent box for testing IHHNV of prawns with RAM including a DNA pick up reagent, a crossbreed solution, a probe capture reagent, a washing liquid, a connecting- liquor, a RAM reaction liquor, a color developing reagent and especially a marker probe for testing IHHNV. A method for testing IHHNA with the reagent box is provided, which has the same high flexibility as the PCR test method but not need expensive PCR instrument, yet only cheap constant-temperature metal bath or water bath is enough.

Description

technical field [0001] The present invention relates to a kit for detecting infectious subcutaneous and hematopoietic necrosis virus (IHHNV) of prawns by isothermal branch amplification (Isothermal Ramification Amplification, referred to as RAM), and further relates to the rapid detection of infectious subcutaneous and hematopoietic necrosis virus (IHHNV) of prawns using the kit. Methods of hematopoietic necrosis virus. Background technique [0002] Shrimp aquaculture is the pillar industry of marine aquaculture in my country. In 1993, the outbreak of aquaculture prawn diseases in my country brought serious economic losses to the entire prawn aquaculture industry. Related industries such as shrimp processing and foreign trade exports were also seriously affected. In recent years, my country has introduced new species of prawns such as Litopenaeus v vannamei (commonly known as Litopenaeus vannamei) and Litopenaeus stylirostris (commonly known as blue prawn), which are native ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
Inventor 任春华胡超群沈琪
Owner SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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