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Method for detecting WSSV and IHHNV of prawn simultaneously and detection kit used by same

A technology for detection kits and kits, applied in biochemical equipment and methods, microbial measurement/inspection, etc., can solve problems such as reaction principles, complex primer design, etc.

Inactive Publication Date: 2010-08-04
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

LAMP is a new type of constant temperature nucleic acid amplification method. Compared with other nucleic acid amplification techniques, its reaction principle and primer design are more complicated.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Embodiment 1, a kind of simultaneously detects prawn WSSV and IHHNV nucleic acid multiplex isothermal amplification detection kit (5 samples, can be used for the detection of 5 samples), and it is made up of following contents:

[0065] (1) Grinding solution tube, 1 tube, 3ml.

[0066] Grinding solution preparation: 10 parts of 1M Tris-HCl solution (Tris-HCl, pH8.0), 0.25M ethylenediaminetetraacetic acid disodium solution (EDTANa 2 ) 2.0 parts, 10 parts of sodium dodecylsulfonate solution (SDS) with a mass fraction of 5%, 0.5 parts of mercaptoethanol, 0.01 part of 8-hydroxyquinoline, 7 parts of balanced phenol, 8 parts of chloroform, fixed with double distilled water Can hold up to 100 copies.

[0067](2) Tube A of nucleic acid extraction solution, 1 tube, containing 400 μl of 3M sodium acetate solution (NaAc, pH 5.2).

[0068] (3) Tube B of nucleic acid extraction solution, 1 tube, filled with 10ml of absolute ethanol.

[0069] (4) Tube C of nucleic acid extraction ...

Embodiment 2

[0106] Embodiment 2, a multiple isothermal amplification detection kit (5 samples) for simultaneous detection of prawn WSSV and IHHNV nucleic acid, which is different from embodiment 1 only in the following content, and the rest are the same as embodiment 1.

[0107] Grinding solution preparation: 5 parts of 2M Tris-HCl (Tris-HCl, pH8.0), 0.5M disodium ethylenediaminetetraacetic acid (EDTANa 2 ) 1 part, 7 parts of sodium dodecylsulfonate (SDS) with a mass fraction of 7%, 1 part of mercaptoethanol, 0.02 part of 8-hydroxyquinoline, 5 parts of balanced phenol, 10 parts of chloroform, constant volume with double distilled water to 100 servings.

[0108] The multi-LAMP reaction solution consists of the following components: multi-LAMP primers WSSV-FIP, WSSV-BIP, IHHNV-FIP and IHHNV-BIP each 1 μM, multi-LAMP primers WSSV-F3, WSSV-B3, IHHNV-F3 and IHHNV -B3 each 0.1μM, dATP, dGTP and dCTP each 2mM, dTTP, dUTP each 1mM, Tris-HCl 10mM, KCl 10mM, (NH 4 ) 2 SO 4 5mM, MgSO 4 4mM, T...

Embodiment 3

[0109] Embodiment 3, a multiple isothermal amplification detection kit (5 samples) for simultaneous detection of prawn WSSV and IHHNV nucleic acid, which is different from Example 1 only in the following content, and the rest are the same as Example 1.

[0110] Grinding solution preparation: 7 parts of 1.5M Tris-HCl solution (Tris-HCl, pH8.0), 1.0M ethylenediaminetetraacetic acid disodium solution (EDTANa 2 ) 0.5 part, 5 parts of sodium dodecylsulfonate solution (SDS) with a mass fraction of 10%, 0.01 part of mercaptoethanol, 0.015 part of 8-hydroxyquinoline, 10 parts of balanced phenol, 10 parts of chloroform, fixed with double distilled water Can hold up to 100 copies.

[0111] The multi-LAMP reaction solution consists of the following components: multi-LAMP primers WSSV-FIP, WSSV-BIP, IHHNV-FIP and IHHNV-BIP 4 μM each, multi-LAMP primers WSSV-F3, WSSV-B3, IHHNV-F3 and IHHNV -B3 each 0.4μM, dATP, dGTP and dCTP each 0.5mM, dTTP, dUTP each 0.25mM, Tris-HCl 40mM, KCl 20mM, (NH...

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PUM

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Abstract

The invention discloses a nucleic acid multiplex isothermal amplification detection kit for detecting WSSV and IHHNV of a prawn simultaneously, which comprises a grinding liquid tube filled with a grinding liquid, a nucleic acid extraction liquid tube A filled with the solution of sodium acetate, a nucleic acid extraction liquid tube B filled with absolute ethanol, a nucleic acid extraction liquid tube C filled with ethanol of which the mass concentration is 70 percent, a buffer solution E filled with TE buffer solution, a UNG enzyme tube filled with uracil-DNA glycosylase, a multi-LAMP reaction liquid tube filled with a multi-LAMP reaction liquid, a Bst DNA polymerase tube filled with Bst DNA polymerase, a color developing agent tube filled with a nucleic acid dye SYBR Green I, and positive control nucleic acid tube filled with positive DNA of the WWS and the IHHNV, of the prawn, in a copy number ratio of 1 to 1, and a negative control tube filled with sterilized double stilled water. The invention also provides a method detecting the WSSV and the IHHNV of the prawn simultaneously, which has high detection sensitivity.

Description

technical field [0001] The invention relates to an aquatic animal pathogen detection technology, in particular to a nucleic acid multiple isothermal amplification detection kit and a detection method for prawn white spot syndrome and infectious subcutaneous and hematopoietic tissue necrosis virus. Background technique [0002] Shrimp white spot syndrome virus WSSV first appeared in the early 1990s. It is a highly infectious and lethal virus, and it is the most important virus that endangers the global shrimp farming industry. WSSV can infect a wide range of hosts, including all species of prawns and most other crustaceans, and has even been found in insects, and can be transmitted vertically. Infected prawns exhibit very high mortality, with cumulative mortality usually reaching 100% 3-10 days after the first overt signs of infection are observed. [0003] Infectious hypodermic and hematopoietic necrosis virus (IHHNV) was first discovered in the cultured Litopenaeus styliro...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68
Inventor 徐海圣何琳王美珍
Owner ZHEJIANG UNIV
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