Detection method of prawn IHHNV (infectious hypodermal and hematopoietic necrosis virus) and used nucleic acid isothermal amplification detection kit

A detection kit and isothermal amplification technology, which are applied in the determination/inspection of microorganisms, biochemical equipment and methods, etc., can solve the problems of limiting the popularization and application of PCR detection methods, inability to detect viruses, and low detection sensitivity, so as to reduce false positives. Positive results, less error-prone, good detection specificity

Inactive Publication Date: 2010-08-04
ZHEJIANG UNIV
View PDF0 Cites 8 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although electron microscopy can directly observe the presence of virus particles, the operation is complicated, time-consuming and costly
The HE staining method is based on pathological techniques, which cannot directly detect the virus, but can only be detected by using the histopathological signs of the disease
Antibody detection method and nucleic acid probe hybridization method are used to detect the protein or nucleic acid components of the virus itself. The detection sensitivity is low and time-consuming, and can only be used for the detection of diseased shrimp or shrimps that are about to become diseased; IHHNV has not yet caused infection Or it is difficult to detect with the above methods in the very early stage of infection
Although the PCR detection method of IHHNV overcomes the shortcomings of the first four methods, it can achieve relatively fast and accurate detection of IHHNV under laboratory conditions, but because conventional PCR detection requires expensive PCR machines, gel electrophoresis and imaging systems , and the detection time is slightly longer, which also greatly limits the popularization and application of PCR detection method in production practice

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Embodiment 1, a kind of prawn IHHNV nucleic acid isothermal amplification detection kit (5 samples, can be used for the detection of 5 samples), it is made up of following content:

[0049] (1) Grinding solution tube, 1 tube, 3ml.

[0050] Grinding solution preparation: 10 parts of 1M Tris-HCl solution (Tris-HCl, pH8.0), 0.25M ethylenediaminetetraacetic acid disodium solution (EDTANa 2 ) 2 parts, 10 parts of sodium dodecylsulfonate solution (SDS) with a mass fraction of 5%, 0.5 part 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. The above parts are all parts by volume.

[0051] (2) Tube A of nucleic acid extraction solution, 1 tube, containing 400 μl of 3M sodium acetate solution (NaAc, pH 5.2).

[0052] (3) Tube B of nucleic acid extraction solution, 1 tube, filled with 10ml of absolute ethanol.

[0053] (4) Tube C of nucleic acid extraction solutio...

Embodiment 2

[0077] Embodiment 2. A prawn IHHNV nucleic acid isothermal amplification detection kit (5 samples), which is different from embodiment 1 only in the following content, and the rest are the same as embodiment 1.

[0078] Grinding solution preparation: 5 parts of 2M Tris-HCl solution (Tris-HCl, pH8.0), 0.5M ethylenediaminetetraacetic acid disodium solution (EDTANa 2 ) 1 part, 7 parts of sodium dodecylsulfonate solution (SDS) with a mass fraction of 7%, 0.01 part of mercaptoethanol, 0.02 part of 8-hydroxyquinoline, 5 parts of balanced phenol, 10 parts of chloroform, fixed with double distilled water Can hold up to 100 copies. The above parts are all parts by volume.

[0079] The composition of the LAMP reaction solution is as follows: LAMP primers IHHNV-FIP and IHHNV-BIP each 4 μM, LAMP primers IHHNV-F3 and IHHNV-B3 each 0.5 μM, dATP, dGTP and dCTP each 2 mM, dTTP, dUTP each 1.0 mM, Tris- HCl 40mM, KCl 20mM, (NH 4 ) 2 SO 4 15mM, MgSO 4 4mM, Triton X-100 1.0%, Betaine 0.8M...

Embodiment 3

[0080] Embodiment 3. A prawn IHHNV nucleic acid isothermal amplification detection kit (5 samples), which is different from Example 1 only in the following content, and the rest are the same as Example 1.

[0081] 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. The above parts are all parts by volume.

[0082] The composition of the LAMP reaction solution is as follows: LAMP primers IHHNV-FIP and IHHNV-BIP each 1 μM, LAMP primers IHHNV-F3 and IHHNV-B3 each 0.25 μM, dATP, dGTP and dCTP each 0.5 mM, dTTP, dUTP each 0.5 mM, Tris -HCl 40mM, KCl 10mM, (NH 4 ) 2 SO 4 5mM, MgSO 4 1mM, Triton X-1000.05%, Betaine 0...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention discloses a prawn IHHNV (infectious hypodermal and hematopoietic necrosis virus) nucleic acid isothermal amplification detection kit which comprises a grinding fluid tube containing a grinding fluid, a nucleic acid extracting solution tube A containing a sodium acetate solution, a nucleic acid extracting solution tube B containing absolute ethyl alcohol, a nucleic acid extracting solution tube C containing an alcohol solution with a mass concentration of 70 percent, a TE (tellurium) buffer solution tube containing a TE buffer solution, a UNG (uracil-DNA glycosidase) enzyme tube containing uracil DNA (deoxyribonucleic acid) glycosylase, an LAMP (Loop-mediated isothermal amplification) reaction liquid tube containing an LAMP reaction liquid, a BstDNA polymerase tube containing Bst DNA polymerase, a color-developing agent tube containing nucleic acid dye SYBR Green I, a positive control nucleic acid tube containing prawn IHHNV positive DNA and a negative control tube containing sterilizing double distilled water. The invention also provides a method for detecting the prawn IHHNV by utilizing the detection kit. The method has the characteristics of low cost, high detection sensitivity and easy site operation.

Description

technical field [0001] The invention relates to an aquatic animal pathogen detection technology, in particular to a nucleic acid isothermal amplification detection kit and a detection method for infectious subcutaneous and hematopoietic tissue necrosis virus of prawns. Background technique [0002] Infectious hypocutaneous and hematopoietic necrosis virus (IHHNV) was first discovered in the cultured Litopenaeus stylirostris in Hawaii. It is widely distributed and seriously harmful, causing a mortality rate of over 90%. It can also infect the cultured shrimps around the world. , is one of the other important diseases of crustaceans delineated by the International Office of Epizootics (OIE), and has a great impact on the development of shrimp farming in the world. Among the various viruses that can infect Penaeus vannamei, IHHNV can cause Runt-deformity syndrome (RDS for short). Although it will not cause a large number of prawns to die, the virus infection will actually cause...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68
Inventor 徐海圣何琳王美珍戎华南
Owner ZHEJIANG UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap