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Shrimp white spot syndrome virus detection reagent kit and detecting method

A technology for vitiligo syndrome and viral disease, which is applied in the fields of biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc. It can solve the problems of uncommon applicability, complicated purification steps, and high equipment requirements. Low, suitable for batch testing, easy to operate

Inactive Publication Date: 2009-04-29
SUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In 2004, Tomoya Kono purified WSSV from kuruma shrimp, then extracted the nucleic acid of WSSV, and used LAMP technology to amplify the nucleic acid. The results confirmed that LAMP can be used for the amplification of the corresponding sequence of WSSV; but the purification steps in this method are complicated and The requirements for equipment are high, the equipment is expensive, and the applicability is not universal. In addition, when LAMP technology is applied to actual detection, problems that need to be solved include the design of primers and the optimization of experimental conditions.

Method used

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  • Shrimp white spot syndrome virus detection reagent kit and detecting method

Examples

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Effect test

Embodiment 1

[0041] Embodiment one: the diagnosis of natural infection WSSV crayfish

[0042] A method for detecting shrimp WSSV, the kit used in the method is composed of a set of primers, DNA extraction buffer, DNA polymerase, loop-mediated isothermal amplification reaction solution, positive control, negative control, betaine and dNTP; The set of primers described above is composed of a pair of outer primers and a pair of inner primers, wherein a pair of outer primers is composed of front outer primer F3-234 and back outer primer B3-234; a pair of inner primers is composed of front inner primer FIP-234 and back primer Composition of the back inner primer BIP-234;

[0043] The composition of described extraction buffer comprises: 10mM tris (hydroxymethyl) aminomethane hydrochloride (Tris-HCl), 0.05mM ethylenediaminetetraacetic acid (EDTA), 5mM ammonium sulfate ((NH 4 ) 2 SO 4 ), 0.1% mass percent concentration Triton X-100 (Triton X-100); solution pH is 7.8;

[0044] The DNA polymera...

Embodiment 2

[0052] Embodiment two: the crayfish diagnosis of artificial infection WSSV

[0053] 1. Extraction of template DNA (the DNA extraction method in the common PCR method was adopted, which took about 110 minutes): take 0.2 g of crawfish tissue suspected of being naturally infected with WSSV, grind it on ice and press 1 / 6 Add TN buffer solution (50mM Tris-HCl, 0.4M NaCl, pH7.6) at a (W / V) ratio, and divide the virus-containing tissue grinding solution into two centrifuge tubes, 500 μl each. Add an equal volume of phenol and mix by inversion. Centrifuge at 12,000 rpm for 10 min at 4°C. Transfer the supernatant to a new centrifuge tube, add an equal volume of phenol / chloroform, mix by inverting, centrifuge at 12,000 rpm at 4°C for 10 min. Transfer the supernatant to a new centrifuge tube, add an equal volume of chloroform, mix by inverting, centrifuge at 12,000 rpm at 4°C for 10 min. Transfer the supernatant to a new centrifuge tube, add 1 / 10 volume of NaAC (3mol / L), twice the vol...

Embodiment 3

[0056] Embodiment three: the diagnosis of natural infection WSSV Macrobrachium rosenbergii

[0057] The technical operation process is as follows:

[0058] 1. Extraction of template DNA: Extract template DNA from the cultured Macrobrachium rosenbergii to be tested according to Step 1 of Implementation Example 3, but the template is not diluted.

[0059] 2. Target sequence amplification is the same as step 2 in Example 1.

[0060] 3. Detection of the amplification product is the same as step 3 in Example 1. The results were positive for WSSV and were supported by electron microscopic observations.

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Abstract

The invention relates to a shrimp white spot syndrome virus disease detection reagent kit and a detection method thereof. The reagent kit consists of a set of primers, a DNA extracting buffer solution, DNA polymerase, a loop-mediated isothermal amplification reaction liquid, positive contrast, negative contract, betaine and dNTP. The reagent kit can detect whether a shrimp to be detected is infected by a shrimp white spot syndrome virus. The reagent kit utilizes the strand displacement characteristic of Bst DNA enzymes, designs four primers to identify six regions of a target sequence, generates a large number of stem-loop sample structures under the isothermal condition, and does not require any special equipment under the condition of qualitative detection, thereby having the characteristics of specificity, high sensitivity and short detection time compared with common PCR.

Description

technical field [0001] The invention relates to a kit and a detection method for detecting viruses, in particular to a kit and a detection method for detecting shrimp white spot syndrome virus. Background technique [0002] Since 1993, the coastal prawn aquaculture industry in my country has often been attacked by outbreaks of epidemic diseases. The disease is highly contagious, has a wide range of hosts, and has a high fatality rate, which seriously restricts the sustainable development of my country's shrimp farming industry. It has been confirmed that the pathogen of shrimp outbreak is white spot syndrome virus (WSSV). At present, WSSV has spread to major shrimp farming countries and regions in Asia, and has spread to North America. Among all the shrimp viruses that have been reported, the virus is the most virulent and the most harmful. Penaeus vannamei, Penaeus monodon, Penaeus japonicus, Penaeus moji, Penaeus longhair, Penaeus China, Penaeus india, Penaeus pink, Pena...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
Inventor 薛仁宇贡成良曹广力魏育红沈卫德张伟明顾金寿杨贺
Owner SUZHOU UNIV
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