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Macrobrachium rosenbergii microsporidia visualized quick detection kit and detection method thereof

A detection kit, the technology of Macrobrachium rosenbergii, which is applied in the field of rapid detection of target gene fragments, can solve the problems of lack of high-sensitivity detection kits for Microsporidium rosenbergii, which cannot meet the needs of disease quarantine and prevention work, and achieve the goal of overcoming detection Long time, reduced background effect, high yield effect

Pending Publication Date: 2017-08-18
ZHEJIANG INST OF FRESH WATER FISHERIES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is a lack of highly sensitive detection kits for M. rosenbergii, which can no longer meet the quarantine and prevention of diseases caused by this pathogen.

Method used

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  • Macrobrachium rosenbergii microsporidia visualized quick detection kit and detection method thereof
  • Macrobrachium rosenbergii microsporidia visualized quick detection kit and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 Visual Rapid Detection Kit for Macrobrachium rosenbergii Microsporidia

[0046] Macrobrachium rosenbergii visual rapid detection kit includes Microsporidia DNA extraction reagents and reaction reagents. The kit only needs a conventional centrifuge and a water bath to complete nucleic acid extraction and detection, and the entire detection process takes less than 2 hours , The detection product is closed and observed, and will not pollute the environment. In addition, the detection result can be judged by observing the color change with the naked eye, which is very practical.

[0047] Wherein, the Microsporidia DNA extraction reagent is a pH 8.0 lysate composed of 60mM Tris, 800mM NaCl, 0.5% by mass SDS, and 1% by volume NP-40.

[0048] Reagents include the following components:

[0049] a) Pre-reaction solution: 20mM Tris-HCl with pH 8.8, 8mM magnesium sulfate, 15mM potassium chloride, 10mM sodium sulfate, 0.12% Tween-20, 1.4mM dNTP, 0.4M betaine, 0.2μM primer ...

Embodiment 2

[0053] Embodiment 2 Utilize the method for the rapid detection kit of Macrobrachium rosenbergii described in Example 1 to detect

[0054] (1) DNA extraction:

[0055] Take Macrobrachium rosenbergii larvae or seedlings or gill filaments or liver tissue 30-80mg in a 1.5mL centrifuge tube, and add 200μL of the above-mentioned Microsporidia DNA extraction reagent lysate to the centrifuge tube, grind it on ice with a grinding rod, and grind evenly , heated above 95°C for about 10 minutes, centrifuged at 10,000 rpm for 10 minutes, the supernatant was the DNA template to be tested, and stored at -80°C for later use.

[0056] (2) Rapid amplification of Macrobrachium rosenbergii Microsporidium gene:

[0057] According to the number of samples to be detected, the required quick reaction tube number N is set, and N=sample number+2, wherein 1 tube is a positive control (plasmid containing the M. rosenbergii gene), and 1 tube is a negative control (no nucleic acid deionized water); absor...

Embodiment 3

[0061] Example 3 Kit Specific Detection

[0062] (1) DNA extraction:

[0063] Take 30-80 mg of positive tissue samples of Macrobrachium rosenbergii into 1.5 mL centrifuge tubes, and add 200 μL of DNA extraction reagent lysate to each centrifuge tube, grind with a grinding rod on ice, after grinding evenly, heat at 95°C for 10 After centrifugation at 10,000 rpm for 10 minutes, the supernatant is the DNA template to be tested and stored at -80°C for later use.

[0064] (2) Rapid amplification of Macrobrachium rosenbergii Microsporidium gene:

[0065] Set up 9 quick reaction tubes; draw the pre-reaction solution to a volume of 9×22 μL, add it to a clean 1.5mL centrifuge tube, then add 9 μL of reaction enzyme, mix well, centrifuge at 1500-2000 rpm for 10 seconds, and pour to the set Add 23 μL of the mixed solution to the 9 designated reaction tubes, and add the negative control, M. rosenbergii DNA template, M. rosenbergii bicistronic virus nucleic acid, M. Noda village virus nu...

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Abstract

The invention discloses a macrobrachium rosenbergii microsporidia visualized quick detection kit and a detection method thereof. The macrobrachium rosenbergii microsporidia visualized quick detection kit comprises a microsporidia DNA extraction reagent and a reaction reagent, and the reaction reagent contains a nucleic acid primer set used for detecting macrobrachium rosenbergii microsporidia proliferation, wherein a primer group comprises a primer MrMSP-F3, a primer MrMSP-B3, a primer MrMSP-FIP, a primer MrMSP-BIP, a primer MrMSP-LpF and a primer MrMSP-LpB. According to the macrobrachium rosenbergii microsporidia visualized quick detection kit, a set of optimized quick proliferation reaction system is established, so that not only is macrobrachium rosenbergii microsporidia qualitative detection simpler and faster, high in specificity and high in sensitivity, but also the kit can be used for field detection, and has very high scientific study and economic value.

Description

technical field [0001] The invention belongs to the field of rapid detection of target gene fragments, and in particular relates to a visual rapid detection kit and detection method for Macrobrachium rosenbergii. Background technique [0002] Macrobrachium rosenbergii Microsporidium (MrMSP) is a pathogenic parasite that seriously affects the intestinal nutrient absorption of larvae and adults of Macrobrachium rosenbergii. It can parasitize the intestinal and liver tissues of shrimp, and can cause intestinal epithelial tissue damage, thereby causing malabsorption of nutrients in Macrobrachium rosenbergii, affecting the health and growth of shrimp. The pathogen can indirectly lead to smaller individuals of Macrobrachium rosenbergii due to impaired nutrient absorption and low aquaculture output. Macrobrachium rosenbergii is a new pathogen that was recently discovered and prevalent in the seedlings of Macrobrachium rosenbergii. The carry rate is close to 50%. At this stage, t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/90
CPCC12Q1/6844C12Q1/6893C12Q2563/107C12Q2527/101
Inventor 潘晓艺蔺凌云袁雪梅沈锦玉徐洋姚嘉赟尹文林郝贵杰
Owner ZHEJIANG INST OF FRESH WATER FISHERIES
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