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Real-time fluorescence quantification PCR detection primer of Honghu lake mysidium, kit and detection method

A real-time fluorescence quantification, detection kit technology, applied in the direction of microbial determination/inspection, biochemical equipment and methods, DNA/RNA fragments, etc., can solve the problems of cumbersome operation, easy pollution, low sensitivity, etc. Simple, improved detection efficiency, and high amplification efficiency

Inactive Publication Date: 2018-08-17
INST OF AQUATIC LIFE ACAD SINICA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although these methods can achieve the purpose of detection, there are still some shortcomings, such as high missed diagnosis rate, prone to false positives, cumbersome operation, easy contamination, relatively low sensitivity, and long time-consuming, etc. degree to quantify

Method used

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  • Real-time fluorescence quantification PCR detection primer of Honghu lake mysidium, kit and detection method
  • Real-time fluorescence quantification PCR detection primer of Honghu lake mysidium, kit and detection method
  • Real-time fluorescence quantification PCR detection primer of Honghu lake mysidium, kit and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] 1. Experimental materials

[0058] 1. Honghu iodine worm and Wuli iodine worm suspension

[0059] Myxobolus honghuensis and Myxobolus wulii suspensions were provided by the Healthy Breeding Laboratory of the Institute of Hydrobiology, Chinese Academy of Sciences.

[0060] 2. Main reagents and equipment

[0061] Reagents: DL marker 2000, pMD-18T vector, Escherichia coli DH5α competent cells, QIAGEN DNeasyBlood&Tissue kit (Wuhan Baiteng Ruida Biotechnology Co., Ltd., catalog number: 69504), EZNA Water DNAKit (Wuhan Baiteng Ruida Biotechnology Co., Ltd. company, product number: D5525-01), EZNA Soil DNA Kit (Wuhan Baiteng Ruida Biotechnology Co., Ltd., product number: D5625), Real-time PCR Mix (Wuhan Baileizhen Biotechnology Co., Ltd., product number: 1708882AP), ddH 2 O (Wuhan Baiteng Ruida Biotechnology Co., Ltd., article number: BTH9988).

[0062] Equipment: Bio-Rad T100 PCR instrument, Nano Drop 2000, Bio-rad CFX956 fluorescent quantitative PCR instrument.

[0063] ...

Embodiment 2

[0100] Example 2 Detection of Honghu Iodia in Honghu Lake Tissue Samples, Water Samples and Mud Samples

[0101] 5 crucian carp throat tissue samples, 5 water samples and 5 mud samples were collected from the ponds of Chia Tai Aquatic Products (Hubei) Co., Ltd. in Wulitun, Qichun County, Huanggang City, Hubei Province where throat sporidiosis occurred every year. , after using QIAGEND Neasy Blood & Tissue, EZNA Water DNA Kit, and EZNA Soil DNA Kit kit to extract DNA respectively, use the real-time fluorescence quantitative PCR method optimized in step 5 of the above-mentioned Example 1 to detect crucian carp throat tissue samples, water samples and mud samples Honghu iodine worm, double distilled water was used as a negative control. Repeat 3 wells for each sample, average the 3 Ct values ​​obtained by the fluorescent quantitative PCR instrument to obtain the ΔCt value, and substitute the detected ΔCt value into the above-mentioned regression equation Y=-3.2577X+39.043, The c...

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Abstract

The invention discloses a real-time fluorescence quantification PCR detection primer of Honghu lake mysidium. The invention further discloses a real-time fluorescence quantification PCR detection kitof Honghu lake mysidium and a detection method thereof. The primer, the kit and the detection method disclosed by the invention have high flexibility, strong specificity, good repeatability, quicknessand convenience on detecting Honghu lake mysidium and can detect 302 copies at the minimum extent. The primer, the kit and the detection method not only can detect out Honghu lake mysidium from tissues of infected fish, but also can detect out Honghu lake mysidium from environmental samples of a water sample, a mud sample and the like of a pond with disease all the year around; thus, spatiotemporal dynamic quantitative monitoring on the whole life history stage of the Honghu lake mysidium in the cultivation environment is achieved. The primer, the kit and the detection disclosed by the invention can provide technological support for early rapid diagnosis, monitoring, epidemiological investigation, fingerling production area quarantine, throat sporozoosis occurrence risk assessment and early warning, pharmacological effect assessment and reasonable control countermeasure making of crucian carp throat sporozoosis.

Description

technical field [0001] The invention relates to the field of fish parasite detection, in particular to a real-time fluorescence quantitative PCR detection primer, a detection kit and a detection method for Honghu iodine parasite. Background technique [0002] Myxozoa are a class of metazoan parasites that mainly parasitize fish, and to a lesser extent amphibians, reptiles, birds and mammals. According to literature records, there are more than 40 species of myxospores that parasitize heterogeneous gibel carp, and more than 10 species can cause diseases. Allogenetic gibel carp is a new breeding object for crucian carp successfully developed by fish breeding experts from the Institute of Hydrobiology, Chinese Academy of Sciences in 1976-1981. The male parent, the offspring bred by artificial insemination. Because of its advantages of fast growth, easy feeding, strong stress resistance and delicious meat, it is an important species of crucian carp cultured in my country. In ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6893C12Q1/6851C12Q1/06C12N15/11
CPCC12Q1/6851C12Q1/6893C12Q2545/114C12Q2563/107C12Q2561/113
Inventor 章晋勇罗丹赵媛莉刘新华吴伟
Owner INST OF AQUATIC LIFE ACAD SINICA
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