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Primer for discriminating schistosoma japonicas, corresponding kit and detection method thereof

A schistosomiasis and kit technology, applied in the field of primers for identifying Schistosoma japonicum, can solve problems such as epidemic spread and recovery, and achieve the effects of objective judgment, optimized reaction system, and simple operation.

Inactive Publication Date: 2012-12-26
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In recent years, global climate warming, frequent outbreaks of debris flows and floods, and changes in ecological environment elements such as water level, climate, vegetation, and microorganisms caused by the implementation of the South-to-North Water Diversion Project have had a major impact on the distribution of the only intermediate host of schistosome snails, making the epidemic spread. and the rising trend, bringing new severe challenges to the prevention and control of schistosomiasis

Method used

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  • Primer for discriminating schistosoma japonicas, corresponding kit and detection method thereof
  • Primer for discriminating schistosoma japonicas, corresponding kit and detection method thereof
  • Primer for discriminating schistosoma japonicas, corresponding kit and detection method thereof

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Experimental program
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Effect test

Embodiment 1

[0043] The composition of embodiment 1 kit

[0044]The kit contains 27.5mL of DNA lysis solution, which contains Nuclei lysis solution, 0.5M EDTA at pH8.0, proteinase K (20mg / mL) and RNase A solution (4mg / mL); PCR reaction solution for 100 reactions (25μL / reaction), dATP, dTTP, dGTP, dCTP with a final concentration of 200 μM each, upstream and downstream primers with a final concentration of 0.25 pmol / μL, and 2 mM MgCl 2 , Taq Enzyme 25 μL (5U / μL); 1 branch of Schistosoma japonicum mountain type DNA control and lake type Schistosoma japonicum DNA control.

Embodiment 2

[0045] Embodiment 2 kit specificity test

[0046] Use 1 μl of DNA from each of six control samples of Schistosoma mansoni, Schistosoma haematobium, Fasciola hepatica, Fasciola large, Clonorchis sinensis and Opisthorchis felis that have been verified for DNA validity as templates, and perform specific PCR according to the reaction conditions of the kit Amplification, while setting a blank control.

[0047] Table 1 PCR amplification system

[0048]

[0049] PCR amplification conditions are: 94°C pre-denaturation for 5 minutes

[0050] Denaturation at 94℃ for 30sec

[0051] Annealing at 56℃ for 30sec

[0052] Extend at 72°C for 30sec

[0053] After extension at 72°C for 10 min,

[0054] Among them, 35 cycles of denaturation, annealing and extension were performed.

[0055] After the PCR products were electrophoresed in 1.0% TBE agarose gel, the results were observed under the ultraviolet transilluminator and photographed by the gel imaging system.

[0056] Results The...

Embodiment 3

[0057] The PCR sensitivity test of embodiment 3 kit

[0058] First, the DNA of Schistosoma japonicum was extracted, diluted, vortexed and mixed, and the total DNA content was detected according to the operating procedures of the Eppendorf Biophotometer Nucleic Acid Protein Analyzer. Dilute the DNA according to 8, 6, 4, 2, 0.8, 0.6, 0.4, 0.2, 0.1, and 0.08 ng / μL. The PCR amplification conditions are the same as above, and a blank control is set at the same time. PCR products were detected by 1.0wt% agarose gel electrophoresis to determine their sensitivity. The test results show that the PCR detection method has high sensitivity, and can detect 0.2ng DNA of Schistosoma japonicum ( figure 2 ).

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Abstract

The invention discloses a primer for discriminating schistosoma japonicas, a corresponding kit and a detection method thereof. The sequence of an upstream primer for discriminating the schistosoma japonicas is shown as SEQIDNO:1, and the sequence of a downstream primer is shown as SEQIDNO:2. In the invention, a method for fast detecting the schistosoma japonica is also established, and the schistosoma japonicas with different types can be discriminated accurately. The primer disclosed by the invention is prepared into the kit, has the advantages that the operating procedure is simplified, the detecting specificity is strong, the sensitivity is high and the result judgment is objective and the like and is suitable for being promoted and applied.

Description

technical field [0001] The invention relates to the field of inspection and detection, in particular to a primer for identifying Schistosoma japonicum, a corresponding kit and a detection method. Background technique [0002] Schistosomiasis japonicum is a zoonotic parasitic disease caused by Schistosoma japonicum, which is distributed worldwide, causing great economic losses and having important public health and socioeconomic significance. In my country, the disease is mainly prevalent in the Yangtze River Basin and the areas south of it. In addition to Shanghai, Zhejiang, Fujian, Guangxi and Guangdong provinces meeting the schistosomiasis interruption standards, Jiangsu, Anhui, Jiangxi, Hubei, Hunan, Yunnan and Sichuan 7 provinces are still serious. According to statistics, the population threatened by this disease reaches 60 million, of which only chronic patients reach more than 800,000. In 2004, my country listed schistosomiasis, tuberculosis and AIDS as the three mos...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 林瑞庆李娟赵光辉陈芬邹丰才翁亚彪宋慧群袁子国朱兴全
Owner SOUTH CHINA AGRI UNIV
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