Rapid 1-type dengue gold-marking diagnosis test paper strip
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A technology of dengue fever and test strips, applied in the field of antigens in disease diagnosis, to achieve high sensitivity, control the source of infection, and reduce costs
Inactive Publication Date: 2012-11-14
严延生
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[0006] Retrieval of relevant materials including Chinese patents shows that there is no gold-labeled rapid method for simultaneous detection of IgM and IgG antibodies in the serum of dengue virus-infected patients using genetic engineering methods to obtain highly expressed and highly active recombinant antigens from the DIII region of the outer membrane protein. Related reports on diagnostic test strips
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Embodiment 1
[0060] 1. Preparation of recombinant antigen
[0061] 1. The type 1 dengue virus strain used in this study is the Hawaii strain;
[0062] 2. Take the virus infection supernatant and use Viral RNA Mini Kit (QIAGEN) to extract viral RNA.
[0063] 3. Using P1 / R1 as PCR amplification primers, one-step RT-PCR was used to amplify the full-length E gene 2065bp from the type 1 dengue virus strain genome. The one-step RT-PCR reaction system is as follows:
[0066] 4. Purify the amplified product with QIAquick Gel extraction kit. Use TA cloning technology to clone the purified PCR product directly into the TA vector pMD 18-T to obtain a recombinant cloning plasmid, named T-DEN1E;
[0067] 5. Using the recombinant cloning plasmid T-DEN1E as a template, a fragment DEN1EDIII with a length of about 378 bp was amplified with primers F1D / R1D, and the amplified fra...
Embodiment 2
[0111] The production of type 1 dengue fever gold standard rapid diagnosis test strip
[0112] (1) Take 1000ml of 0.03% tetrachloroauric acid, heat it to boiling, add 25ml of 1% trisodium citrate solution, continue to boil for 5 minutes, wait until the color of the colloidal gold solution changes from blue to purple to red, cool it naturally for use;
[0113] (2) Take 1ml of colloidal gold solution and add 0.2M K 2 CO 3 10μl, 50μg purified antigen, stir well and let stand for 1h;
[0114] (3) Add 25μl of 20% bovine serum albumin, 20% PEG 20,000 15μl, 8,000r / min, centrifuge for 8min, discard the supernatant;
[0115] (4) Suspend the precipitate in 1ml of basic buffer, which is the gold-labeled antigen solution;
[0116] (5) Label mouse IgG in the same way;
[0117] (6) After mixing the two gold-labeled antigen solutions, fully soak the glass fiber paper, and freeze-dry it in a lyophilizer;
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Abstract
The invention discloses an recombinant antigen and a rapid gold-marking diagnosis paper strip for 1-type dengue. The invention mainly aims to provide an outer-membrane protein DIII region recombination antigen for the 1-type dengue diagnosis and can simultaneously aims to a rapid gold-marking diagnosis test paper strip used for detecting IgM, IgG antibodies in sera of 1-type dengue virus infected persons. The test paper strip utilizes a gene clone technology to obtain the outer-membrane protein DIII region recombination antigen of a 1-type dengue virus, and the outer-membrane protein DIII region recombination antigen can be used for immunology diagnosis after purification; the rapid gold-marking diagnosis test paper strip can simultaneously be used for detecting the IgM, IgG antibodies in the sera of the 1-type denguen virus infected persons, so as to judge the infection states of patients, to do early diagnosis on the patients, so that the important action for controlling an infection source and reducing disease spread; in addition, according to the gold-marking diagnosis test paper strip, a specific instrument is not required, and well-trained professional staff is not required, so that requirements of clinic detection in substrate hospitals and rural health centers can be met; and the cost is greatly reduced, the convenience is brought to the patients, and the test paper strip is suitable for large-scale seroepidemiological survey.
Description
Technical field [0001] The invention relates to an antigen for disease diagnosis, in particular to a recombinant antigen for serological diagnosis of type 1 dengue fever and a rapid gold-labeled diagnostic test strip. Background technique [0002] Dengue fever is a viral infectious disease transmitted by the Aedes mosquito. It is mainly prevalent in tropical and subtropical countries and regions such as Southeast Asia and the United States. Endemic dengue fever is transmitted in more than 100 countries, with millions of cases occurring every year. Sexual public health issues. In recent years, my country’s southeast coastal provinces have reported imported cases throughout the year, and the resulting local spread and outbreaks have become more frequent. As the main transmission vector of dengue fever-Aedes albopictus is widely distributed in the country, it spreads quickly and easily causes adverse social impacts. At the same time, it has an impact on local economic development, ...
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