Semi-nested PCR method testing toxoplasma gondii

A technology of toxoplasma gondii and detection kit, which is applied in the direction of biochemical equipment and methods, microbiological measurement/inspection, etc., can solve the problems of long time and achieve the effects of long time, improved diagnostic accuracy and high cost

Inactive Publication Date: 2008-08-20
ZHEJIANG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, these methods have shortcomings, such as serological methods require sufficient serum titers or pathogens, and mouse inoculation requires fresh disease materials, which takes a long time

Method used

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  • Semi-nested PCR method testing toxoplasma gondii
  • Semi-nested PCR method testing toxoplasma gondii
  • Semi-nested PCR method testing toxoplasma gondii

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] 1. Sample collection: Strictly aseptically collect no less than 500 μl of blood samples from suspicious pigs, or liver, spleen, lymph nodes and other tissues, and store them at 4°C or frozen;

[0029] 2. DNA extraction from the sample to be tested: take 200 μl of sterile anticoagulated blood (or take 2 g of samples such as liver, spleen and lymph nodes, put them in a mortar, add 2 ml of normal saline, grind them into a paste, and transfer them to a centrifuge tube), add 100 μl For Trizol reagent, mix well and add an equal volume of Tris saturated phenol / chloroform mixture for extraction. The supernatant is precipitated with 2 times the volume of absolute ethanol for 2 hours, washed with 70% ethanol and dried, and the precipitate is buffered with 10-20 μl TE After the solution is dissolved, store it at 4°C for later use;

[0030] 3. Set up the PCR detection kit, which includes:

[0031] (1) PCR tube: the tube contains 10×buffer, dNTP, primer 1, primer 2 and primer 3, Mg...

Embodiment 2

[0043] The method is the same as in Example 1, randomly aseptically collect pig blood samples, extract DNA, and amplify by PCR. For the results, see figure 2 , where 1 is positive control, 2 is negative control, 3, 11, 13, 14, 23 samples are positive, 4, 5, 6, 7, 8, 9, 10, 12, 15, 16, 17, 18, 19 , 20, 21, 22, and 24 samples were negative. The positive rate was 22.72%.

[0044] Sequences involved in the present invention

[0045]

[0046]

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Abstract

The invention provides a semi-nested PCR method for detecting toxoplasma gondii, which determines whether a sample is toxoplasma gondii infection positive or negative by arranging a PCR detection kit, extracting the DNA of a test sample and carrying out the semi-nested PCR amplification and the analysis of the amplified products. The method of the invention can rapidly and accurately detect the toxoplasma gondii in the test sample, and can also be used for the molecular epidemiological investigation and the efficacy monitoring of toxoplasmosis. The template DNA preparation steps of the method are simple, the cost is low, however, the conventional method needs to be processed by lysozyme, protease K, sodium dodecyl sulfate, cetyltrimethyl ammonium bromide and other reagents, the time is long and the cost is high. The method is established on the molecular biology, which can exclude the interferences of bacteria and impurity particles, thus greatly improving diagnostic accuracy and reducing false positive rate.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a semi-nested PCR method for detecting toxoplasma gondii. Background technique [0002] Toxoplasma gondii is an intracellular parasitic protozoan, and the toxoplasmosis caused by it is a worldwide public health problem. In my country, pork is one of the main sources of meat for people, and eating pork containing Toxoplasma gondii is the main way for humans to be infected with Toxoplasma gondii. At present, the detection of Toxoplasma gondii mainly relies on methods such as serology, pathogenic form identification, or intraperitoneal inoculation of mice with suspicious disease materials. However, these methods have disadvantages, for example, serological methods require sufficient serum titers or pathogens, and mouse inoculation requires fresh disease materials, which takes a long time. The PCR detection method only needs to have the nucleic acid of the pathogen to confirm the diagn...

Claims

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Application Information

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IPC IPC(8): C12Q1/04C12Q1/68
Inventor 杜爱芳曲道峰孔得翔
Owner ZHEJIANG UNIV
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