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Method for detecting PCR of toxoplasmas

A detection method and technology for Toxoplasma gondii, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of affecting diagnosis, polluted reaction system, time-consuming and laborious, etc.

Inactive Publication Date: 2010-02-17
SHANGHAI RES CENT FOR MODEL ORGANISMS
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Time-consuming, labor-intensive, careless human operation, equipment at the operating site (such as failure of the ultra-clean workbench), and even improper layout of the PCR operation area will greatly increase the probability of contamination of the reaction system, which will directly change the PCR results and affect the diagnosis.
[0005] In addition, in this revised manuscript, animals still need to be sacrificed to take ascites, other tissues, or a large amount of blood (0.2 ml has exceeded the maximum safe blood collection volume of mice) as a sample. Every time an animal is tested, it must be sacrificed or blood collection will cause animal injury

Method used

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  • Method for detecting PCR of toxoplasmas

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Effect test

Embodiment 1

[0062] Experimental mice were artificially infected with Toxophasma gondii (Toxophasma gondii) international standard strain RH strain (inbred line C57, 12 male and female mice, outbred line ICR, 24 male and female mice, and nude mice, 6 male and female mice, all Bought from SCXK (Shanghai) 2003-003 of Shanghai Slack Experimental Animal Co., Ltd., the animal experiment was carried out in the animal room SYXK (Shanghai) 2003-019 of Shanghai Institute of Family Planning Science), and a normal saline control group (inbred Line C57, outbred line ICR and nude mice, each with 6 males and 50 males). From the 4th to the 120th hour after infection, the urine and feces of the mice were collected every 4 hours (products of mouse metabolic cage Suzhou Fengshi Experimental Animal Equipment Co., Ltd.). PCR reaction tubes for Toxoplasma gondii PCR detection. At 120 hours after infection, blood was collected from the animals for serological testing. Toxoplasma gondii indirect hemagglutinatio...

Embodiment 2

[0064] The urine and feces of mice were collected from the sampled experimental mice of six different inspected units during the five-year re-evaluation test of the Shanghai experimental animal production license in 2007, and a total of 6 strains (KM, ICR, nude) were collected. Mouse, BALB / c, transgenic mouse, C57) 148 nucleic acid samples from 78 mice. After the nucleic acid was extracted, the self-made Toxoplasma gondii PCR rapid detection kit was used for detection. At the same time, the Toxoplasma gondii PCR detection kit produced by German genekam was used for comparison. In addition to the kit comparison, a 3-person comparison was also carried out. As a result, the PCR reactions were all negative, and the results of the comparison among the mouse samples detected by the self-made PCR rapid detection kit were consistent, and the results of the German genekam product were completely consistent with the self-made Toxoplasma gondii PCR rapid detection kit.

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Abstract

The invention discloses a method for detecting PCR of toxoplasmas. The detecting method comprises the following steps: (1) mixing a primer, Taq DNA polyase, a polyase stabilizer, four mononucleotides,a buffer solution and a sampling buffer solution before a PCR product is subjected to electrophoresis, and lyophilizing the mixture into powder; (2) mixing the powder obtained in the step (1) and a sample to perform a PCR reaction so as to obtain the PCR product; and (3) performing electrophoresis detection on the PCR product. The sample comprises nucleic acid which is extracted from mammalian urine and / or faeces.

Description

technical field [0001] The invention relates to the technical field of biomedicine and experimental animal parasite quality control, in particular to a PCR detection method for toxoplasma gondii. Background technique [0002] The detection of Toxoplasma gondii in the current national standard relies on serological detection, but serological detection has unavoidable lag and it is difficult to detect pathogens in the environment. [0003] The PCR method for the detection of Toxoplasma gondii is listed in the revised draft of the new national standard for quality control of experimental animals. This method is an effective supplement to the original serological methods (IHA, IFA, ELISA, etc.). However, animals still need to be sacrificed, and the detection method is relatively cumbersome. [0004] Since PCR is amplified in an exponential manner, a very small amount of DNA contamination may cause false positive results, so the cleanliness of the reaction system is absolutely ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04
Inventor 胡建华濮俊毅高诚
Owner SHANGHAI RES CENT FOR MODEL ORGANISMS
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