Animals toxoplasmosis PCR detection reagent kit

A detection kit and technology for toxoplasmosis are applied in the field of diagnosis and detection of animal toxoplasmosis to achieve the effects of objective judgment, simple operation and strong specificity

Inactive Publication Date: 2008-05-21
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is currently no detection method and kit for Toxoplasma gondii designed and invented by using this scientific research achievement

Method used

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  • Animals toxoplasmosis PCR detection reagent kit
  • Animals toxoplasmosis PCR detection reagent kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] The composition of embodiment 1 kit

[0049] The kit contains 30ml of DNA lysate, which contains 100mM NaCl, 10mM Tris-Cl at pH 8.0, 25mM EDTA at pH 8.0, 1% (W / V) SDS and 4μg / μL proteinase K; Red blood cell lysate 100ml, containing 10mM NaCl, pH 8.0 10mM Tris-Cl, 5mM MgCl 2 100ml of white blood cell lysate containing 100mM NaCl, 10mM Tris-Cl of pH8.0, EDTA of pH8.0 imM, 0.5% (W / V) SDS; 100 reactions of PCR reaction solution (25 μ L / reaction ), dATP, dTTP, dGTP, dCTP at a final concentration of 200 μM each, primers TOX4 and TOX5 at a final concentration of 0.5 pmol / μL, and MgCl at 2 mM 2 , Taq enzyme was added in 1.25 U for each PCR reaction, and 1 Toxoplasma gondii genomic DNA positive control.

Embodiment 2

[0050] Embodiment 2 kit specificity test

[0051] Use 1 μL each of DNA from six control samples, including Neospora caninum, Trichomonas, Cryptosporidium parvum, Plasmodium falciparum, Eimeria tenella, and Ascaris suum, which have been verified for DNA validity, as templates, and follow the kit’s reaction Conditions for specific PCR amplification, while setting a blank control and a positive control kit.

[0052] The PCR amplification conditions are:

[0053] Pre-denaturation at 94°C for 5 minutes

[0054]

[0055] Extend at 72°C for 10 minutes

[0056] After the PCR products were electrophoresed in 0.8% TBE agarose gel, the results were observed under an ultraviolet transilluminator and photographed by a gel imaging system.

[0057] As a result, only a band of about 530 bp was amplified in the positive control DNA sample of the Toxoplasma gondii kit, while no band appeared in the other six control parasites and the negative control.

Embodiment 3

[0058] The sensitivity test of embodiment 3 kit

[0059] First, the Toxoplasma gondii tachyzoite DNA extracted after counting was diluted, vortexed and mixed, and the total DNA content was detected according to the operating procedures of the Eppendorf Biophotometer nucleic acid and protein analyzer. The tachyzoite DNA was diluted 10×, 100×, 1000×, 2000×, 4000× by the doubling dilution method. The PCR amplification conditions were the same as above, and a blank control was set at the same time. PCR products were detected by 0.8% agarose gel electrophoresis to determine their sensitivity. Through the PCR reaction of Toxoplasma gondii DNA in 5 concentration gradients, it is shown that the PCR detection method can detect the DNA of 0.5 Toxoplasma gondii tachyzoites at least.

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Abstract

The invention discloses a PCR detection reagent box of animal toxoplasmosis; the box contains DNA lysate, red blood cell lysate, white blood cell lysate, PCR reaction liquid and toxoplasmosis gene DNA positive control liquid; the 529bp repeated DNA segment of toxoplasmin is taken as genetic mark and special primer is taken to optimize the PCR reaction conditions such as the magnesium ion consistency, the anneal temperature and the regulation of the cycle number. The invention can detect the toxoplasmosis infection of pig, dog and cat in a fast special sensible way; the operation is convenient and the sensibility is high; the invention can be used for diagnosing the toxoplasmosis of pig, dog and cat and detecting and testing the drug effect appraisal of the selection of the toxoplasmosis medicines.

Description

technical field [0001] The invention relates to the technical field of diagnosis and detection of animal toxoplasmosis, in particular to a PCR detection kit for animal toxoplasmosis and a use method thereof. Background technique [0002] Toxoplasmosis is a zoonotic parasitic disease caused by Toxoplasma gondii infecting humans and various animals. It is widely distributed in my country and all over the world. The average human infection rate in western countries is as high as about 30% (Tenter et al, 2000; Xie Mingquan and Li Guoqing, 2003), the average infection rate of the human body in my country is 7.88%, and there are about 100 million people infected in the country (Lu Yuancong and Cui Junzhao, 1994; Chen Zhaoyi et al., 2005; Feng Yueju et al., 2005; Xu Longqi et al., 2005; Xu Xiangzhen et al., 2005; Zhang Jing et al., 2006), and the animal infection rate is higher (Xie Dehua et al., 2004). [0003] Toxoplasmosis is not only one of the most common infectious diseases in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 宋慧群朱兴全翁亚彪林瑞庆
Owner SOUTH CHINA AGRI UNIV
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