Reagent kit for detecting Toxoplasma Gondii and detection method thereof

A detection kit and a technology for Toxoplasma gondii are applied in the field of detection kits for Toxoplasma gondii, and can solve problems such as discomfort in grassroots applications and the like

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

AI Technical Summary

Problems solved by technology

The molecular biology diagnosis method is mainly polymerase chain reaction (PCR), which is faster and more sensitive than the first two methods, but it needs to use a special PCR instrument, which is not suitable for grassroots applications

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0128] Kit preparation:

[0129] (1) select primer set 1;

[0130] (2) Preparation of LAMP reaction solution: 0.5 μmol Tris-HCl, 0.25 μmol KCl, 0.25 μmol (NH 4 ) 2 SO 4 , 0.04 μmol Triton x-100, 0.1 μmol MgSO 4 , 5 μmol betaine (Betaine), 0.02 μmol deoxynucleotides (dNTPs), 0.04 μmol upstream internal primer (FIP), 0.04 μmol downstream internal primer (BIP), 0.004 μmol upstream external primer (F3), 0.004 μmol downstream external primer (B3), 8U of Bst DNA polymerase, and the rest in sterilized double distilled water, mix and configure 23 μL LAMP reaction solution;

[0131] (3) Positive control: 2 μL Toxoplasma gondii DNA;

[0132] (4) Negative control: 2 μL sterilized double distilled water;

[0133] (5) 1 μL of fluorescent dye SYBR GREEN I.

[0134] The 0.02 μmol of deoxyribonucleotide used in this example includes 0.02 μmol of deoxyribose adenine nucleotide, 0.02 μmol of deoxyribose guanine nucleotide, 0.02 μmol of deoxyribocytosine nucleotide and 0.02 μmol of deoxyr...

Embodiment 2

[0136] Kit preparation:

[0137] (1) select primer set 2;

[0138] (2) Preparation of LAMP reaction solution: according to 0.5 μmol Tris-HCl, 0.25 μmol KCl, 0.25 μmol (NH 4 ) 2 SO 4 , 0.04 μmol Triton x-100, 0.2 μmol MgSO 4 , 35 μmol betaine (Betaine), 0.04 μmol deoxynucleotides (dNTPs), 0.06 μmol upstream inner primer (FIP), 0.06 μmol downstream inner primer (BIP), 0.008 μmol upstream outer primer (F3), 0.008 μmol downstream outer primer (B3), 8U of Bst DNA polymerase, the rest is sterilized double-distilled water, and 23 μL of LAMP reaction solution is prepared;

[0139] (3) Positive control: 2 μl of Toxoplasma gondii DNA;

[0140] (4) Negative control: 2 μl of sterilized double distilled water;

[0141] (5) 3 µl of fluorescent dye SYBR GREEN I.

[0142] The 0.04 μmol of deoxyribonucleotide used in this example includes 0.04 μmol of deoxyribose adenine nucleotide, 0.04 μmol of deoxyribose guanine nucleotide, 0.04 μmol of deoxyribocytosine nucleotide and 0.04 μmol of d...

Embodiment 3

[0144] Kit preparation:

[0145] (1) Select primer sets 3 and 4;

[0146] (2) Preparation of LAMP reaction solution: according to 0.5 μmol Tris-HCl, 0.25 μmol KCl, 0.25 μmol (NH 4 ) 2 SO 4, 0.04 μmol Tritonx-100, 0.2 μmol MgSO 4 , 20 μmol betaine (Betaine), 0.03 μmol deoxynucleotides (dNTPs), 0.03 μmol upstream internal primer (FIP), 0.03 μmol downstream internal primer (BIP), 0.006 μmol upstream external primer (F3), 0.006 μmol downstream external primer (B3) and 8U of Bst DNA polymerase;

[0147] (3) Positive control: 2 μL of Toxoplasma gondii DNA;

[0148] (4) Negative control: 2 μL of sterilized double distilled water;

[0149] (5) 2 μL of fluorescent dye SYBR GREEN I.

[0150] The 0.03 μmol of deoxyribonucleotide used in this example includes 0.03 μmol of deoxyribose adenine nucleotide, 0.03 μmol of deoxyribose guanine nucleotide, 0.03 μmol of deoxyribocytosine nucleotide and 0.03 μmol of deoxyribose thymidine acid.

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Abstract

The invention provides a detecting reagent box of toxoplasmin; the box comprises the following components of LAMP reaction liquid, positive control, negative control and fluorescence visualization reagent. The invention also provides a detection method with the detection reagent box of the toxoplasmin. By adopting the toxoplasmin detection reagent box and method of the present invention, the detection sensibility is high and only 6 to 10 copies are needed for detecting the object DNA; moreover, the operation is simple; therefore, the invention is especially fit for clinical medical detection at grass-roots level and on-the-spot instant detection.

Description

technical field [0001] The invention relates to a detection kit for toxoplasma gondii and a detection method thereof, in particular to a detection kit for toxoplasma gondii based on a loop-mediated isothermal amplification technique and a corresponding detection method thereof. Background technique [0002] Toxoplasmosis is a zoonotic protozoan disease caused by Toxoplasma gondii. Toxoplasma can infect a variety of animals: 45 mammals, 70 birds and 5 cold-blooded animals. Cats and felines are the final hosts of Toxoplasma gondii. In the final host, Toxoplasma gondii mainly invades the epithelial cells of the small intestine, causing intestinal diseases. In intermediate hosts, such as pigs, cattle, sheep, etc., Toxoplasma gondii invades the intestinal wall, enters the cells of the mononuclear macrophage system through the blood or lymph, and spreads to tissues and organs throughout the body, such as the brain, lymph nodes, and liver. , heart, lungs, muscles, etc., causing m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04
Inventor 吴文学索勋朱引洁邹杰师凯张跃伟郭盼盼王文欢
Owner CHINA AGRI UNIV
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