Toxoplasma gondii fluorogenic quantitative PCR (polymerase chain reaction) specific primer, kit and detecting method of toxoplasma gondii fluorogenic quantitative PCR specific primer

A fluorescence quantitative and detection method technology, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve the problems of low detection efficiency and insufficient detection sensitivity, and achieve high sensitivity and results. Judging the effect of objective and simple operation

Inactive Publication Date: 2016-09-28
广州海沥生物科技有限公司
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  • Abstract
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  • Application Information

AI Technical Summary

Problems solved by technology

[0021] The purpose of the present invention is to provide a pair of specific primers that can be used to detect Toxoplasma gondii in order to overcome the problems of insufficient detection sensitivity and low detection efficiency in the prior art

Method used

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  • Toxoplasma gondii fluorogenic quantitative PCR (polymerase chain reaction) specific primer, kit and detecting method of toxoplasma gondii fluorogenic quantitative PCR specific primer
  • Toxoplasma gondii fluorogenic quantitative PCR (polymerase chain reaction) specific primer, kit and detecting method of toxoplasma gondii fluorogenic quantitative PCR specific primer
  • Toxoplasma gondii fluorogenic quantitative PCR (polymerase chain reaction) specific primer, kit and detecting method of toxoplasma gondii fluorogenic quantitative PCR specific primer

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Effect test

Embodiment 1

[0069] The composition of embodiment 1 kit

[0070] The kit contains:

[0071] 1) DNA extraction solution, which contains 50mL of Buffer GA, 50mL of Buffer GB, 52mL of Buffer GD, 50mL of Buffer PW, 60mL of Buffer TE, 4mL of Proteinase K, and 10mL of absolute ethanol that have been reacted 200 times;

[0072] 2) Fluorescent quantitative PCR reaction solution is SYBR Green I reagent reacted 200 times, Toxoplasma gondii-specific primer with a final concentration of 10 pmol / μL, sterilized double distilled water, MgCl2, dNTPs, Taq enzyme.

[0073] 3) The quality control reaction solution is a negative control reaction solution and a positive control reaction solution that have been reacted 200 times.

Embodiment 2

[0074] Embodiment 2 kit specificity test

[0075] Using DNA from RH strain, DNA from PRU strain, DNA from VEG strain, DNA from Isospora suis, DNA from Epierythropodium, and DNA from Neospora as templates, carry out specific fluorescent quantitative PCR amplification according to the reaction conditions of the kit, and set a blank at the same time control.

[0076] The reaction system was: SYBR Green I 10 μL, upstream and downstream primers 0.4 μL each, double distilled water 7.2 μL, template DNA 2 μL.

[0077] Fluorescent quantitative PCR amplification conditions are:

[0078]

[0079] The results showed that the DNA of different strains of Toxoplasma gondii had specific amplification, and the negative control and other DNAs had no amplification ( figure 1 ).

Embodiment 3

[0080] Standard curve and sensitivity analysis of embodiment 3 kit

[0081] respectively by 10 10 ~10 0 The plasmid standard product of copy / μL was used as a template for real-time fluorescent quantitative PCR amplification, and 5 consecutive points were selected to make a standard curve ( figure 2 ) and the standard equation (Table 1) and analyze its sensitivity, the results show that the fluorescent quantitative PCR has no amplification when the template concentration is below 10 copies / μL, and it can be concluded that the sensitivity of the fluorescent quantitative PCR is 10 copies of the target DNA .

[0082] Table 1 Toxoplasma gondii fluorescence quantitative PCR standard equation

[0083]

[0084] Note: x=lg (initial template concentration), CT value is the cycle number when the fluorescence signal reaches the set threshold.

[0085] In order to more truly reflect the gap between the theoretical lower limit of detection and the actual lower limit of detection, th...

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Abstract

The invention discloses a toxoplasma gondii fluorogenic quantitative PCR specific primer, a kit and a detecting method of the toxoplasma gondii fluorogenic quantitative PCR specific primer. The kit comprises the toxoplasma gondii fluorogenic quantitative PCR specific primer, a DNA extracting solution, a fluorogenic quantitative PCR reacting solution, a quality-controlling reacting solution, Spin Columns CB3 and Collection Tubes 2ml. According to the kit, GRA7 gene is utilized as a genetic marker, and the specific primer is used for optimizing PCR reacting system conditions like reagent concentration and temperature. Thus, the kit can quickly, specifically and flexibly detect whether animals are infected by toxoplasmosis, is convenient to operate and can be applied to diagnosis of various kinds of animal toxoplasmosis.

Description

technical field [0001] The invention relates to the technical field of molecular biology diagnosis, in particular to a fluorescent quantitative PCR specific primer and its application detection technology. Background technique [0002] Toxoplasma gondii is an obligate intracellular parasite. The life cycle of Toxoplasma gondii includes five forms, namely trophozoite (also known as tachyzoite), cyst (which can survive in the tissue for a long time, and can release bradyzoite after rupture), schizont, gametophyte and cyst zygote, The first 3 periods are asexual reproduction, and the last 2 periods are sexual reproduction. The completion of the life cycle of Toxoplasma gondii requires an intermediate host and a final host. Toxoplasma parasitizes in the intermediate host for asexual reproduction, and can only reproduce sexually in the final host. [0003] Cats and other cats are the ultimate hosts of Toxoplasma gondii. Sexual reproduction of Toxoplasma gondii only develops in ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
Inventor 廖昕萌袁子国谢国清
Owner 广州海沥生物科技有限公司
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