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Fluorescent PCR (polymerase chain reaction) method and kit for specifically detecting Toxoplasma gondii nucleic acid

A Toxoplasma-specific technology, applied in microorganism-based methods, biochemical equipment and methods, and microbial determination/inspection, etc., can solve the problems of easy contamination, weak antibody response, poor specificity, etc., and achieve high reaction efficiency. Effect

Pending Publication Date: 2017-08-04
ZHANGJIAGANG LANSU BIOLOGICAL ENG
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the difficulty in the source of Toxoplasma gondii antigens, serological tests are still difficult to popularize, and some people have weak or no antibodies after infection with the virus. Therefore, the results of antibody testing alone cannot accurately determine the infection of Toxoplasma gondii. Antigens or nucleic acids are needed. The test results are used as the basis for diagnosis and treatment
At present, the relatively mature nucleic acid detection method is the fluorescent PCR method, which overcomes the shortcomings of conventional PCR methods such as poor specificity, easy contamination, and many steps in result analysis. The TaqMan method or double-labeled probe method (fluorescent quenched probe or FQ probe), that is, a fluorescent reporter group and a fluorescent quencher group are labeled at its 5' end and 3' end respectively

Method used

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  • Fluorescent PCR (polymerase chain reaction) method and kit for specifically detecting Toxoplasma gondii nucleic acid
  • Fluorescent PCR (polymerase chain reaction) method and kit for specifically detecting Toxoplasma gondii nucleic acid
  • Fluorescent PCR (polymerase chain reaction) method and kit for specifically detecting Toxoplasma gondii nucleic acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Embodiment 1: the preparation of kit

[0070] 1. Design and synthesis of specific primer pairs and probes

[0071] Use the Primer Express 3.0 software to design specific primer pairs and specific probes using the DNA sequence of the Toxoplasma gondii B1 gene. Reporter and fluorescent quencher labels. Among them, the primers were purified by PAGE, and the probes were purified by HPLC.

[0072] The designed specific primer pairs and probes are as follows:

[0073] Forward primer: 5'-CCGGGTGAAACAATAGAGAGTACTG-3' (SEQ ID NO.1);

[0074] Reverse primer: 5'-GGTCTACGTCGATGGCATGA-3' (SEQ ID NO.2);

[0075] The probe is: 5'-AACGTCGCCGCTACTGCCCAGTT-3' (SEQ ID NO.3).

[0076] After the primers and probes were synthesized, they were prepared into lyophilized powder, then diluted with 1×TE buffer to a concentration of 100 μM as a mother solution, and stored at -20°C. The working solution is prepared by diluting the mother solution 10 times with sterile water for routine use. ...

Embodiment 2

[0083] Embodiment 2: Extraction of sample nucleic acid

[0084] Use the blood / tissue / cell genome extraction kit (DP304) of Tiangen Biochemical Technology (Beijing) Co., Ltd., and follow the steps in the kit instructions to extract nucleic acid from the sample. After extraction, the product was stored at -20°C.

Embodiment 3

[0085] Embodiment 3: the making of standard curve

[0086] Select the positive quality control product as the standard substance, dilute the concentration to 1 μg / mL, and then continue to dilute five concentrations in a 10-fold gradient, a total of 6 concentration gradients, namely 1 μg / mL, 10 -1 μg / mL, 10 -2 μg / mL, 10 -3 μg / mL, 10 -4 μg / mL, 10 -5 μg / mL. Then, the samples of these six concentration gradients were used as the samples to be tested according to the reaction system and reaction conditions of the following example 4, and the fluorescent PCR amplification reaction was performed, and the standard curve was generated with the software matched with the ABI7500 instrument. The result is as figure 1 and figure 2 As shown, and the system of the instrument shows that the reaction efficiency reaches 88.5%.

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Abstract

The invention relates to a fluorescent PCR (polymerase chain reaction) kit for specifically detecting Toxoplasma gondii nucleic acid, comprising a specific primer pair and a probe; the specific primer pair includes a forward primer: 5'-CCGGGTGAAACAATAGAGAGTACTG-3' and a reverse primer: 5'-GGTCTACGTCGATGGCATGA-3'; the probe is 5'-AACGTCGCCGCTACTGCCCAGTT-3'. The kit of the invention comprises the specific primer pair and probe, has the advantages of high sensitivity, high specificity and high reaction efficiency, can provide qualitative detection for Toxoplasma gondii, and may act as an effective auxiliary detection tool.

Description

technical field [0001] The invention belongs to the field of in vitro nucleic acid detection of microorganisms, and in particular relates to a fluorescent PCR method and a kit for specifically detecting the nucleic acid of Toxoplasma gondii, which can qualitatively detect the DNA of Toxoplasma gondii and can be used as an effective detection tool. Background technique [0002] Toxoplasmosis is a zoonotic parasitic disease caused by Toxoplasma gondii (Toxoplasma gondii), which is distributed worldwide and seriously endangers human health. Toxoplasma gondii is an obligate intracellular parasite that infects humans and most mammals. According to incomplete statistics, about one-third of adults in the world are infected with Toxoplasma gondii. The vast majority of adults infected by Toxoplasma gondii will not cause serious symptoms, but for organ transplants, malignant tumors, AIDS and other immunocompromised persons, as well as pregnant women, Toxoplasma infection can lead to ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/90
CPCC12Q1/686C12Q1/6893C12Q2563/107C12Q2545/101
Inventor 余圣良方园周健
Owner ZHANGJIAGANG LANSU BIOLOGICAL ENG
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