Specific primers, probes, kit and chip for gene detection of Toxoplasma gondii

A technology for detecting chips and Toxoplasma gondii is applied in the field of bioengineering, which can solve the problems of labor and time consumption, low throughput, complicated process of Toxoplasma gondii, etc., and achieve the effects of simple operation, accurate results and controllable cost.

Pending Publication Date: 2020-04-10
中国疾病预防控制中心寄生虫病预防控制所国家热带病研究中心
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Problems solved by technology

[0006] The object of the present invention is to provide a kind of specific primer, probe, kit and chip of Toxoplasma gondii gene detection, described a kind of specific primer, probe, The kit and chip should solve the technical problems of complex detection process, low throughput, manpower and time-consuming in the prior art for detecting Toxoplasma gondii

Method used

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  • Specific primers, probes, kit and chip for gene detection of Toxoplasma gondii
  • Specific primers, probes, kit and chip for gene detection of Toxoplasma gondii
  • Specific primers, probes, kit and chip for gene detection of Toxoplasma gondii

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Embodiment 1

[0029] The preparation of embodiment 1 gene chip

[0030] The following specific probes were artificially synthesized, dissolved in water to a concentration of 100 pmol / μL, and then mixed in equal proportions with 2× spotting buffer (product number: BST02010, Shanghai Bio Technology Co., Ltd.). Next, use a spotting instrument to spot on the aldehyde-modified glass slide (product number: BST03011, Shanghai Bio Technology Co., Ltd.) according to the method described in the instructions. figure 1 array of . Leave overnight at room temperature.

[0031] Each specific probe sequence is shown in SEQ ID No.3-6.

[0032] Such as figure 1 As shown, quality control probes, negative control probes, positive control probes and Toxoplasma gondii probes were spotted on the slide. Among them, QC is a quality control probe, NC is a negative probe, PC is a positive probe, and T. gondii 1-4 are Toxoplasma gondii probes.

[0033] SEQ ID No. 3: gttcttaatg ccggctttgt acggaggata tggtaacttc

...

Embodiment 2

[0037] Preparation of embodiment 2 chromosomal DNA

[0038] Use the QIAmp DNA Mini kit250 blood sample DNA extraction kit (Qiagen) according to the instructions as follows:

[0039] (1) Take 100 μL of the blood sample to be tested, add 100 μL of PBS, and then add 20 μL of proteinase K;

[0040] (2) Add 200 μL of AL buffer, mix well and place in a 56°C water bath for 10 minutes;

[0041] (3) Add 200 μL absolute ethanol and mix well;

[0042] (4) Move the above reaction system to a filter tube, centrifuge at 8000rpm for 1min, and transfer the adsorption column to a new 2ml empty tube;

[0043] (5) Add 500 μL of AW1 solution, centrifuge at 8000 rpm for 1 min, and discard the filtrate;

[0044] (6) Add 500 μL of AW2 solution, centrifuge at 14000 rpm for 3 minutes, and discard the filtrate;

[0045] (7) No addition of washing solution, 14000rpm, centrifuge for 1min, spin to remove excess solution on the column; transfer the adsorption column to a 1.5mL empty tube, add 150μL Buf...

Embodiment 3

[0046] Embodiment 3 amplifies Toxoplasma gondii coxI gene by PCR method with the primer provided by the present invention

[0047] Entrust Shanghai Sangon Bioengineering Technology Service Co., Ltd. to synthesize primers, and the primer information is as follows.

[0048] The primer sequences used to amplify the coxI gene of Toxoplasma gondii are:

[0049]SEQ ID No.1: Upstream 5'-GATAATTACAATACATGGTC-3',

[0050] SEQ ID No.2: Downstream 5'-TAGAAGACAAATCCACAT-3'.

[0051] Also, the 5' end of the primer was modified with biotin.

[0052] Then dissolve and dilute to 50 pmol / μL with water. The purchased Taq enzyme (TaKaRa), 10×PCR buffer (TaKaRa), MgCl 2 (TaKaRa), dNTPs(TaKaRa), ddH 2 0 and the PCR template that embodiment 2 obtains configures PCR amplification system by following table 1 formula:

[0053] Table 1 PCR amplification system

[0054]

[0055] Use a PCR instrument (TC-96 / G / H PCR amplification instrument, Hangzhou Bioer) to amplify according to the following ...

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Abstract

The invention relates to the field of molecular biology, and provides specific primers for detecting Toxoplasma gondii. The sequence of an upstream primer in the specific primers is as shown in SEQ IDNo. 1; the sequence of a downstream primer in the specific primers is as shown in SEQ ID No. 2. The invention also provides probes for detecting Toxoplasma gondii. The sequences of the probes are asshown in SEQ ID No. 3-6. The invention further provides a kit for detecting Toxoplasma gondii. The kit comprises the primers and the probes for detecting Toxoplasma gondii. The invention further provides a chip for detecting Toxoplasma gondii. The chip comprises the probes for detecting Toxoplasma gondii. According to the invention, gene chip detection is carried out on Toxoplasma gondii by usingthe primers and the probes, and differential diagnosis can be specifically carried out on Toxoplasma gondii infection conditions.

Description

technical field [0001] The invention belongs to the field of bioengineering and relates to the detection of toxoplasma gondii, in particular to a specific primer, probe, kit and chip for the detection of toxoplasma gondii gene. Background technique [0002] Toxoplasmosis is a zoonotic parasitic protozoan disease caused by Toxoplasma gondii. The parasite mainly invades the eyes, brain, heart and other organs, parasitizes in nucleated cells, and the human body usually manifests as recessive infection. Toxoplasma gondii is one of the important pathogens causing fetal malformation caused by intrauterine infection during pregnancy. [0003] Toxoplasma is widely found in many mammals, and humans are also important hosts of the parasite. It is estimated that about 1 billion people in the world are infected with Toxoplasma gondii, and the antibody-positive rate of the population is 25%-50%, most of which are recessive infections. Toxoplasma gondii infection and toxoplasmosis are ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6837C12Q1/6893C12Q1/04C12N15/11
CPCC12Q1/6837C12Q1/6893C12Q2531/113C12Q2565/501C12Q2563/107
Inventor 陈家旭胡薇周晓农艾琳陈木新陈韶红宋鹏李浩卢艳蔡玉春田利光储言红吴秀萍俞英昉
Owner 中国疾病预防控制中心寄生虫病预防控制所国家热带病研究中心
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