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Specific primers, probe, kit and chip for Trypanosoma gene detection

A kit and probe technology, applied in the field of bioengineering, can solve the problems of complex, high-throughput, labor-consuming, and time-consuming detection of trypanosomes, and achieve the effects of controllable cost, accurate results, and easy operation.

Pending Publication Date: 2020-04-07
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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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0008] The object of the present invention is to provide a kind of specific primer, probe, test kit and chip for trypanosome gene detection, described this specific primer, probe, test kit and chip for trypanosome gene detection To solve the technical problems of complex, high-throughput, labor-intensive and time-consuming processes for detecting trypanosomes in the prior art

Method used

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  • Specific primers, probe, kit and chip for Trypanosoma gene detection
  • Specific primers, probe, kit and chip for Trypanosoma gene detection
  • Specific primers, probe, kit and chip for Trypanosoma gene detection

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Embodiment 1

[0040] The preparation of embodiment 1 gene chip

[0041] 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.

[0042] like figure 1 As shown, quality control probes, negative control probes, positive control probes and trypanosome probes were spotted on the slide. Among them, QC is quality control probe, NC is negative probe, PC is positive probe, Trypanosoma 1~12 is trypanosoma probe; T.cruzi1~3 is Trypanosoma cruzi probe; T.b.rhodesiense 1~3 is trypanosoma probe Probe for Trypanosoma brucei Rhodesia; T.b.gambiense 1~3 are probes for Trypanos...

Embodiment 2

[0065] Preparation of embodiment 2 chromosomal DNA

[0066] Use the QIAmp DNA Mini kit250 blood sample DNA extraction kit (Qiagen) according to the instructions as follows:

[0067] (1) Take 100 μL of the blood sample to be tested, add 100 μL of PBS, and then add 20 μL of proteinase K;

[0068] (2) Add 200 μL of AL buffer, mix well and place in a 56°C water bath for 10 minutes;

[0069] (3) Add 200 μL absolute ethanol and mix well;

[0070] (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;

[0071] (5) Add 500 μL of AW1 solution, centrifuge at 8000 rpm for 1 min, and discard the filtrate;

[0072] (6) Add 500 μL of AW2 solution, centrifuge at 14000 rpm for 3 minutes, and discard the filtrate;

[0073] (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

[0074] Embodiment 3 uses the primer provided by the invention to amplify trypanosome gene by PCR method

[0075] Entrust Shanghai Sangon Bioengineering Technology Service Co., Ltd. to synthesize primers, and the primer information is as follows.

[0076] The primer sequences used to amplify the 18S gene of Trypanosoma cruzi are:

[0077] SEQ ID NO.1: Upstream 5'-CGAACAACTGCCCTATCA-3',

[0078] SEQ ID NO. 2: Downstream 5'-ACGACTTTTGCTTCCTCT-3'.

[0079] The primer sequences used to amplify the 18S gene of Trypanosoma brucei are:

[0080] SEQ ID NO.3: Upstream 5'-TTCTACGGAGGGCAGCAGGC-3',

[0081] SEQ ID NO.4: Downstream 5'-CTGTAACCTCAAAGCTTTCGCGT-3'.

[0082] The primer sequences used to amplify the P67 gene of Trypanosoma brucei are:

[0083] SEQ ID NO.5: Upstream 5'-CTCTGCGCTTTTGTCTGA-3',

[0084] SEQ ID NO. 6: Downstream 5'-TACGCCCCACTGCTTTCC-3'.

[0085] The primer sequences used to amplify the HP gene of Trypanosoma brucei gambiae are:

[0086] SEQ ID NO.7: Upstream...

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Abstract

The invention provides specific primers for the detection of Trypanosoma cruzi with the sequences as shown in SEQ ID No.1 and SEQ ID No.2, provides specific primers for the detection of Trypanosoma brucei with the sequences as shown in SEQ ID No.3 and SEQ ID No.4, provides specific primers for the detection of Trypanosoma brucei rhodesiense with the sequences as shown in SEQ ID No.5 and SEQ ID No.6, provides specific primers for the detection of Trypanosoma brucei gambiense with the sequences as shown in SEQ ID No.7 and SEQ ID No.8, provides a probe for the detection of Trypanosoma with the sequences as shown in SEQ ID No.9 to 26, and also provides a kit and a chip for the detection of Trypanosoma. In the invention, differential diagnosis for Trypanosoma infection can be specifically carried out.

Description

technical field [0001] The invention belongs to the field of bioengineering and relates to the detection of trypanosome genes, in particular to a specific primer, probe, kit and chip for the detection of trypanosome genes. Background technique [0002] Trypanosomiasis is a blood protozoan disease caused by African and American trypanosomiasis. African trypanosomiasis is caused by Trypanosoma brucei gambiense (Trypanosoma brucei gambiense) and Trypanosoma brucei rhodesiense (Trypanosoma brucei rhodesiense). The medium of transmission is the tsetse fly. [0003] African trypanosomiasis, also known as sleeping sickness, caused by Trypanosoma brucei gambiae accounts for more than 95% of all reported cases and is often a chronic infection. Patients can be infected for months or even years without noticeable symptoms. By the time symptoms appear, patients are often in advanced disease and the central nervous system is affected. Trypanosoma brucei usually causes an acute infect...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6893C12Q1/6837C12N15/11
CPCC12Q1/6893C12Q1/6837
Inventor 陈家旭周晓农胡薇陈木新艾琳陈韶红李浩宋鹏蔡玉春卢艳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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