Specific primer for detecting trypanosome protozoan, detection method and application

A specific, trypanosomal technology, applied in the fields of molecular biology and pathogenic biology, can solve the lack of specificity in the detection of trypanosoma protozoa, the lack of versatility of trypanosoma protozoa, and the detection results are affected by other blood-borne protozoa to achieve the effects of high versatility within the genus, improved efficiency, and strong specificity between genus

Active Publication Date: 2018-04-13
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Existing reports in the prior art adopt PCR technology to detect the method for detecting trypanosoma protozoa, but still there are following deficiencies: 1) existing method is not strong for the universality that trypanosoma protozoa detects, can only be to trypanosoma protozoa 2) The specificity of the existing method for the detection of Trypanosoma protozoa is still lacking, and the test results will be affected by other blood samples. Influence of source protozoa

Method used

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  • Specific primer for detecting trypanosome protozoan, detection method and application
  • Specific primer for detecting trypanosome protozoan, detection method and application
  • Specific primer for detecting trypanosome protozoan, detection method and application

Examples

Experimental program
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Effect test

Embodiment 1

[0043] Embodiment 1 The generality within the genus of the specific primers of the special detection trypanosoma protozoa of the present invention:

[0044] Use the following methods to detect:

[0045] (1) extract the genomic DNA of the target sample;

[0046] DNA extraction was carried out using DNeasy Blood & Tissue Kit (QIAGEN) according to the instructions. In addition, this method can also detect the Whatman Classic FTA card (WB120205) that contains the worm body, and the specific processing steps are as follows: punch a diameter of 1.2mm from the target area on the FTA card by a puncher (1.2mm in diameter, produced by Whatman, UK). Place the wafer in a PCR tube, soak it with water for 2-3 times, 15 minutes each time, and then put it in an oven at 80°C for 10 minutes, and then add the PCR reaction system into the PCR tube to achieve DNA amplification.

[0047] The target samples include T.b.brucei samples, T.b.rhodesiense samples, T.congolense samples, and T.evansi sam...

Embodiment 2

[0054] Embodiment 2 The intergenus specificity of the specific primers specifically detecting Trypanosoma protozoa of the present invention:

[0055] Use the following methods to detect:

[0056] (1) extract the genomic DNA of the target sample;

[0057] DNA extraction was carried out using DNeasy Blood & Tissue Kit (QIAGEN) according to the instructions.

[0058] Target samples included the following 5 other genera of blood-borne protozoa common in blood parasite examination, and the test results were all negative. The five other genera of blood-borne protozoa include: Babesia microti, Leishmania donovani, Plasmodium vivax, Plasmodium falciparum, Toxoplasma gondii Insects (Toxoplasma gondii) were provided by the China Center for Disease Control and Prevention (CDC) Parasite Prevention and Control.

[0059] (2) Using the genomic DNA of each target sample extracted in step (1) as a template, carry out PCR amplification with primers SEQ ID NO:1 and SEQ ID NO:2 provided by the...

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Abstract

The invention discloses a specific primer for detecting trypanosome protozoan. The sequences of the specific primer are as follows: the sequence of an upstream primer is 5'-CCTGATGAAAGGTGCAATG (SEQ IDNO: 1) or a complementary sequence thereof; the sequence of a downstream primer is 5'-CGTTTTCGCCTTCTTGTGGA (SEQ ID NO: 2) or a complementary sequence thereof. The invention further discloses a methodfor detecting the specificity of trypanosome protozoan. The method comprises the following steps: (1), extracting genome DNA of a target sample; (2), taking the extracted genome DNA of the target sample in the step (1) as a template, and performing PCR amplification through the specific primer; (3), performing agarose gel electrophoresis experiment on a PCR product, and observing whether a specific band exists in a target position. The specific primer and the detection method can rapidly and accurately detect whether trypanosome protozoan is contained in the target sample, the detection method is high in species universality, and strong in intergeneric specificity, and can specially perform effective detection on trypanosome protozoan. The invention further provides application of the primer.

Description

technical field [0001] The invention relates to the fields of molecular biology and pathogenic biology, in particular to a specific primer designed using the partial sequence of the 60S RPL44 gene of Trypanosoma brucei subspecies, a detection method and an application. Background technique [0002] There are about 20 kinds of protozoa of the genus Trypanosoma (Trypanosoma sp.), which commonly parasitize in the blood or tissue cells of fish, amphibians, reptiles, birds, mammals and humans. Trypanosomes that parasitize humans can be divided into two categories according to their infection routes, namely salivary trypanosomes that are transmitted through saliva and fecal trypanosomes that are transmitted through feces. Trypanosomes that are seriously pathogenic to livestock include T.brucei, T.congolense, T.evansi, etc., and those that are seriously pathogenic to humans Species include T. brucei and T. cruzi. Trypanosomiasis is listed as one of the key prevention and control ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6893C12N15/11
CPCC12Q1/6893
Inventor 胡薇殷明波刘骁洪清华王韵丞张瑞祥李健李鸿雁陈木新蔡玉春徐斌刘秀凤陈家旭王敬文
Owner FUDAN UNIV
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