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Primer pair and kit for detecting mouse trypanosome

A kit and primer pair technology, applied in the field of primer pairs and kits for detecting mouse trypanosomes, to achieve the effects of improved operability, fast and convenient detection, and high sensitivity

Active Publication Date: 2017-08-18
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, using ITS to identify trypanosomes in mice, due to the technical flaws of ITS and 18S, only other subgenera can be distinguished. Although the wide distribution of the protozoa is known, due to public neglect, the epidemiological data are outdated. At this stage Epidemiological investigations are necessary to assess the risk of infecting people

Method used

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  • Primer pair and kit for detecting mouse trypanosome
  • Primer pair and kit for detecting mouse trypanosome
  • Primer pair and kit for detecting mouse trypanosome

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Primer Design

[0037] Mouse Trypanosoma (KY426815) and Trypanosoma luzi ( T. lewisi CPO02, KR072974), Trypanosoma cruzi (GenBank: FJ203996.1; GenBank: DQ343646.1; GenBank: DQ343645.1) were compared to design primers. The primer sequences are shown in Table 1; among them, Tubulin is an internal reference gene.

[0038]

Embodiment 2

[0039]Embodiment 2 Determination of PCR detection system temperature

[0040] (1) Establish a PCR amplification reaction system: 12.5 μl of PrimerSTAR Max Premix (TAKARA, Japan), 6.25 pmol / L of forward and reverse primers, 100 ng of extracted DNA, filled up to 25 μl, the negative control is sterile double distilled water.

[0041] (2) PCR amplification reaction conditions: 30 reaction cycles: denaturation at 98°C for 10 s, annealing at (50-60°C) for 15 s, extension at 72°C for 8 s.

[0042] (3) Analysis of amplified products: The amplified products were detected by agarose gel electrophoresis. Take 5 μl of 25 μl of the final reaction product amplified by polymerase chain reaction PCR, add 6×loading buffer, spot on 1% agarose gel containing GOLD VIEW dye, electrophoresis at 220 v for 20 min, and run on the gel Imaging observation results on the imaging system. For the amplification of TM2, the target band can be amplified at 50°C to 54°C, and the brightness of the band has b...

Embodiment 3

[0043] Example 3 PCR detection method specificity experiment

[0044] Perform PCR amplification according to the method in Example 2.

[0045] (1) Establish a PCR amplification reaction system: 12.5 μl of PrimerSTAR Max Premix (TAKARA, Japan), 6.25 pmol / L of primer TM2-F, 6.25 pmol / L of primer TM2-R (or 6.25 pmol / L of primer TM1 -F, 6.25 pmol / L primer TM1-R), 100 ng DNA (15 samples), make up to 25 μl with sterilized double distilled water, and the negative control is sterilized double distilled water.

[0046] (2) PCR amplification reaction conditions: 30 reaction cycles: denaturation at 98°C for 10 s, annealing at 52°C for 15 s, extension at 72°C for 8 s.

[0047] (3) Analysis of amplified products: The amplified products were detected by agarose gel electrophoresis. Take 5 μl of 25 μl of the final reaction product amplified by polymerase chain reaction PCR, add 6×loading buffer, spot on 1% agarose gel containing GOLD VIEW dye, electrophoresis at 220 v for 20 min, and run o...

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Abstract

The invention discloses a primer pair and kit for detecting a mouse trypanosome. By comparing a macrocyclic sequence of the mouse trypanosome with macrocyclic sequences of other similar trypanosomes, one primer pair TM2-F and TM2-R which can specifically detect the mouse trypanosome is screened, and sequences of the primer pair are shown in SEQ ID NO:1 and SEQ ID NO:2; the primer pair can specifically amplify a partial segment of the mouse trypanosome, is high in sensitivity and can detect a sample which contains 10 trypanosomes only or 1 ng DNA sample; and by utilizing the primer pair, the mouse trypanosome can be rapidly and accurately identified from other trypanozoons and leishmania amazonesis without an expensive microscope and professional etiology knowledge, detection is quick and convenient, and the primer pair can be used for detecting early infection, is also applicable to detection of animal trypanosome mixed infection, is beneficial to development of epidemiological investigation and can be applicable to detection of human mouse trypanosome infection.

Description

technical field [0001] The invention relates to the technical field of parasite molecular detection, in particular to a primer pair and a kit for detecting mouse trypanosoma. Background technique [0002] In nature, mouse trypanosomes ( Trypanosoma musculi ) is a globally distributed parasitic protozoan, which is transmitted among mammals by means of the vector insect flea, belonging to the subgenus Trypanosoma ( Subgenus Herpetosoma ), for the detection of mouse trypanosoma, many modern detection methods have been developed in the prior art. For example, using ITS to identify trypanosomes in mice, due to the technical flaws of ITS and 18S, only other subgenera can be distinguished. Although the wide distribution of the protozoa is known, due to public neglect, the epidemiological data are outdated. At this stage It is necessary to conduct epidemiological investigations to assess the risk of infecting people. Contents of the invention [0003] The technical problem 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
CPCC12Q1/6893
Inventor 赖德华洪晓昆张玄温砚子伦照荣
Owner SUN YAT SEN UNIV
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