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Theileria annulata SYBR Green I real-time fluorescence PCR (Polymerase Chain Reaction) specific primer as well as detection kit and detecting method

The technology of a Theileria annulus and detection kit is applied in the field of biology, which can solve the problems of epidemic disease general survey and import and export quarantine difficulties, low sensitivity of serological diagnosis, and easy false positives, etc., to achieve convenient and fast detection and low resolution. Specificity and low sensitivity, the effect of improving specificity and sensitivity

Inactive Publication Date: 2018-06-19
王素华
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Problems solved by technology

Direct microscopic examination can only be used for the detection of diseased animals, but it is difficult for general epidemic disease and import and export quarantine; serological diagnosis also has the problem of low sensitivity
A method with higher sensitivity, such as conventional PCR detection technology is also the most commonly used method for diagnosing Theileria annuli, but this method has a big disadvantage—low specificity and low sensitivity, and it is easy to produce false positives during amplification

Method used

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  • Theileria annulata SYBR Green I real-time fluorescence PCR (Polymerase Chain Reaction) specific primer as well as detection kit and detecting method
  • Theileria annulata SYBR Green I real-time fluorescence PCR (Polymerase Chain Reaction) specific primer as well as detection kit and detecting method
  • Theileria annulata SYBR Green I real-time fluorescence PCR (Polymerase Chain Reaction) specific primer as well as detection kit and detecting method

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Embodiment Construction

[0027] Set up the SYBR Green I Real-Time PCR Detection Kit, which includes:

[0028] (1) EP dof tube: the reaction tube contains Premix Ex Taq TM II (2x).

[0029] (2) EP dof tube: the reaction tube contains ROX Reference Dye (50×).

[0030] (3) Primer Tams-F and Primer Tams-R. Primer Tams-F and primer Tams-R are DNA fragments synthesized by a DNA synthesizer according to the following base sequences:

[0031] Tams-F: 5'-CAA-ATT-CGA-GAC-CTA-CTA-CGA-TC-3';

[0032] Tams-R: 5'-CCA-CTT-ATC-GTC-CTT-AAG-CTC-G-3'.

[0033] (4) Positive control: This control is a recombinant plasmid containing the target gene fragment, which is constructed by our laboratory. The method is as follows: clone the amplified product into pUCm-T to transform DH5-α Escherichia coli competent cells, obtain a positive recombinant plasmid after identification by enzyme digestion and sequencing, and the plasmid contains a 1014bp gene fragment of Theileria annulus.

[0034] (5) Negative control, which is...

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Abstract

The invention discloses a theileria annulata SYBR Green I real-time fluorescence PCR (Polymerase Chain Reaction) specific primer as well as a detection kit and a real-time fluorescence PCR detecting method. The method is very convenient and quick in detecting, can improve specificity and sensibility of the detecting method, takes trace pathogen genome DNA (deoxyribonucleic acid) extracted from blood or lymph as a template to authenticate, designs two specific primers for a specific area which takes Tams1 as a target gene of theileria annulata, and solves the defects that an existing PCR detecting technology is low in specificity and sensibility, and easily causes leakage detection of false positive and when content of theileria annulata is relatively low during amplification. The method isstronger in reaction specificity, is higher in sensibility, is specially used for precise detection of theileria annulata, and has a wide application prospect.

Description

technical field [0001] The invention relates to the technical field of biology, in particular to a specific primer for real-time fluorescent PCR of Theileria annulus SYBR Green I, a detection kit and a detection method. Background technique [0002] Theileria annulus is a cattle and sheep parasite transmitted by Hyalomia ticks, and the disease caused by it is called Theileriasis. Theileria annulare mainly parasitizes macrophages, lymphocytes and red blood cells of cattle and sheep. Hyalomia tick is a kind of double-host tick, which spreads Theileria annulare in the way of interim transmission, that is, after the larva or nymph sucks the blood of the carrier, Theileria develops and reproduces in the tick, and when the adult tick sucks blood, it can spread the disease. Sick animals showed persistent high fever, rapid weight loss, severe anemia, and enlarged lymph nodes. Microscopic examination of smears after lymph node puncture revealed that theileria ring schizonts could b...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6893C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/6893C12Q2561/113C12Q2563/107
Inventor 王素华吴绍强蔡军吕继洲帅江冰张晓峰
Owner 王素华
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