Bovine babesiosisbigemina nested PCR specific primer as well as detection kit and nested PCR detection method

A technology of Babesia double buds and a detection kit, which is applied in the field of biology, can solve the problems of high false positive rate, achieve high sensitivity, improve specificity and sensitivity, and detect conveniently and quickly

Inactive Publication Date: 2018-05-15
王素华
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Methods with higher sensitivity and specificity, such as reverse linear dot hybridization (RBL) and PCR-ELISA, have also been reported, but these methods have the disadvantage of high false positive rate

Method used

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  • Bovine babesiosisbigemina nested PCR specific primer as well as detection kit and nested PCR detection method
  • Bovine babesiosisbigemina nested PCR specific primer as well as detection kit and nested PCR detection method
  • Bovine babesiosisbigemina nested PCR specific primer as well as detection kit and nested PCR detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment Construction

[0038] Set up a nested PCR detection kit that includes:

[0039] (1) EP dof tube: The reaction tube contains 10×PCR Buffer, deoxyribonucleoside triphosphate (dNTP), magnesium chloride (Mgcl 2 ).

[0040] (2) Primer F1, primer R1, primer F2, primer R2. Primer F1, primer R1, primer F2, and primer R2 are DNA fragments synthesized by a DNA synthesizer according to the following base sequences:

[0041] Primer BF1: 5'-CAT-CTA-ATT-TCT-CTC-CAT-ACC-CCT-CC-3';

[0042] Primer BR1: 5'-CCT-CGG-CTT-CAA-CTC-TGA-TGG-CAA-AG-3';

[0043] Primer BF2: 5'-CGC-AAG-CCC-AGC-AGG-CCC-CGG-TGC-3';

[0044] Primer BR2: 5'-CCG-ACC-TGG-ATA-GGC-TGT-GTC-ATG-3'.

[0045] (3) 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 competent cells, obtain a positive recombinant plasmid after enzyme digestion identification and sequencing, and the ...

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Abstract

The invention provides a bovine babesiosisbigemina nested PCR specific primer as well as a detection kit and a nested PCR detection method. The method is simple in template DNA preparation steps and low in cost; the detection of the method is extremely convenient and quick; specificity and sensitivity of the detection method can be improved; trace genome DNA extracted by single tick and even partof tissue and organs of the tick can be used as a template for identifying; four specific primers are designed for the specific area of bovine babesiosisbigemina taking RAP-1 as target gene; the defect of cross reaction between bovine babesiosisbigemina and bovine babesia when diagnosing the babesia in the tick body through the existing PCR detection technology is solved. The method is higher in reaction specificity and higher in sensitivity, is specially applied to accurate detection of the bovine babesiosisbigemina, and has broad application prospects.

Description

technical field [0001] The invention specifically relates to the technical field of biology, in particular to a nested PCR specific primer, a detection kit and a nested PCR detection method for Babesia double buds. Background technique [0002] Babesia bovis is a blood protozoa that is transmitted by ticks and parasitizes in red blood cells. Babesia disease caused by this type of parasite is an acute seasonal disease that occurs and prevails in many parts of the world. my country It also often occurs in various places, causing huge losses to animal husbandry and the national economy. After cattle are infected with the worm, symptoms such as anemia, fever, hemoglobinuria, and ataxia often occur, which seriously affect the health of cattle. The disease has caused huge economic losses and is expanding worldwide, thus attracting widespread attention from scholars at home and abroad. Babesia bovis is a blood protozoan transmitted by ticks, parasitic in red blood cells, lymphocyt...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6893C12Q1/6848C12Q1/04C12N15/11
CPCC12Q1/6848C12Q1/6893C12Q2549/119C12Q2545/101
Inventor 王素华帅江冰张晓勇吕继洲袁淑辉吴绍强张晓峰
Owner 王素华
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