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Polymerase chain reaction (PCR) method for quickly detecting canine babesiosis and use thereof

A technology for Babesia canis and Babesia canis, which is applied in the direction of biochemical equipment and methods, and the determination/testing of microorganisms, which can solve the problem that serological diagnostic methods cannot distinguish between current infection and past infection, and achieve detection speed Fast, sensitive results

Inactive Publication Date: 2011-11-02
HENAN UNIV OF SCI & TECH
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Problems solved by technology

Serological diagnostic tests cannot differentiate between current infection and past infection

Method used

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Embodiment 1

[0037] 1. Incidence

[0038] On July 16, 2010, a German shepherd dog, male, 4 months old, was immunized according to the normal procedure in a factory in the suburbs of Luoyang. It is understood that there are many grass sports fields in the field. Dogs often play on the grass, and ticks can often be found on the dogs. There is a dog suffering from blood worm disease in the adjacent factory. According to the clinical observation, the dog was depressed, eating little or no food, the urine was yellowish brown, and the body temperature was as high as 40 degrees Celsius. The visible mucous membrane was anemia and mild jaundice, and the urine was yellow. Microscopic examination did not find parasites, it is recommended to carry out PCR detection. The operation process is as follows:

[0039] (1) Sample DNA extraction

[0040] First, 300 μl of anticoagulated blood was collected from the saphenous vein of the forelimb of the dog to be tested, and then the whole blood DNA extractio...

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Abstract

The invention discloses a PCR method for quickly detecting canine babesiosis and use thereof. The method comprises: preparing 1U of Taq enzyme, 2.5 microliters of Taq enzyme with 10*PCRBuffer, 3.0 microliters of Taq enzyme with 25mmol.L<-1>Mg<2+> salt, 0.5 microliter of 10mmol.L<-1>mixdNTP, 0.5 microliter of 10pmol.microliter <-1> canine babesiosis specific PCR upstream primer, 0.5 microliter of 10pmol.microliter <-1> canine babesiosis specific PCR downstream primer and 4.0 microliters of DNA sample to be detected; adding water till the total volume reaches 25.0 microliters; performing pre-degeneration at 95 DEG C for 5 minutes; and performing degenerating, annealing and extending circularly for 35 times, extending at 72 DEG C for 7 minutes, cooling, and ending the reaction. When the method is used, the detection speed is high, the result can be obtained within 2 hours, the anine babesiosis can still be detected when the sample is diluted by 1,000 folds, the selectivity of the primersis high, and the primers do not react with other DNA.

Description

[0001] technical field [0002] The invention relates to a bioengineering technology, in particular to a PCR method and application for rapid detection of Babesia in dogs, a pair of specific diagnostic primers designed and synthesized based on the Babesia 18s RNA gene sequence, and a PCR diagnosis method disclosed at the same time The operation of the method. Background technique [0003] Babesiosis is a blood-borne protozoan disease that widely occurs in various livestock and is transmitted by ticks. It parasitizes in red blood cells singly or in pairs in domestic animals, causing severe anemia and hemoglobinuria. There are three kinds of pathogens that cause babesiosis in dogs, namely, Babeisa canis, B. vitlli and B. gibsoni. Babesia canis is found in Europe, the Americas and India, Babesia veschneri is found in South America, and Babesia gizzardi is found in Asia. The main insect species prevalent in my country is Babesia gibsonii. This disease is widely distribut...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04
Inventor 张才李小康何万领杨自军王雪莹尚泽松朱重伟王亚垒
Owner HENAN UNIV OF SCI & TECH
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