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Nested PCR (polymerase chain reaction) detection kit for 11 types of rabbit Eimeria

A detection kit and coccidia technology, applied in the field of molecular biology detection, can solve the problems of insufficient DNA extraction, non-specific bands, false positive results, etc. Effect

Inactive Publication Date: 2017-01-11
JILIN UNIV
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  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the complex composition of stool samples, which contain a large number of bacteria, non-specific bands or false positive results often appear when using ordinary PCR methods for detection
In particular, the general fecal genome extraction kit is not thorough enough for sample processing and DNA extraction in coccidia sporangia

Method used

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  • Nested PCR (polymerase chain reaction) detection kit for 11 types of rabbit Eimeria
  • Nested PCR (polymerase chain reaction) detection kit for 11 types of rabbit Eimeria
  • Nested PCR (polymerase chain reaction) detection kit for 11 types of rabbit Eimeria

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Experimental program
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Effect test

Embodiment 1

[0034] Example 1 Eimeria rabbit specific gene selection

[0035] Rabbit Eimeria species-specific diagnostic sequence: select the highly conserved 18S rRNA ITS-1 gene sequence, which is unique to Eimeria in NCBI, and various coccidia are within this segment There are differences and meet the standards of species-specific detection research.

Embodiment 2

[0036] Embodiment 2.nestPCR specificity test:

[0037] The first purpose of the experiment is to distinguish the differences between Eimeria rabbits and other pathogenic infections, and the second is to be able to detect 11 different Eimeria species. Therefore, common parasite DNA samples (Giardia, Neospora, and Toxoplasma) were selected as controls for specific detection. Taking Eimeria stutzeri as an example, the results are as follows: the method can accurately detect Eimeria rabbiti, and it is Eimeria stutzeri, other species (Giardia G, Neospora N, Isospora I, Toxoplasma gondii tachyzoites G.t, Toxoplasma gondii bradyzoites G.b) Specific bands cannot be displayed by this detection method.

Embodiment 3

[0038] Embodiment 3.nestPCR sensitivity test:

[0039] The number of coccidia obtained per μl was calculated by the McFarland counting method, and the final result is shown in the figure below, that is, the presence of Eimeria rabbits can be successfully detected when each μl contains at least 100 coccidia.

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Abstract

The invention aims to disclose a nested PCR (polymerase chain reaction) detection kit for 11 types of rabbit Eimeria. The nested PCR detection kit is characterized in that with an ITS-1 sequence of 18S rRNA gene of the Rabbit Eimeria as a target sequence, a pair of genus-specific nested PCR primers are designed, 11 pairs of species-specific nested PCR primers are designed for each Eimeria body, and a nested PCR detection method is established and used for the detection of 11 types of rabbit Eimeria in a clinical fecal sample. The nested PCR detection kit is rigorous in design, easy to operate, high in flexibility, high in specificity, convenient to operate in batch, capable of identifying infections of specific types of rabbit Eimeria and accurate and objective in result judgment.

Description

technical field [0001] The invention provides a nested PCR detection kit for 11 kinds of Eimeria rabbits, which is used for the detection of 11 different kinds of Eimeria rabbits infection. The invention also discloses the above-mentioned 11 kinds of Eimeria rabbits The invention discloses a preparation method of a nested PCR detection kit for coccidia, belonging to the technical field of molecular biology detection. Background technique [0002] Rabbits are often used as experimental animals in veterinary and human medical research due to their small size and strong fecundity. One of the most common diseases in its breeding is coccidiosis, especially the most common Eimeria infection. Internationally, there are as many as 11 kinds of Eimeria rabbits, and new drug-resistant strains are easy to produce, so this is also the main reason why rabbit coccidiosis is difficult to treat and prevent, and the disease is not easy to control. Like the life cycle of other coccidia, rabb...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6893C12Q1/6848C12Q2549/119C12Q2537/143C12Q2531/113C12Q2565/125
Inventor 宫鹏涛林永超张西臣李巍李建华杨举李赫李棕松
Owner JILIN UNIV
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