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SNP molecular markers related to chicken coccidioides decoquinate resistance and their application

A decoquinate, molecular marker technology, applied in recombinant DNA technology, microbial determination/inspection, DNA/RNA fragments, etc., can solve the problems of many influencing factors, long experiment time, cumbersome process, etc., and achieve high detection. The effect of accuracy, low detection cost and simple operation

Active Publication Date: 2022-05-13
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the detection method of decoquinate resistance is still in the cage feeding experiment. Although the detection results of this method are relatively reliable, the experiment takes a long time, the process is cumbersome, there are many influencing factors, and a large number of experimental animals and a large number of factors are required. Viable oocyst
Therefore, there is an urgent need to develop a method that can quickly detect decoquinate-resistant chicken coccidia, so as to better guide coccidiosis medication and reduce the economic losses caused by coccidia infection to the breeding industry

Method used

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  • SNP molecular markers related to chicken coccidioides decoquinate resistance and their application
  • SNP molecular markers related to chicken coccidioides decoquinate resistance and their application
  • SNP molecular markers related to chicken coccidioides decoquinate resistance and their application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037]Example 1 Acquisition of SNP molecular markers related to chicken coccidian decoxyquinate resistance and design of detection primers

[0038] Chicken coccidia strains from Shandong, Yunnan, Sichuan, Inner Mongolia, Hunan and other regions were collected, and 7 decoquinate-resistant strains were obtained through cage feeding experiments. However, the seven drug-resistant strains were all mixed strains. In order to ensure the accuracy of subsequent resequencing analysis and reduce the random error of next-generation sequencing, intestinal isolation and single oocyst isolation techniques were used to isolate decoquinate-resistant insects. Eimeria acumenia in the strain was isolated, and 7 Eimeria acumenia isolates resistant to decoquinate were obtained.

[0039] When performing high-throughput sequencing, use 100× sequencing depth to minimize random errors caused by random primer amplification. Different analysis strategies such as Fisher's exact test and hidden Markov mod...

Embodiment 2

[0046] Example 2 SNP molecular markers are used to detect the difference between the decoquinate-resistant strains produced in the experimental evolution system and their parent strains

[0047] 3 strains (Howden strain, Xinjiang strain, Zhangjiakou strain) of different genetic backgrounds (Howden strain, Xinjiang strain, Zhangjiakou strain) produced by the SNP molecular markers obtained in Example 1 and their amplification primers were resistant to Eimeria decoquinate. The decoquinate drug resistance of the drug insect strain was detected, and the specific method was as follows:

[0048] 1. The experimental evolution system induces decoquinate-resistant strains

[0049] In this example, the method of increasing drug concentration was used to induce decoquinate-resistant strains, that is, the induction was started at a dose lower than the recommended drug dose, and then the drug dose was increased generation by generation, and drug-resistant strains were obtained after multipl...

Embodiment 3

[0065] Example 3 SNP molecular markers are used to detect decoquinate resistance in mixed chicken coccidia samples collected in the field

[0066] Utilize the SNP molecular marker that embodiment 1 obtains and its amplification merger primer (SEQ ID NO.16-17) for 7 kinds of chicken coccidia to carry out the decoquinate drug resistance of 15 isolated worm strains collected from different regions Detection, the specific method is as follows:

[0067] 1. Preparation of DNA template

[0068] The 15 field mixed chicken coccidia samples after sporulation were inoculated into coccidia-free chickens of the right age (7-14 days old), and the inoculation dose was 5×10 3 -2×10 4 oocyst. Oocysts in feces were collected 4-9 days after inoculation.

[0069] After 48 hours of sporulation, the oocysts were purified. Pour the potassium dichromate containing oocysts into a clean centrifuge tube, centrifuge at 3600rpm for 5min, pour off the upper liquid, resuspend the pellet with PBS, repea...

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Abstract

The invention relates to the technical field of coccidian drug resistance detection, in particular to a SNP molecular marker related to chicken coccidian decoxyquinate drug resistance and an application thereof. The SNP molecular marker provided by the present invention contains a nucleotide sequence whose polymorphism is G / C at position 379 of the sequence shown in SEQ ID NO.1. The SNP molecular marker can be used to distinguish decoquinate-sensitive and resistant chicken coccidia, and has high detection accuracy of decoquinate resistance, and the detection results are consistent with the cage feeding experiment. The present invention also provides detection primers for the SNP molecular marker shown in SEQ ID NO.16-17 and a method for detecting chicken coccidian decoxyquinate resistance by using the primers in combination with PCR technology. The method has the advantages of short detection time, simple operation and low detection cost, and can be widely used in the clinical detection of coccidioides decoxate resistance in chickens.

Description

technical field [0001] The invention relates to the technical field of detection of coccidia drug resistance, in particular to a SNP molecular marker related to chicken coccidia drug resistance to decoxyquinate and an application thereof, and a detection method for chicken coccidia drug resistance to decoxyquinate. Background technique [0002] Coccidia are intracellular parasites of the genus Aimeria and Eimeria. There are 7 species of coccidia that can infect chickens, including Eimeria acervulina and E. brunetti), E.maxima, E.mitis, E.necatrix, E.praecox and Eimeria tenella. They reproduce in different parts of the intestinal tract, destroy intestinal epithelial cells, cause digestive and absorption dysfunction, reduce feed conversion rate, slow growth of chickens, etc., and cause mass death of chicks in severe cases. [0003] Since the use of anticoccidial drugs, the economic loss caused by coccidiosis has been effectively reduced, and it has played a very important ro...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6893C12Q1/6858C12N15/11
CPCC12Q1/6893C12Q1/6858C12Q2600/156C12Q2600/106C12Q2600/172C12Q2531/113C12Q2565/125
Inventor 刘贤勇郝振凯索勋毕菲菲孙霈段春慧张思新胡丹丹王思汤新明于咏兰
Owner CHINA AGRI UNIV
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