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ARMS-PCR primer of sulfanilamide drug-resistant eimeria tenella and molecular detection method thereof

A technology for Eimeria and molecular detection, applied in microorganism-based methods, biochemical equipment and methods, and microbial determination/inspection, etc. methods, etc., to achieve the effect of high accuracy, simple operation and low cost

Inactive Publication Date: 2019-11-22
HUAZHONG AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] There are currently no molecular assays capable of detecting sulfa-resistant Eimeria tenella

Method used

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  • ARMS-PCR primer of sulfanilamide drug-resistant eimeria tenella and molecular detection method thereof
  • ARMS-PCR primer of sulfanilamide drug-resistant eimeria tenella and molecular detection method thereof
  • ARMS-PCR primer of sulfanilamide drug-resistant eimeria tenella and molecular detection method thereof

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Experimental program
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Effect test

Embodiment 1

[0033] Embodiment 1: Carry out the detection of sulfa drug-resistant Eimeria tenella to chicken manure sample

[0034] 1. Extraction of total DNA from clinical chicken feces samples: put 500ul of sporulated oocyst suspension into the sample rapid preparation system, then place the oocyst suspension on a centrifuge and centrifuge at 10000r / min for 1min, retain the precipitate for later use; Dissolve the precipitate in 200ulGA, then add 20ul protein kinase K and digest it in a water bath at 56°C for 8h; follow-up DNA extraction kit (spin column type, purchased from QIAGEN, catalog number: No.51304) followed by Tiangen Blood DNA extraction.

[0035] 2. Gene selection

[0036] We selected dihydrofolate synthase gene and according to the published Eimeria tenella strain dhps gene (2713bp) in Gene Bank, and then used Snapgene software for sequence alignment, the results found that Eimeria tenella dhps gene (2713bp) It has high specificity for protozoa and various hosts. Therefore...

Embodiment 3

[0054] Embodiment 3: Sensitivity test of sulfonamide-resistant Eimeria tenella detection method

[0055] 1. Amplify the target fragment with the ordinary PCR method: select the total DNA amplification containing the sulfa drug-resistant worm strain to amplify the partial gene fragment of Eimeria tenella comprising the dhps of the mutation, and the nucleotide sequence of the described primer pair is as follows shown.

[0056] Upstream primer F2: 5'-GTTGCGGCATTCTGCATCAAGTTCAGGA-3';

[0057] Downstream primer R2: 5'-CTGCATGCGGCAACAACTTATAAGAGGAC-3';

[0058] The reaction system is: Phanta Max Super-Fidelity DNA Polymerase 1.0ul, 2×PhantaMax Buffer25ul, dNTP Mixture 1ul, total DNA template 2ul, ddH 2 O 17ul, 2ul each of upstream and downstream primers. The total reaction system is 50ul. Add 5ul of the above PCR final product to 2% agarose gel containing 0.5ug / mL ethidium bromide (EB) dye, electrophoresis at 120V for 30min, and observe the amplified product by imaging on a gel ...

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Abstract

The invention belongs to the technical field of parasite molecular detection, and relates to a molecular detection method of sulfanilamide drug-resistant eimeria tenella, in particular to a method fordetecting sulfanilamide drug-resistant eimeria tenella by using a four-primer amplification refractory system-PCR. According to the invention, two specific primer pairs are designed according to thedhps gene sequence of the eimeria tenella; the nucleotide sequences of the primer pair are shown as SEQ ID NO: 2 to SEQ ID NO: 5 respectively, then total DNA of a sample is extracted, an ARMS-PCR amplification reaction is carried out, and finally whether the sample contains a sulfanilamide drug-resistant insect strain or not is determined through agarose gel electrophoresis. The method is high insensitivity and strong in specificity, and can be used for quickly identifying the sulfanilamide drug-resistant eimeria tenella.

Description

technical field [0001] The invention belongs to the technical field of parasite molecular detection, and relates to an ARMS-PCR primer of sulfonamide-resistant Eimeria tenella and a molecular detection method thereof. It is a method for rapidly detecting sulfonamide-resistant Eimeria tenella by using four-primer amplification retardation system-PCR (amplification refractory mutation system-PCR) technology. [0002] technical background [0003] Chicken coccidiosis is a disease caused by a variety of coccidia of the genus Eimeria parasitizing in intestinal epithelial cells, which is very harmful to chickens. Chicks aged 15-50 days have a high morbidity rate, and the mortality rate can reach 80%. The annual cost of preventing coccidiosis worldwide exceeds hundreds of millions of dollars. There are 7 common types of coccidia in chickens, among which Eimeria tenella is the most widely distributed coccidia in China, and Eimeria tenella is also one of the chicken coccidia with st...

Claims

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Application Information

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IPC IPC(8): C12Q1/6893C12Q1/6858C12Q1/04C12N15/11C12R1/90
CPCC12Q1/6858C12Q1/6893C12Q2600/106C12Q2600/156C12Q2531/113C12Q2535/137C12Q2565/125
Inventor 周艳琴耿天天雷振宇叶成申邦方瑞胡敏赵俊龙贺兰
Owner HUAZHONG AGRI UNIV
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