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PCR specific primer and detection method for detecting Sanguilla anguilla

A technology of dermatosporidium eel and specific primer pair, which is applied in the field of PCR primers for detection of dermatosporidium eel and the parasitic dermatosporidium eel, can solve the problems of undetectable, achieve high specificity and sensitivity, and predict dermatosporidium eel Effect of disease

Inactive Publication Date: 2021-05-28
QINGDAO AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The technical problem to be solved by the present invention is: how to provide a PCR primer and a method for detecting the parasitic eel Dermosporidium eel and Siniperca sinensis, so as to solve the problem that existing microscopic examination can only detect diseased fish and cannot be detected in the early stage of infection question

Method used

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  • PCR specific primer and detection method for detecting Sanguilla anguilla
  • PCR specific primer and detection method for detecting Sanguilla anguilla

Examples

Experimental program
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Effect test

Embodiment 1

[0018] (1) Primer design and screening: A 1856bp total sequence of Dermatospora anguillaris spliced ​​according to the sequence of seven Dermatospora anguillarum, which is compatible with Anguilla rostrata (FM946071.1) and Sinipercachuatsi (AY452490.1) The 18S rRNA sequences were compared to find the difference sites, primers were designed and screened in the variable regions, and finally a pair of specific primers were obtained. The upstream primer sequence of the primer pair is: 5'-TTCGCTTCTCGAAAGCGGC-3'(SEQ IDNo.1) , the downstream primer sequence is: 5'-TTACCCATACCTTCCGGTACAGGTG-3' (SEQ ID No.2).

[0019] (2) PCR detection: extract the DNA of the specimen to be detected and use the DNA as a template, and use the primer pair shown in SEQ ID No.1 and SEQ ID No.2 as primers to carry out PCR amplification. If the amplification product of 836bp can be obtained , the specimen contains Dermatospora anguillarum. In PCR amplification, the reaction system is: 1 μl of DNA amplificat...

Embodiment 2

[0020] Embodiment 2 specific detection

[0021] Utilize the pair of primers designed by the present invention to amplify the DNA of D. eel, healthy American eel and Siniperca sinensis DNA. The amplification reaction system is: 1 μl of DNA amplification template, 0.5 μl of upstream and downstream primers, 2 × Taq Plus PCR Master Mix 10μl, sterilized ddH 2 O was added to 20 μl; the reaction conditions were: pre-denaturation at 94°C for 4 min, denaturation at 94°C for 30 s, annealing at 59°C for 30 s, extension at 72°C for 1 min, 35 cycles; extension at 72°C for 5 min, and storage at 12°C. After the amplification, 5 μl of the PCR product was taken for 1% agarose gel electrophoresis, and the bands were observed. Check the specificity of the primers. The result is as figure 1 Shown: Only the D. eel DNA as a template can amplify a single target band of the expected length, while the American eel and Siniperca sinensis DNA did not amplify the band. It shows that the screening pri...

Embodiment 3

[0022] Example 3 Sensitivity Detection

[0023] The D. eelensis DNA that extracts is carried out 10 times ratio dilutions, obtains different concentration (1,10 -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 、10 -7 、10 -8 ng / μl), add 100ng healthy American eel DNA and mandarin mandarin mandarin DNA to each reaction, use the mixture of D. 2 O was used as a blank control, and PCR amplification was performed with specific primers. After the amplification, 5 μl of the PCR product was taken for 1% agarose gel electrophoresis, and the bands were observed to detect the sensitivity of the primers. The result is as figure 2 Shown: under the interference of host DNA, the template concentration of D. eelis is 10 -6 ng / μl can still amplify bands, indicating that the established PCR detection method has a minimum detection limit of 10 -6 ng / μl, high sensitivity.

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Abstract

The invention discloses a PCR specific primer and a detection method for detecting Schizospora anguilla. The sequence of an upstream primer of the primer pair is shown as SEQ ID No.1, and the sequence of a downstream primer is shown as SEQ ID No.2. DNA of a sample to be detected is extracted, PCR amplification is carried out by taking the DNA as a template and taking the primer pairs as shown in SEQ ID No.1 and SEQ ID No.2 as primers, and if an 836bp amplification product can be obtained, the sample contains the Schelyriasis anguilla. The primer is designed by using the Schizospora anguilla 18S rDNA as a molecular target, the Schizospora anguilla can be successfully expanded from eel and siniperca chuatsi tissues infected with the Schizospora anguilla, and the primer has high specificity and sensitivity, is simple and rapid to operate, can accurately detect and identify the early stage of the Schizospora anguilla, and the eel skin sporidiosis can be effectively predicted.

Description

technical field [0001] The invention belongs to the field of biological detection, and in particular relates to a PCR primer and a method for detecting the parasitic Dermatosporidium eel and Siniperca sinensis. Background technique [0002] Dermocystidium belongs to Neomonada, Mesomycetozoea, and Dermocystida. insect. Dermatosporidium forms a large number of cysts in the target organs of the host, which affects the growth and development of the host, reduces the value of commercial fish, and can lead to a large number of deaths of farmed fish in severe cases. The value of commercial fish can lead to a large number of deaths of farmed fish in severe cases. [0003] Eel is a rare and high-quality aquaculture species in my country, and mandarin duck is an important economic aquaculture species in my country. With the improvement of aquaculture system and the upgrading of aquaculture technology in recent years, the scale of aquaculture in my country has gradually expanded, but...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6893C12Q1/686C12Q1/04C12N15/11C12R1/90
CPCC12Q1/6893C12Q1/686
Inventor 柳阳李丹
Owner QINGDAO AGRI UNIV
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