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Primer, kit and method for identifying eel germplasm

A technology for identifying eels and eels, applied in biochemical equipment and methods, microbiological determination/inspection, DNA/RNA fragments, etc., can solve problems such as high cost, need for sequencing, loss of restriction enzyme sites, etc., and achieve practicality Strong performance, easy operation, good stability and repeatability

Inactive Publication Date: 2017-12-19
JIMEI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the RFLP method is cumbersome, relatively time-consuming, requires a large amount of DNA, and may cause the loss or acquisition of restriction enzyme sites due to base mutations.
The results of SSCP are easily affected by many factors, and when the difference or mutation of the single-stranded DNA base sequence itself has no effect on the single-stranded conformation, it may cause false negative results and lead to missed detection
The specific PCR method requires sequencing, which takes a long time and cannot meet the requirements of on-site detection
[0005] The above-mentioned methods all have problems such as cumbersome operation, long time-consuming, high cost, or high requirements for technicians and equipment, and are not suitable for production applications.

Method used

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  • Primer, kit and method for identifying eel germplasm

Examples

Experimental program
Comparison scheme
Effect test

no. 1 Embodiment

[0034] A primer for identifying eel germplasm, including one or more of the following 4 pairs of primers, the sequences of the 4 pairs of primers are:

[0035] aj S1:TTATGGCTGATTCATCCGAAATT, aj A1 CCTGCTAATGGGTTGAGTACTAAA, fragment size 889;

[0036] r-a S1 TGATGCTGACTTTGCCACTGAT; r-a A2 GTTCGTTCCCTTGAAAACCTTGT, fragment size 341;

[0037] bp-m S5 TCCATACTTCTCATACAAAGACCTAT, bp-m A3CTAGTCAACCTACTAATGGGTTTAAT, fragment size 457;

[0038] m-a S4 CCGCCGTCCCATACGTAGGAG, m-a A4 TATTTTGTTTTTCTAGTCAACCTACTAAT, fragment size 678.

[0039] In the process of applying the primers described in this example to identify eel germplasm, multiple primers are used for amplification in one PCR process, which avoids waste of resources and time caused by multiple amplifications, and can effectively improve efficiency. And the primers will not interact with each other.

no. 2 Embodiment

[0041] A kit for identifying eel germplasm, said kit comprising one or more of the following 4 pairs of primers, said 4 pairs of primer sequences being:

[0042] aj S1:TTATGGCTGATTCATCCGAAATT, aj A1 CCTGCTAATGGGTTGAGTACTAAA, fragment size 889;

[0043] r-a S1 TGATGCTGACTTTGCCACTGAT; r-a A2 GTTCGTTCCCTTGAAAACCTTGT, fragment size 341;

[0044] bp-m S5 TCCATACTTCTCATACAAAGACCTAT, bp-m A3CTAGTCAACCTACTAATGGGTTTAAT, fragment size 457;

[0045] m-a S4 CCGCCGTCCCATACGTAGGAG, m-a A4 TATTTTGTTTTTCTAGTCAACCTACTAAT, fragment size 678.

[0046] In the process of using the kit described in this example to identify eel germplasm, multiple primers are used for amplification in one PCR process, which avoids waste of resources and time caused by multiple amplifications, and can effectively improve efficiency , and the primers will not interact with each other.

no. 3 Embodiment

[0048] A method for identifying eel germplasm, comprising the steps of:

[0049]1. Extract the genomic DNA of any seed or grilled eel product of Japanese eel, American eel, flower eel, Pacific bicolor eel, and European eel;

[0050] 2. Carry out PCR amplification of the eel DNA sample with primers, and the PCR reaction conditions specifically include:

[0051] Pre-denaturation at 94°C for 5 minutes;

[0052] 27 cycles of denaturation at 94°C for 45 seconds, annealing at 58°C for 45 seconds, and extension at 72°C for 45 seconds;

[0053] Extension at 72°C for 5 minutes.

[0054] 3. Using agarose gel electrophoresis to detect the negative and positive bands and the size can quickly identify eel germplasm, the results are as follows:

[0055] Table PCR results of different primers

[0056]

[0057] Note: + means positive, - means negative

[0058] The size of fragments amplified by different primers of this method is different. The Japanese eel is 889bp, the South America...

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Abstract

The invention discloses a primer for identifying eel germplasm and also discloses a kit containing the primer, and a method for identifying eel germplasm by using the primer or the kit. According to the primer, kit or method disclosed by the invention, the time of detecting samples at a time is 2-3 hours only, many samples can be simultaneously detected, and the primer, kit or method disclosed by the invention has the characteristics of high efficiency and convenience. More conveniently, a large instrument is not needed by the method, field detection can be performed at any time in case of electrification, and the primer, the kit and the method are simple and convenient in operation, excellent in stability and repeatability and high in practicality.

Description

technical field [0001] The invention is used for identification or auxiliary identification of eel germplasm, relates to the technical field of molecular markers, and particularly relates to the identification and detection technology of eel. Background technique [0002] Over the past two decades, the eel farming industry has occupied a large market in my country, but at present, the eel breeding seedlings are completely dependent on natural eel seedlings, and the catch of natural eel seedlings is decreasing sharply due to overfishing and water pollution. , causing the price of eel fry to continue to rise. Due to natural or human factors, it is easy to cause the mixing of different species of eel fry. In addition, due to the large difference in market prices of different species of eel fry, some merchants add low-priced eel fry to high-priced eel fry to obtain greater benefits. However, white eel seedlings are very similar in morphology, and it is difficult to distinguish ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 赖晓健江兴龙罗碧莲
Owner JIMEI UNIV
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