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PCR-RFLP rapid detection method for common sturgeons

A PCR-RFLP, detection method technology, applied in the direction of determination/inspection of microorganisms, biochemical equipment and methods, etc., can solve the problems of long identification time, expensive equipment, high technical requirements of operators, and achieve increased accuracy and credibility. speed, improve work efficiency, convenient, quick and easy to operate

Inactive Publication Date: 2013-12-11
徐鹏
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although these technical means can carry out relatively accurate identification of sturgeon and sturgeon products to varying degrees, there are also disadvantages such as cumbersome technical operations, long identification time, expensive equipment, and high technical requirements for operators.

Method used

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  • PCR-RFLP rapid detection method for common sturgeons
  • PCR-RFLP rapid detection method for common sturgeons
  • PCR-RFLP rapid detection method for common sturgeons

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] To extract the genomic DNA of the sample to be tested, it is recommended to use a DNA extraction kit to extract the genomic DNA:

[0041] a) Cut off a small part of the caudal fin of the known Chinese sturgeon, Sturgeon's sturgeon, Siberian sturgeon, sterlet, huso, Ouhuang, flashing sturgeon or Russian sturgeon, cut about 0.5g of fin rays, and cut into pieces, Put them into 1.5mL centrifuge tubes and number them from 1 to 8;

[0042] b) Add 0.45mL trismethylolmethethanesulfonic acid (TES) and mix well, then add 50μl of 10% sodium dodecylsulfonate (SDS), 5.0μl of 20mg / mL proteinase K , After fully mixing, incubate at 56°C for 4-6h, shake once every 2h.

[0043] c) After incubating for 4-6 hours, place the 1.5mL centrifuge tube containing the mixed solution in step b) at room temperature, add an equal volume of saturated phenol (500μl), mix by inverting, centrifuge at 12000rpm for 10min, and separate the aqueous phase and the organic phase. Carefully pipette the upper n...

Embodiment 2

[0051] Rapid detection of common sturgeon species

[0052] The rapid detection of common sturgeon species using the method of the present invention and the kit includes the following specific steps:

[0053] Step 1, the above-mentioned genomic DNA extracted in Example 1 is prepared into a PCR system using the primer pair shown in SEQ ID No.1 and SEQID No.2 and the reagents contained in the kit, that is, adding the above-mentioned DNA into a 200 μl PCR reaction tube The PCR amplification reaction system is 15 μl: add 0.1 μl of Taq DNA polymerase with a concentration of 2,500 units / mL, 1.5 μl of 10×PCR buffer, 1.5 μl of dNTPs with a concentration of 10 pm / μl, and 1.2 μl of 25 mM MgCl 2 , 2 μl of the genomic DNA, 0.5 μl of the upstream primer at a concentration of 10 pm / μl, 0.5 μl of the downstream primer at a concentration of 10 pm / μl, and 7.7 μl of sterile water.

[0054] Put the above PCR system in a 200μl PCR tube, put the PCR tube containing the PCR system into a PCR amplif...

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Abstract

The invention relates to the field of molecular biology and taxonomy and discloses a PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism) rapid detection method for common sturgeons as well as a primer and a kit for the rapid detection method. The PCR-RFLP rapid detection method for the common sturgeons comprises the following steps: firstly, extracting a genome DNA (Deoxyribose Nucleic Acid) of a sample to be detected as a template; secondly, carrying out PCR amplification reaction to obtain a PCR product by taking the genome DNA as the template; thirdly, digesting the PCR product by using restriction endonuclease to obtain a digested product; fourthly, carrying out agarose gel electrophoresis detection on the PCR product and the digested product and identifying species according to electrophoretogram. According to the PCR-RFLP rapid detection method disclosed by the invention, the simplicity in operation is realized; small amount of fin rays or muscle is needed to be sheared on the basis of ensuring the survival of fingerlings; the identification of fingerlings of the common sturgeons is quickly and accurately carried out; the accuracy and the reliability of the identification result are improved; the working efficiency is greatly increased.

Description

technical field [0001] The invention relates to the fields of molecular biology and taxonomy, in particular to a PCR-RFLP rapid detection method for common sturgeons. Background technique [0002] Sturgeons belong to Osteichthyes, Actinopterygii, Ganoidomorpha, and Acipenseriformes. Now there are 2 families, 6 genera and 27 species in the world. There are 2 families, 3 genera and 8 species in my country, namely Acipenser schrenckii, Huso dauricus, Acipenser sinensis, Acipenser dabryanus, Psephurus gladius, Siberian sturgeon (Acipenser baerii), small sturgeon (Acipenser ruthenus), and naked sturgeon (Acipenser nudiventris). Acipenser is a kind of ancient cartilaginous scale fish, known as "living fossil". These ancient sturgeons are all endangered to varying degrees, and some are even in danger of extinction. Sturgeon roe is rich in nutrition. Sturgeon roe sauce processed from its roe is expensive and is internationally recognized as a luxury food, and is compared to "black ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 徐鹏刘园园刘晓勇孙效文
Owner 徐鹏
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