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PCR-FRLP quick detecting method of common sturgeons

A technology of PCR-RFLP and detection method, which is applied to the determination/inspection of microorganisms, biochemical equipment and methods, etc., which can solve the problems of long identification time, expensive equipment, and high technical requirements for operators, and achieve convenient, fast and easy operation, increase Accuracy and reliability, the effect of improving work efficiency

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-FRLP quick detecting method of common sturgeons
  • PCR-FRLP quick detecting method of common sturgeons
  • PCR-FRLP quick detecting method of common sturgeons

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] 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:

[0043] a) Cut about 0.5g of fin rays from the known Chinese sturgeon, Sturgeon's sturgeon, Siberian sturgeon, sterlet, sturgeon, Acipenser auranthus, Acipenser schneidi and Russian sturgeon, cut them into pieces, and put them into 1.5mL centrifuge tubes And numbered as 1~8;

[0044] b) Add 0.45mL trismethylolmethethanesulfonic acid (TES) and mix well, then add 50μl sodium dodecylsulfonate (SDS) with a mass concentration of 10%, and 5.0μl concentration of 20mg / mL proteinase K , After fully mixing, incubate at 56°C for 4-6h, shake once every 2h.

[0045] 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. Carefull...

Embodiment 2

[0053] Rapid detection of common sturgeon species

[0054] The rapid detection of common sturgeon species using the method of the present invention and the kit includes the following specific steps:

[0055] 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 SEQ ID No.2 and the reagents contained in the kit, that is, adding to 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.

[0056] Put the above PCR system in a 200μl PCR tube, put the PCR tube containing the PCR system into a PCR amplification instrument for PC...

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Abstract

The invention relates to the field of molecular biology and systematy, and discloses a PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism) quick detecting method of common sturgeons, and a primer pair and a kit which are used in the quick detecting method. The PCR-RFLP quick detecting method of common sturgeons comprises the following steps: step 1, extracting genome DNA of a sample to be detected as a template; step 2, carrying out PCR amplification reaction to obtain a PCR product by taking the genome DNA as a template; step 3, carrying out enzyme digestion on the PCR product by using restriction enzyme so as to obtain an enzyme digestion product; step 4, carrying out agarose gel electrophoresis detection on the PCR product and the enzyme digestion product and identifying species according to electrophotogram. The method provided by the invention is simple to operate, and can quickly and accurately realize identification of fish fingerling of common sturgeons only needing shearing less pterygiophore or muscle on the basis of ensuring the survival of the fish sample, so that the, accuracy and confidence level of an identification result are enhanced, and the work efficiency is improved greatly.

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 cartilage and hard 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 "b...

Claims

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Application Information

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