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Primer pair and kit for distinguishing Channa argus and Channa maculata, and PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) detection method

A technique of PCR-RFLP and a detection method, which is applied in the field of primer pairs for distinguishing snakeheads and snakeheads, can solve problems such as difficult supervision by management departments and deceiving consumers, and achieves convenient, fast and easy operation, increased accuracy and credibility, The effect of improving work efficiency

Inactive Publication Date: 2013-09-11
徐鹏 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Many vendors sell snakeheads as snakeheads, defrauding consumers and bringing regulatory difficulties to the management department

Method used

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  • Primer pair and kit for distinguishing Channa argus and Channa maculata, and PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) detection method
  • Primer pair and kit for distinguishing Channa argus and Channa maculata, and PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) detection method
  • Primer pair and kit for distinguishing Channa argus and Channa maculata, and PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) detection method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] The primer pair was synthesized, and the polynucleotide sequence of the primer pair was designed based on the mitochondrial DNA of snakehead and snakehead, and was synthesized by Sangon Bioengineering (Shanghai) Co., Ltd. using a synthesizer model MERMADE192E DNA synthesizer.

[0044] The synthesized primer is, and the upstream primer is SEQ ID No. The polynucleotide sequence shown in 1: 5. CTAAGCCCTT TCCACAGAGG TTCA3; the downstream primer is SEQ ID No. The polynucleotide sequence shown in 2: 5. GCCCAAAAG CTTGTGTTAG CTG3.

Embodiment 2

[0046] As shown in Figure 1, use kit of the present invention to distinguish the PCR-RFLP detection method of snakehead and spotted snakehead,

[0047] Step 1: Firstly, the genomic DNA of the test sample was extracted as a template, and a small part of the caudal fin of 10 known snakeheads and 10 snakeheads were cut respectively. A total of 20 samples were put into 1.5 mL centrifuge tubes and numbered 1 -20, samples 1-10 are snakeheads, and samples 11-20 are snakeheads. Genomic DNA of 20 samples was extracted using a DNA extraction kit. The steps are as follows:

[0048] a) Cut about 0.5 g of fin rays, put them into a 1.5 mL centrifuge tube, and cut them into pieces.

[0049] b) Add 0.45 mL of trimethylolmethylaminoethanesulfonic acid (TES) and mix well, then add 50 μl of 10% sodium dodecylsulfonate (SDS), 5. Oμl concentration is 20 mg / mL proteinase K, mix well, at 56. C for 4-6 h, shake every 2 h.

[0050] c) After incubating for 4-6 h, place the 1.5 mL centrifuge tube con...

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Abstract

The invention discloses a primer pair and kit for distinguishing Channa argus and Channa maculata, and a PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) detection method. The primer pair comprises a forward primer and a reverse primer, wherein the polynucleotide sequence of the forward primer is disclosed as SEQ ID NO.1, and the polynucleotide sequence of the reverse primer is disclosed as SEQ ID NO.2. The kit containing the primer pair also comprises a PCR conventional reagent, a Taq DNA (deoxyribonucleic acid) polymerase, a 100*PCR buffer, dNTPs (deoxyribonucleotide triphosphates), a digestion reagent, a 10*buffer, a digestion buffer and an EcoRI restriction enzyme. The invention also discloses a PCR-RFLP detection method for distinguishing Channa argus and Channa maculata by using the primer pair and the kit. The method disclosed by the invention is simple to operate, only needs to cut a small amount of fin rays or muscles on the premise of ensuring the survival of the fish species, quickly and accurately identifies the fish species, enhances the accuracy and reliability of the identification result, and greatly enhances the work efficiency.

Description

technical field [0001] The invention relates to the fields of molecular science and taxonomy, in particular to a pair of primers, a kit and a PCR-RFLP detection method for distinguishing snakeheads and snakeheads. Background technique [0002] Channa argus (Channa argus), a fish of the genus Channa (Channidae) of the order Perciformes (Perciformes), commonly known as black fish, also known as northern snakehead fish, snakehead fish, mullet fish, black stick, snakehead fish, salmon and scorpion fish , is a famous and high-quality freshwater fish unique to our country. It is widely distributed in our country except in plateau areas. Snakehead has tender meat, delicious taste, and high nutritional value. It is very popular in the domestic market and is currently widely cultivated in my country, with high economic value. [0003] Channa maculata and Channa maculata belong to the Channaidae genus, and are similar to Channa maculata in terms of morphology. Sometimes the morpholog...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
Inventor 徐鹏董传举陈坤慈孙效文
Owner 徐鹏
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