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PCR (polymerase chain reaction) identification method for young little yellow croakers and large yellow croakers and primer

An identification method and technology for large yellow croaker, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve problems such as difficulty, small size, unfavorable protection of germplasm resources, etc. , the effect of the method is simple and easy to implement

Inactive Publication Date: 2017-06-23
ZHEJIANG OCEAN UNIV
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  • Claims
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Problems solved by technology

Large yellow croakers and small yellow croakers are similar in size, and are generally distinguished by the ratio of tail peduncle length to tail peduncle height. However, for juvenile large yellow croakers and small yellow croakers, small yellow croakers and large yellow croakers are very similar in shape at the juvenile stage. There are large errors, and it is very difficult to distinguish these two species only from the external morphology, which is not conducive to the protection of germplasm resources

Method used

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  • PCR (polymerase chain reaction) identification method for young little yellow croakers and large yellow croakers and primer

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Embodiment

[0026] 1. Use phenol / chloroform method for DNA extraction: For small yellow croaker and large yellow croaker samples stored in 95% alcohol or -20°C cryopreservation, take 50-100 mg of back muscle tissue, and use traditional method (phenol / chloroform extraction) to extract Genomic DNA.

[0027] The detailed method and steps are as follows:

[0028] (1) Put the sample into a 1.5ml centrifuge tube and cut it into pieces. Add 600 μL of STE (10 mmol / L TriS-Cl, pH 8.0; 0.1 mol / L EDTA, pH 8.0; 1% m / v SDS), shake evenly on a shaker. Add 6 ul of RNase (4 μg / μL), the final concentration is 0.04 μg / μL, and incubate in a 37°C oven for 1 hour.

[0029] (2) Add 15 μL of proteinase K (20 μg / μL) to a final concentration of 0.5 μg / μl, mix by inverting, and incubate overnight (>8 hours) in an oven at 50°C.

[0030] (3) Take it out of the oven and cool to room temperature: add 600 μL (pH 8.0) to balance the phenol, and gently invert and mix until it becomes milky (≥30min). Centrifuge (8000g,...

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Abstract

The invention discloses a PCR (polymerase chain reaction) identification method for young little yellow croakers and large yellow croakers and a primer. The PCR identification method comprises the following steps: extracting genome DNA of a sample, performing PCR amplification on the extracted DNA with the identification primer, and performing agarose gel electrophoresis detection on the PCR amplification product, wherein the sample is a little yellow croaker when one amplification strip occurs and is a large yellow croaker when two amplification strips occur. The identification method disclosed by the invention is simple and easy to implement and high in specificity, and can clearly identify and distinguish plesiomorphous young little yellow croakers and large yellow croakers.

Description

technical field [0001] The invention relates to a species identification method, in particular to a PCR identification method and primers for small yellow croaker and large yellow croaker juveniles. Background technique [0002] Both the small yellow croaker (Larimichthys polyactis) and the large yellow croaker (Larimichthys crocea) belong to the order Perciformes, Perciformes, Totoabaidae, and Yellow croaker, and were once the four famous marine products in my country. Large yellow croakers and small yellow croakers are similar in size, and are generally distinguished by the ratio of tail peduncle length to tail peduncle height. However, for juvenile large yellow croakers and small yellow croakers, small yellow croakers and large yellow croakers are very similar in shape at the juvenile stage. There are large errors, and it is very difficult to distinguish the two species only from the external morphology, which is not conducive to the protection of germplasm resources. ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6888C12Q2600/124
Inventor 刘洋蔡珊珊韩志强
Owner ZHEJIANG OCEAN UNIV
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