Female specific primer of Penaeus chinensis and application thereof

A Chinese prawn, specific technology, applied in the direction of microbial determination/inspection, biochemical equipment and methods, DNA/RNA fragments, etc., can solve the problems of distinguishing between male and female, hindering the development of parthenogenesis and breeding industry, and avoiding mistakes Judgment, beneficial to China's prawn single-sex breeding and aquaculture industry, and improving the effect of correct rate

Inactive Publication Date: 2021-07-23
YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, it is difficult to distinguish males and females of Chinese shrimp juveniles, especially early-stage larvae, and there is no method for gender...

Method used

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  • Female specific primer of Penaeus chinensis and application thereof
  • Female specific primer of Penaeus chinensis and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] A pair of female-specific primers (Female-specific-F / Female-specific-R) of Penaeus chinensis were designed according to the specific DNA fragment of female Penaeus chinensis, and its nucleotide sequence is as follows:

[0031] Female-specific-F: 5'-GTTTCACCTACGCTTCACCC-3' (SEQ ID No.1);

[0032] Female-specific-R: 5'-TCCTTAATCCCTGCTGCATCCA-3' (SEQ ID No. 2).

[0033] In order to avoid the situation of not amplifying the band due to the template problem, resulting in misjudgment of the result, the present invention also designed a pair of internal reference primers as a control, the nucleotide sequence of which is as follows:

[0034] Control-F: 5'-GGGTGGGTGGTATGGAAAGT-3' (SEQ ID No. 3);

[0035] Control-R: 5'-TCCTTAATCCTGCTGCATCCA-3' (SEQ ID No. 4).

Embodiment 2

[0037] Samples for test: 20 "Huanghai No. 1" live Penaeus prawns of known sex, including 10 male and 10 male.

[0038] 1. Take the muscle tissue of the test sample, extract the genomic DNA, measure the concentration and dilute to 100ng / μL;

[0039] The extraction method of the genomic DNA is as follows: homogenate the muscle tissue of the sample in the lysate; add proteinase K to digest the tissue, and digest it in a water bath at 55°C until clarified; use a standard phenol-chloroform extraction method or use a commercial Kit to extract DNA.

[0040] 2. Using the genomic DNA obtained in step 1 as a template, carry out PCR amplification; wherein, the sequence of the Chinese prawn female-specific primer pair used in PCR amplification is as follows:

[0041] Female-specific-F: 5'-GTTTCACCTACGCTTCACCC-3' (SEQ ID No.1);

[0042] Female-specific-R: 5'-TCCTTAATCCTGCTGCATCCA-3' (SEQ ID No.2);

[0043] The sequence of the control primer pair used in PCR amplification is as follows: ...

Embodiment 3

[0050] Samples to be tested: 12 sea-caught wild Penaeus sinensis frozen samples of known sex, including 6 male and 6 male.

[0051] Implementation steps and experimental conditions are the same as in Example 2.

[0052] Electrophoresis results see figure 2 , where M is Marker, the 6 lanes on the left are female Penaeus chinensis, two bands are amplified, the sizes are 392bp and 210bp respectively, and the 6 lanes on the right are male Penaeus chinensis, only one band is amplified with a size of 392bp Bands.

[0053] It can also be shown from the above results that the Chinese prawn female-specific primers (Female-specific-F / Female-specific-R) obtained by the present invention have strong specificity, and the primer pair and the corresponding PCR detection method can quickly and accurately detect Chinese prawns. The gender identification of living or frozen samples of shrimp is simple and easy to operate, and is not limited to the developmental period of Penaeus chinensis, a...

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Abstract

The invention discloses a female specific primer of Penaeus chinensis and application thereof. The sequence of the female specific primer is as follows: Femal-specific-F: 5'-GTTTCACCTACGCTTCACCC-3', and Femal-specific-R is: 5'-TCCTTAATCCTGCTGCATCCA-3'. The primer is used for carrying out PCR (Polymerase Chain Reaction) amplification on genome DNA (Deoxyribose Nucleic Acid) of Penaeus orientalis to be detected, if a electrophoresis result shows that two bands of 210 bp and 392 bp appear, the Penaeus orientalis are judged to be female Penaeus chinensis, and if only the band of 392 bp appears, the Penaeus orientalis are judged to be male Penaeus chinensis. The invention also utilizes a designed contrast primer to avoid misjudgment of the identification result caused by the problem of the template, the accuracy is improved. The specific primer provided by the invention has strong specificity, and the identification method is simple, rapid and accurate, and is especially suitable for early genetic sex identification of the penaeus chinensis.

Description

technical field [0001] The invention belongs to marine shrimp sex identification technology, in particular to a Chinese prawn female-specific primer and application thereof. Background technique [0002] Chinese prawn (Fenneropenaeus chinensis) belongs to Arthropoda, Crustacea, Decapoda, Penaeidae, also known as Oriental prawn, commonly known as prawn, prawn, mainly distributed in the Yellow Sea and the East China Sea The Shengsi Islands and Zhoushan Islands in the north are one of the most representative seawater economically farmed animals in my country, and enjoy a high reputation in the international market. Penaeus chinensis is a wide-temperature, wide-saline, annual warm-water large-scale migratory shrimp. It has obvious migration habits in the Yellow Sea and Bohai Sea. It is divided into winter migration and reproductive migration. The spawning period is between April and June. The amount is 300,000 to 1,000,000 capsules. In recent years, the annual production of pra...

Claims

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Application Information

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IPC IPC(8): C12Q1/6879C12Q1/6888C12Q1/686C12N15/11
CPCC12Q1/6879C12Q1/6888C12Q1/686
Inventor 王琼何玉英任宪云吕建建王佳佳刘萍李健
Owner YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
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