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SNP marker for distinguishing ability of litopenaeus vannamei against vibrio harveyi infection and detection method thereof and application of SNP marker

An anti-Vibrio harveta and vanabine-resistant technology is applied in the SNP marker for distinguishing the anti-Vibrio harvetii infection ability of Litopenaeus vannamei and its detection and application fields, which can solve the problems of cumbersome operation, high cost and the like, and achieve low cost effect

Active Publication Date: 2020-02-14
SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The four-primer amplification hindered mutation system PCR not only has the accuracy of DNA sequencing, but also overcomes the disadvantages of high cost, cumbersome operation, false positives, etc., and has no special requirements for the detected sequence sites

Method used

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  • SNP marker for distinguishing ability of litopenaeus vannamei against vibrio harveyi infection and detection method thereof and application of SNP marker
  • SNP marker for distinguishing ability of litopenaeus vannamei against vibrio harveyi infection and detection method thereof and application of SNP marker
  • SNP marker for distinguishing ability of litopenaeus vannamei against vibrio harveyi infection and detection method thereof and application of SNP marker

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Embodiment 2

[0040] A prawn farm in Zhanjiang had 1,000 tails of Litopenaeus vannamei with a size of 8-10cm. Swimming feet were taken to extract the genomic DNA of each Litopenaeus vannamei (taking the tissue does not affect the survival of the Litopenaeus vannamei), and carried out the Lyz- i2 gene four-primer amplification hindered mutation system region PCR detection (determining the genotyping of the SNP site), the specific method is as follows:

[0041] 1. Amplification primers:

[0042] LyzSNPF1: 5'-GAGACCGTCCGCCGCTACAT-3'

[0043] LyzSNPR1: 5'-ACCACAAACGACACCCTACCATT-3'

[0044] LyzSNPF2: 5'-TCAACTCCTCACGCGAGTAG-3';

[0045] LyzSNPR2: 5'-GCCGTTGCCGTCGCAATCD-3';

[0046] LyzSNPF3: 5'-TCAACTCCTCACGCGAGTAH-3';

[0047] LyzSNPR3: 5'-GCCGTTGCCGTCGCAATCC-3';

[0048] Among them, D is A, T, G degeneracy, H is A, T, C degeneracy.

[0049] 2. Amplification system:

[0050] (1) Amplification system combination 1:

[0051]

[0052] (2) Amplification system combination 2:

[0053] ...

Embodiment 3

[0062]Vibrio harveyi was isolated from the hepatopancreas of luminescent vibrio prawn Litopenaeus vannamei by the South China Sea Institute of Oceanology, Chinese Academy of Sciences. The strains were stored in 25% glycerol at -80°C, streaked in LB solid medium, inoculated in LB liquid medium and cultured with shaking at 30°C for 24 hours. The bacterial solution was centrifuged at 3000 g for 10 minutes at high speed, and the precipitated bacterial cells were washed 3 times with PBS buffer and then suspended in an equal volume. The concentration of the bacterial solution was determined by a spectrophotometer at 560nm (OD560).

[0063] Then, PBS (50 mM, pH 6.2) was used to resuspend to prepare a Vibrio harveyi suspension, which was measured by a spectrophotometer at 450 nm (OD450), so that its OD450 was 0.6. The long and short Lyz-i2 recombinant proteins (the short Lyz-i2 protein sequence is shown in SEQ ID NO.3, and the long Lyz-i2 protein sequence is shown in SEQ ID NO.5) wer...

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Abstract

The present invention discloses a SNP marker for distinguishing ability of litopenaeus vannamei against vibrio harveyi infection and a detection method thereof and an application of the SNP marker. The SNP marker is located at the 1191th site of a Lyz-I2 gene sequence of litopenaeus vannamei, with mutation type comprising G homozygote, heterozygote of G and three other nucleotides, and homozygoteor heterozygote of three other nucleotides except for G. The invention provides a SNP site for distinguishing ability of litopenaeus vannamei against vibrio harveyi infection, and develops a tetra-primer for amplifying genotype of the SNP site and the detection method for detecting the SNP genotype. The method can simply, rapidly and accurately detects polymorphism forms of the site at low cost, thereby facilitating to rapidly screen individual litopenaeus vannamei and breeding group with high ability against vibrio harveyi, and to maintain antibacterial varieties of litopenaeus vannamei against vibrio harveyi.

Description

technical field [0001] The invention belongs to the field of aquaculture biotechnology, more specifically, relates to the detection of gene single nucleotide polymorphism (SNP), in particular to a kind of i-2 type lysozyme gene of Litopenaeus vannamei, one of which causes different antibacterial ability to lyse bacteria The SNP molecular marker produced by the enzyme protein, and the detection method of the SNP marker genotype detected by the PCR detection method of the four-primer amplification hindered mutation system, and the antibacterial broodstock individual identification, antibacterial prawn population selection and antibacterial prawn strain maintenance. application. Background technique [0002] Litopenaeus vannamei, commonly known as Litopenaeus vannamei, is the first cultured prawn species in the world. In my country, the annual output of Litopenaeus vannamei has reached 1.67 million tons, accounting for 83% of the total output of cultured prawns in the country....

Claims

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Application Information

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IPC IPC(8): C12Q1/6888C12Q1/6858C12N15/11
CPCC12Q1/6858C12Q1/6888C12Q2600/124C12Q2600/156C12Q2531/113C12Q2535/137C12Q2565/125
Inventor 陈廷胡超群任春华王艳红张鑫李小敏江晓黄文
Owner SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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