Microsatellite multiplex-PCR (Polymerase Chain Reaction) method for carrying out paternity testing on crassostrea gigas

A technology for paternity testing and oyster growth, which is applied in biochemical equipment and methods, microbiological measurement/inspection, etc., can solve the problems of easy loss and change of physical markers, low reliability, etc., and achieve the effect of improving analysis accuracy and experimental efficiency

Active Publication Date: 2016-03-30
OCEAN UNIV OF CHINA
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  • Abstract
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AI Technical Summary

Problems solved by technology

For a long time, most aquaculture products have been marked by physical methods

Method used

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  • Microsatellite multiplex-PCR (Polymerase Chain Reaction) method for carrying out paternity testing on crassostrea gigas
  • Microsatellite multiplex-PCR (Polymerase Chain Reaction) method for carrying out paternity testing on crassostrea gigas
  • Microsatellite multiplex-PCR (Polymerase Chain Reaction) method for carrying out paternity testing on crassostrea gigas

Examples

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Embodiment

[0031] (1) Select 1-year-old long oyster broodstock with mature gonads for artificial induction, and establish 12 full-sibling single-pair mating families; collect all parent individuals (24 in total) and some offspring individuals (40 for each family) from each family. D-shaped larva);

[0032] (2) use the phenol chloroform method to extract the DNA of each parent, and use the chelating resin method to extract the DNA of each larva;

[0033] (3) Synthesize primers according to the 18 primer sequences and universal primer sequences screened above;

[0034] (4) Multiplex PCR amplification was carried out using the aforementioned 6 kinds of primer combinations, and the reaction system of multiplex PCR was shown in Table 1;

[0035] The reaction program of multiplex PCR was: 94°C for 3min; denaturation at 94°C for 30s, annealing at annealing temperature for 60s, extension at 72°C for 75s, 35 cycles; denaturation at 94°C for 30s, annealing at 53°C for 60s, extension at 72°C for 7...

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Abstract

The invention discloses a microsatellite multiplex-PCR (Polymerase Chain Reaction) method for carrying out a paternity testing on crassostrea gigas. The microsatellite multiplex-PCR method comprises the following steps of extracting parent and offspring DNA (Deoxyribose Nucleic Acid); carrying out multiplex-PCR amplification on extracted DNA samples by using 18 microsatellite sites and M13 (-21) universal primers with fluorescent marks; detecting a fluorescent signal of an amplified product, and obtaining parent and offspring gene type data; carrying out genetic relationship identification on the parent and the offspring. The microsatellite multiplex-PCR method disclosed by the invention verifies that two groups of multiplex-PCR combinations are used for carrying out the paternity testing on 12 crassostrea gigas full-sib families, so that the identification success rate is up to 100 percent; a set of DNA mark-based paternity testing technology which is economic and effective and is high in accuracy is provided for the crassostrea gigas; by a genealogical relationship record obtained through microsatellite mark identification, producing places of individual crassostrea gigas can be traced back, so that a tracking and source tracing technology of crassostrea gigas products can be established, and powerful technical support can be provided for solving food safety problems which are increasingly concerned by people in nowadays society.

Description

technical field [0001] The invention relates to a method for paternity identification of aquatic animals, in particular to a method for paternity identification of long oyster by using a microsatellite multiplex PCR method. Background technique [0002] The microsatellite marker method has the characteristics of rich polymorphism, high heterozygosity, good stability, following Mendelian segregation law, co-dominant inheritance, and easy PCR amplification. It is one of the most popular molecular markers today. In paternity testing, compared with SNP markers, using microsatellite markers requires fewer loci, but the relative identification power can reach more than 5 times; microsatellite marker method does not require special equipment, and it is easy to operate, easy to detect, time-saving and efficient and high accuracy features. [0003] Using microsatellite markers for paternity identification, the traditional analysis method is to type the PCR amplification products on ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/686C12Q1/6888C12Q2600/156C12Q2600/16C12Q2537/143C12Q2563/107C12Q2565/125
Inventor 李琪刘婷于红
Owner OCEAN UNIV OF CHINA
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