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Method for noninvasive identification of pseudo-female Chinese softshell turtle through fluorescent quantitative PCR

A fluorescence quantitative and Chinese soft-shelled turtle technology, applied in the field of bioinformatics, can solve the problems of result judgment interference, low specificity, identification of Chinese soft-shelled turtle and pseudo-female soft-shelled turtle, etc., and achieve good stability, high specificity, and strong specificity

Inactive Publication Date: 2020-09-15
ANHUI AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there is no big difference in the appearance of the pseudo female soft-shelled turtle from the normal development of the Chinese soft-shelled soft-shelled turtle, and it is difficult to identify the pseudo-female soft-shelled turtle from the appearance
[0004] The patent document with the publication number CN108588237B discloses a method for identifying the sex of the Chinese soft-shelled turtle by using the difference in the genome sequences on the Z and W chromosomes. This identification method extracts the liver tissue of the Chinese soft-shelled turtle, resulting in the death of the Chinese soft-shelled turtle.
Moreover, the sex-linked gene primers are detected by ordinary PCR, and the specificity is relatively low, and it is easy to produce dimers, which will interfere with the result judgment to a certain extent.
In addition, the ordinary PCR amplification reaction time is long, and the PCR product needs to be detected by electrophoresis, which reduces the detection efficiency

Method used

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  • Method for noninvasive identification of pseudo-female Chinese softshell turtle through fluorescent quantitative PCR
  • Method for noninvasive identification of pseudo-female Chinese softshell turtle through fluorescent quantitative PCR
  • Method for noninvasive identification of pseudo-female Chinese softshell turtle through fluorescent quantitative PCR

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Estrogen-induced cultivation of pseudofemale soft-shelled turtles

[0033] 1.1 Incubation of soft-shelled turtle eggs and treatment with estrogen drugs

[0034] 60 soft-shelled turtle eggs were taken as the treatment group. When the eggs were hatched to the 15th stage, they were treated with a 50 μg / μL 17β-estradiol solution of the Chinese soft-shelled turtle sex hormone. The 17β-estradiol solution was made of 95wt% ethanol as a solvent. Then, use the egg canister to determine the distribution position of the soft-shelled turtle embryo in the soft-shelled turtle egg, and drop 5 μL of medicament on the shell where the embryonic surface of the soft-shelled turtle egg is located. For soft-shelled soft-shelled turtle eggs hatched to the 15-18 stage, drug treatment was carried out at each stage, and the position and dosage of each drop were unchanged, and a total of four sex hormone treatments were carried out. The embryos at the 15-18 stage The characteristics of...

Embodiment 2

[0043] Embodiment 2 fluorescent quantitative PCR test

[0044]Fluorescent quantitative PCR test was carried out on soft-shelled soft-shelled turtle, the specific steps are as follows:

[0045] 1.1 Chinese soft-shelled turtle draws blood

[0046] 1) Place the female soft-shelled turtle phenotype selected above on a clean table, control the soft-shelled turtle and straighten its tail.

[0047] 2) Insert the needle into the position on both sides of the tail, stir the needle slightly, and when blood seeps out, slowly pull the syringe to take out the blood.

[0048] 1.2 Extraction of DNA from the blood of soft-shelled turtle

[0049] Total DNA was extracted from the blood of soft-shelled turtles in each group, and the extracted DNA was detected by ddH 2 O for a 50-fold dilution. 18SrRNA was used as the primer of the target gene, Gapdh was used as the internal reference, and the diluted DNA was used as the template for fluorescent quantitative PCR. The genomic DNA extraction p...

Embodiment 3

[0070] Embodiment 3 determines the threshold

[0071] Take the blood of 50 soft-shelled soft-shelled turtles whose gender is determined to be male, and perform fluorescent quantitative PCR with reference to the steps in Example 2 to measure the gene copy number. With the gene copy number of sample number 1 male soft-shelled turtle as a reference, set it as 1, and calculate the other 49 The relative value Y of the gene copy value of a male soft-shelled turtle and the gene copy value of this male soft-shelled turtle, and the relative value of the fluorescence quantitative results of 50 male soft-shelled turtles are as follows: figure 1 As shown, the relative value of the fluorescence quantitative results of 50 male soft-shelled turtles was used to calculate the average value and standard deviation SD to As the threshold, the threshold value is 2.57.

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Abstract

The invention relates to the technical field of biological information, and discloses a method for noninvasive identification of pseudo-female Chinese softshell turtle through fluorescent quantitativePCR. The method comprises the following specific steps of setting a pair of primer groups for sexual identification of the pseudo-female Chinese softshell turtle, wherein the primer groups comprise primer pairs designed by DNA sequences of target genes 18S rRNA and Gapdh; performing fluorescent quantitative PCR data processing by adopting a 2-[delta][delta]Ct method, using the Gapdh as a reference gene, and calculating the relative copy number of the 18S rRNA in a genome; and by utilizing the fact that the DNA sequence copy number of the 18S rRNA on the W chromosome of the Chinese softshell turtle is higher than that on the Z chromosome, screening out the pseudo-female Chinese softshell turtle with the ovarian gonad and the ZZ genotype. According to the identification method, the fluorescent quantitative PCR technology is used for sexual identification of the Chinese softshell turtle, detection can be achieved only through a trace amount of blood, non-invasive collection of tail bloodof the Chinese softshell turtle has no influence on health of the Chinese softshell turtle, the method has the characteristics of being efficient, high in sensitivity, good in stability and high in specificity, the blank of pseudo-female Chinese softshell turtle identification is filled, breeding of all male softshell turtle fries is facilitated, and the breeding benefit is improved.

Description

technical field [0001] The invention relates to the technical field of biological information, in particular to a method for non-invasive identification of Chinese soft-shelled turtles and pseudo-female soft-shelled turtles by fluorescent quantitative PCR. Background technique [0002] Soft-shelled turtle (Pelodiscus sinensis), commonly known as soft-shelled turtle, is an important and valuable aquaculture species in my country. The male soft-shelled soft-shelled turtle grows faster than the female soft-shelled turtle, has a wider skirt, less fat, and higher prices. Therefore, the development of parthenogenesis of all male seedlings of soft-shelled soft-shelled turtles can significantly improve economic benefits, and is of great significance to the development of the Chinese soft-shelled turtle breeding industry. [0003] The sex determination of soft-shelled turtles in normal development is female heterogamy, that is, the sex chromosomes of female soft-shelled turtles are Z...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6851C12Q1/6888C12N15/11
CPCC12Q1/6851C12Q1/6888C12Q2600/166C12Q2531/113C12Q2545/114C12Q2537/16C12Q2563/107
Inventor 张君赵阳周伟尚乔丹章纪龙
Owner ANHUI AGRICULTURAL UNIVERSITY
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