Minimally invasive rapid identification of the sex chromosome type of soft-shelled soft-shelled turtle zw PCR primer set, site, method and kit

A technology of Chinese soft-shelled turtle and chromosome, which is applied in biochemical equipment and methods, microbial measurement/inspection, recombinant DNA technology, etc. It can solve the problems of cumbersome operation, trauma to the individual to be inspected, sacrifice of the individual to be inspected, etc., and achieve accurate and intuitive results , the effect of easy operation

Active Publication Date: 2019-10-18
ZHEJIANG FISHERIES TECH EXTENSION STATION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, these methods are cumbersome to operate, and need to sacrifice the individual to be tested or cause serious trauma to the individual to be tested, so they do not meet the requirements of practical application

Method used

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  • Minimally invasive rapid identification of the sex chromosome type of soft-shelled soft-shelled turtle zw PCR primer set, site, method and kit
  • Minimally invasive rapid identification of the sex chromosome type of soft-shelled soft-shelled turtle zw PCR primer set, site, method and kit
  • Minimally invasive rapid identification of the sex chromosome type of soft-shelled soft-shelled turtle zw PCR primer set, site, method and kit

Examples

Experimental program
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Effect test

Embodiment 1

[0042] The primers provided in this example for quickly identifying the ZW sex chromosome type of soft-shelled turtles include 1 forward primer and 1 reverse primer, wherein:

[0043] The forward primer for identification of the ZW sex chromosome type of soft-shelled turtle is shown in SEQ ID No.1;

[0044] The reverse primer for identifying ZW sex chromosome type of soft-shelled turtle is shown in SEQ ID No.2.

[0045] specific:

[0046] The forward primer sequence is: 5'-TGGTGCTGTRAAGAAGGMRRCT-3',

[0047] The reverse primer sequence is: 5'-TTCTTCATCTTCCTCCTCRGGT-3'

[0048]This method is based on the ZW sex chromosome linked gene sequence database obtained by comparative analysis of the NCBI Chinese soft-shelled turtle genome (Pelsin-1.0), and a pair of Chinese soft-shelled turtle ZW sex-chromotype identification primers were designed and verified according to the differential fragments.

Embodiment 2

[0050] The PCR method for quickly identifying the ZW sex chromosome type of soft-shelled soft-shelled turtle provided in this embodiment includes the following steps:

[0051] (1) Sampling of soft-shelled soft-shelled turtle tissues to be tested

[0052] Randomly select 10 male and female soft-shelled soft-shelled turtles identified by appearance (Samples 1-5 are female soft-shelled turtles of the Japanese strain of Chinese soft-shelled turtle; samples 6-10 are female soft-shelled soft-shelled turtles of Qingxi; samples 11-15 are Japanese strains of Chinese soft-shelled turtles Male soft-shelled soft-shelled turtle; samples 16-20 are Qingxi black soft-shelled soft-shelled soft-shelled soft-shelled soft-shelled soft-shelled soft-shelled soft-shelled soft-shelled soft-shelled soft-shelled soft-shelled soft-shelled soft-shelled soft-shelled soft-shelled soft-shelled soft-shelled soft-shelled soft-shelled soft-shelled soft-shelled soft-shelled soft-shelled turtle (No. 16-20), after...

Embodiment 3

[0073] The PCR method for rapid and minimally invasive identification of the ZW sex chromosome type of soft-shelled soft-shelled turtle provided in this example comprises the following steps: (1) blood sample collection of newly hatched juvenile soft-shelled soft-shelled turtle

[0074] Randomly select 10 juvenile soft-shelled soft-shelled soft-shelled soft-shelled soft-shelled turtles just out of their shells, and use a 10 μL pipette to collect 1 μL of blood samples in the yolk sac after opening the yolk sac with a syringe needle for juvenile soft-shelled turtles 1-5, and transfer them to PCR tubes; 6 -No. 10 only punctured the skin of the hindlimb with a syringe needle, collected 1 μL of peripheral blood with a 10 μL pipette, and transferred it to a PCR tube. After blood collection, each juvenile soft-shelled turtle was placed in a separate water basin, treated with 10 ppm potassium permanganate for 5 minutes, and kept for five days for temporary observation. No wound infecti...

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Abstract

The invention discloses a PCR primer set, site, method and kit for minimally invasive and rapid identification of the ZW sex chromosome type of pelodiscus sinensis. The sequence of the primer set is shown as SEQ ID No.1 and SEQ ID No.2. With a trace amount (0.5-1 microliter) of pelodiscus sinensis blood as a starter template, the primer set is used for carrying out PCR amplification, an amplification product is subjected to electrophoresis detection, and when 2031 bp bands appear in the amplification product, it is determined that the pelodiscus sinensis is in a ZZ sex chromosome type; when 2031 bp and 1624 bp bands appear in the amplification product, it is determined that the pelodiscus sinensis is in a ZW sex chromosome type. With the trace amount of blood obtained by minimally invasivecollection as a starter material, the sex chromosome type of the pelodiscus sinensis is rapidly detected; the detection hardly damages tested individuals, it can be ensured that pelodiscus sinensis in each growth and development stage can survive healthily after sampling, and an important tool is provided for identifying the sex hromosome type in the pelodiscus sinensis monosex culture technologyand sex-controlled breeding.

Description

technical field [0001] The invention belongs to the technical field of molecular biology identification, relates to the field of identification of the sex chromosome type of soft-shelled soft-shelled turtle ZZ / ZW, and in particular relates to a PCR amplification primer set and a positioner for minimally invasive and rapid identification of the sex chromosome type of soft-shelled soft-shelled turtle, which can save the genome extraction operation. Points, methods and kits. Background technique [0002] The Chinese soft-shelled soft-shelled turtle (Pelodiscus sinensis) belongs to the genus Soft-shelled Turtle of the order Turtleidae of the order Reptiles, and is commonly known as soft-shelled turtle, turtle, and tortoise. It is distributed in various provinces in my country except Xinjiang, Tibet, and Qinghai. At present, the Chinese soft-shelled turtle breeding industry has developed into a large-scale and influential special aquaculture industry. In 2017, the total national ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6888C12Q1/6879C12Q1/686C12N15/11
Inventor 许晓军张海琪朱凝瑜孟庆辉
Owner ZHEJIANG FISHERIES TECH EXTENSION STATION
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