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A method for simply and rapidly detecting oyster ploidy

A rapid and oyster technology, applied in the field of simple and rapid detection of oyster ploidy, non-lethal, can solve the problems of complex internal components, inability to ensure accurate extraction of hemolymph, etc., and achieve the effect of simple sampling method

Inactive Publication Date: 2018-12-04
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are still great difficulties in how to perform simple and rapid ploidy detection under the condition of ensuring the survival of oysters
At present, the lethal detection methods of oyster ploidy, such as the method of dissecting gills or other tissues, will inevitably cause the loss of precious experimental samples, making it impossible to carry out subsequent experimental research
[0004] A method for detecting the ploidy of oysters by using lymphocytes in oyster coelom fluid (Patent No. 201510194867.X, Li Jun et al., 2015), which can extract mucus without damage, but its operating environment needs to be in seawater and oysters must be naturally It can only be carried out when the double shell is opened, which cannot meet the needs of sampling and detecting ploidy at any time in the experiment; and the hemolymph in the body cavity fluid is extracted by opening the mouth. A single hemolymph, stimulates the oyster to close the shell immediately during the operation, causing problems such as needle clamping and inability to sample or pull out the needle
That is, the patented method is not practical in actual operation

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] (1) Take 30 common long oysters with a shell height of 6-10 cm.

[0029] (2) At the edge between the two shells of the oyster to be tested near the adductor muscle, use needle-nose pliers to crush the edge of the shell to form a gap of 2-3 mm.

[0030] (3) Insert a 1.5 ml disposable syringe into the sinus of the adductor muscle through the gap, and extract 100-200 mL of hemolymph.

[0031] (4) The extracted hemolymph was dropped drop by drop into -20°C pre-cooled absolute ethanol for fixation, and kept overnight at 4°C.

[0032] (5) After centrifuging the fixed blood lymphocytes to remove the supernatant, add 1 mL 1×PBS to resuspend, then add 1 mg RNase A to react for 30 min.

[0033] (6) Then add 35 mg PI and stain for 30 min.

[0034] (7) After filtering through a 300-mesh sieve, the DNA content was detected by flow cytometry to determine the ploidy.

[0035] (8) The tested individuals are transferred to the breeding sea area to continue to grow, and the survival r...

Embodiment 2

[0039] (1) The triploid experiment induction group of the new species of long oyster "Haida No. 2" with a shell height of 3-4 cm, left and right shells and mantles were taken from the Rongcheng aquaculture area of ​​Weihai, and the number was 45.

[0040] (2) At the edge between the two shells of the oyster to be tested near the adductor muscle, use needle-nose pliers to crush the edge of the shell to form a gap of 2-3mm.

[0041] (3) Insert a 1.5 ml disposable syringe into the sinus of the adductor muscle through the gap, and extract 100-200 mL of hemolymph.

[0042] (4) The extracted hemolymph was dropped drop by drop into -20°C pre-cooled absolute ethanol for fixation, and kept overnight at 4°C.

[0043] (5) After centrifuging the fixed blood lymphocytes to remove the supernatant, add 1 mL 1×PBS to resuspend, then add 1 mg RNase A to react for 30 min.

[0044] (6) Then add 35 mg PI and stain for 30 min.

[0045] (7) After filtering through a 300-mesh sieve, the DNA conten...

Embodiment 3

[0050] (1) The triploid experimental induction group of a new strain of Oyster oyster with a shell height of 3-4 cm and black shells on the left and right shells bred in this laboratory was taken from the Rongcheng aquaculture area of ​​Weihai, and the number was 50.

[0051] (2) At the edge between the two shells of the oyster to be tested near the adductor muscle, use needle-nose pliers to crush the edge of the shell to form a gap of 2-3 mm.

[0052] (3) Insert a 1.5 ml disposable syringe into the sinus of the adductor muscle through the gap, and extract 100-200 mL of hemolymph.

[0053] (4) The extracted hemolymph was dropped drop by drop into -20°C pre-cooled absolute ethanol for fixation, and kept overnight at 4°C.

[0054] (5) After centrifuging the fixed blood lymphocytes to remove the supernatant, add 1 mL 1×PBS to resuspend, then add 1 mg RNase A to react for 30 min.

[0055] (6) Then add 35 mg PI and stain for 30 min.

[0056] (7) After filtering through a 300-mesh...

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Abstract

A method for simply and rapidly detecting oyster ploidy is disclosed. The method includes firstly crumbing shell edges, of two shells of an oyster, close to the adductor muscle with sharp-nose pliersto form a gap having a length of 2-3 mm; drawing hemolymph by inserting a disposable syringe through the gap into a hemolymph sinus of the adductor muscle; after the hemolymph is immobilized with absolute alcohol, performing centrifugation and removing a liquid supernatant; adding PBS and suspending the mixture again; performing RNA enzyme reaction and PI staining in order; and performing filtration with a cloth sieve having a size of 300 meshes; and then detecting the DNA content with a flow cytometer to determine the ploidy. The method performing ploidy detection through drawing the hemolymph in the hemolymph sinus of the adductor muscle of the oyster is non-lethal, simple, rapid, not limited by oyster living environment, and the like, and is of great significance for oyster polyploidy breeding research, especially ploidy detection of live oyster polyploidy and subsequent deep research.

Description

technical field [0001] The invention belongs to the technical field of shellfish polyploid detection, and in particular relates to a non-lethal, simple and rapid method for detecting oyster ploidy. Background technique [0002] The research on oyster polyploidy breeding has always been a hot spot in marine economic shellfish genetics and breeding. Compared with common oyster diploid, oyster polyploid has irreplaceable advantages. Triploid oysters can be supplied to the market throughout the year, which solves the problem that diploid oysters have poor taste and are difficult to supply the market due to the large consumption of glycogen in the body in summer, and triploids also have fast growth and good quality And other advantages, loved by the majority of consumers and respected by the oyster farming industry, has formed industrialization, has very broad development prospects. However, oyster triploids have poor fertility, even in theory, they are sterile, and there are p...

Claims

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Application Information

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IPC IPC(8): G01N15/14A61B10/02
CPCG01N15/14A61B10/02
Inventor 李永国李琪于瑞海
Owner OCEAN UNIV OF CHINA
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