阅读说明：本技术 一种从贝类***物中提取贝类dna的方法 (Method for extracting shellfish DNA from shellfish excrement ) 是由 董志国 张敏 茆双 段海宝 冯森磊 孙泽鹏 李雯倩 张冬冬 于 2019-07-31 设计创作，主要内容包括：本发明公开一种从贝类排泄物中提取贝类DNA的方法,该提取方法为采集贝类排泄物置于筛绢网上,ddH<Sub>2</Sub>O缓慢冲洗,加入ddH<Sub>2</Sub>O,吹打成排泄物匀浆,离心取上清液；加入SDS、蛋白酶K,混匀后水浴；加入RNA酶,混匀后水浴,离心取上清液；加入冰冷饱和酚,混匀后水浴,离心取上清液；加入氯仿,混匀后水浴,离心取上清液；加入异丙醇沉淀DNA,混匀,离心,弃上清；加入乙醇漂洗沉淀DNA,混匀,离心,取沉淀干燥,即提取获得贝类DNA。本发明可应用于贝类DNA的无损伤提取,对贝类无刺激无损伤,操作简单,检测准确性高,宜于推广应用。(The invention discloses a method for extracting shellfish DNA from shellfish excrement, which comprises collecting shellfish excrement, placing on a bolting silk net, and adding ddH 2 Slowly flushing with O, adding ddH 2 Blowing and beating the mixture into a drainage homogenate, and centrifuging to obtain a supernatant; adding SDS and proteinase K, mixing uniformly and then carrying out water bath; adding RNA enzyme, mixing, water bathing, centrifuging, and collecting supernatant; adding ice-cold saturated phenol, mixing, water bathing, centrifuging, and collecting supernatant; adding chloroform, mixing, water bathing, centrifuging, and collecting supernatant; adding isopropanol to precipitate DNA, mixing, centrifuging, and removing supernatant; adding ethanol to rinse and precipitate DNA, mixing uniformly, centrifuging, taking precipitate, and drying to obtain shellfish DNA. The invention can be applied to the nondestructive extraction of shellfish DNA, has no stimulation and no damage to shellfish, has simple operation and high detection accuracy, and is suitable for popularization and application.)

1. A method for extracting shellfish DNA from shellfish excrement comprises the following steps:

(1) placing shellfish excrement on 200 mesh bolting-silk net, adding appropriate amount of ddH₂O, slowly washing the excrement to remove impurities on the surface of the excrement; about 200mg of fecal material was placed in a 1.5mL centrifuge tube and 200. mu.L ddH was added₂O, blowing and beating the mixture into excrement homogenate by a suction pipe, centrifuging the excrement homogenate for 3min at the speed of 800r/min, and taking supernatant;

(2) adding 400 μ L10% SDS and 10 μ L proteinase K, completely digesting in 65 deg.C water bath for 60min, mixing once every 10min, adding 10 μ L20mg/mLRNase, water bath at 37 deg.C for 10min, centrifuging at 12000r/min for 3min, and collecting supernatant;

(3) adding ice-cold saturated phenol with the same volume, reversing the upper part and the lower part, uniformly mixing, standing at room temperature for 5min, centrifuging at 12000r/min for 12min, and taking supernatant; adding equal volume of chloroform, turning upside down, mixing, centrifuging at 12000r/min for 10min, and collecting supernatant; adding isovolumetric isopropanol, reversing for 8 times, mixing well, standing at room temperature for 3min, centrifuging at 12000r/min for 5min, and removing supernatant;

(4) adding 1mL of 70% ice-cold ethanol for rinsing, centrifuging at 12000r/min for 3min, and removing the supernatant; repeatedly rinsing once, centrifuging and discarding the supernatant; the precipitate was dried at room temperature and dissolved in 30. mu.LTE Buffer.

2. The method for extracting shellfish DNA from shellfish excretion according to claim 1, wherein the shellfish excretion of step 1) is 0-5d (summer)/0-10 d (spring, autumn)/0-25 d (winter) excretion from shellfish; ddH is necessary for rinsing₂O slowly washes the surface of the excrement without destroying the excrement structure.

3. The method for extracting shellfish DNA from shellfish excrement according to claim 1 or 2, wherein said shellfish comprises marine shellfish such as Cyclina sinensis, Meretrix meretrix Linnaeus, scallop, oyster, etc.

Technical Field

The invention relates to a DNA extraction method, in particular to a method for extracting shellfish DNA from shellfish excrement.

Background

The shellfish farming industry plays an important role in supplying high-quality protein, increasing fisherman income, improving offshore environment and the like. Meanwhile, shellfish culture also faces the problem of resource failure, more and more scholars obtain shellfish DNA through molecular biology technology and research the genetic information thereof, and the molecular assisted breeding method is used for achieving the purposes of improving shellfish quality and breeding shellfish stress-resistant new products and solving the problem of shellfish resource exhaustion. However, the traditional sampling method mostly focuses on the destructive sampling or the non-destructive sampling, such as direct dissection, shell opening by using a mouth expander, drilling on the shell and the like, which damages the integrity of the internal tissue structure of the shellfish, causes strong mechanical damage to the shellfish and even causes death, and is not beneficial to the shellfish later-stage seed preservation, genetic breeding and the protection of shellfish resources, especially to endangered precious shellfish. Therefore, how to extract the shellfish DNA without damage becomes a technical problem to be solved urgently in the field.

Disclosure of Invention

In order to solve the problems, the invention provides a method for extracting shellfish DNA from excrement so as to realize nondestructive DNA extraction of shellfish, which is realized by the following steps:

a method for nondestructive extraction of shellfish DNA from excrement comprises the following steps:

1. collecting shellfish excrement and pretreating:

(1) starving the shellfish for 2 days to completely discharge impurities in the shellfish. Feeding a proper amount of bait, and collecting excrement from the bottom of the pool by using a siphon method after defecation.

(2) Moving the collected feces to 20On a 0-mesh screen cloth, using ddH₂O carefully and slowly rinse the fecal surface to remove surface impurities. Approximately 200mg of feces were transferred to a 1.5mL centrifuge tube and approximately 200. mu.L of ddH was added₂And O, blowing and beating the mixture into excrement homogenate by using a suction pipe, centrifuging the excrement homogenate for 3min at the speed of 800r/min, and taking supernatant.

2. DNA extraction:

(1) adding 400 μ L10% SDS and 10 μ L of protease K into the supernatant, performing water bath at 65 deg.C for 60min until complete digestion, mixing uniformly every 10min, adding 10 μ L20mg/mLRNase, performing water bath at 37 deg.C for 10min, centrifuging at 12000r/min for 3min, and collecting the supernatant;

(2) adding ice-cold saturated phenol with the same volume, reversing the upper part and the lower part, uniformly mixing, standing at room temperature for 5min, centrifuging at 12000r/min for 12min, and taking supernatant; adding equal volume of chloroform, turning upside down, mixing, centrifuging at 12000r/min for 10min, and collecting supernatant; adding isovolumetric isopropanol, reversing for 8 times, mixing well, standing at room temperature for 3min, centrifuging at 12000r/min for 5min, and removing supernatant;

(3) adding 1mL of 70% ice-cold ethanol for rinsing, centrifuging at 12000r/min for 3min, and removing the supernatant; repeatedly rinsing once, centrifuging and discarding the supernatant; the precipitate was dried at room temperature and dissolved in 30. mu.L of TE Buffer.

The 'shellfish' in the invention refers to marine shellfish including clam, scallop, oyster and the like.

Has the advantages that: the invention can be applied to the nondestructive extraction of shellfish DNA, has no stimulation and no damage to shellfish, has simple operation and high detection accuracy, and is suitable for popularization and application.

Drawings

FIG. 1 is a schematic diagram showing the electrophoresis result of the method of the present invention for extracting DNA in example 1;

FIG. 2 is a schematic diagram of the detection result of the clam DNA extracted from the clam feces by 4 pairs of primers PCR amplification electrophoresis in example 2;

FIG. 3 is a graph showing the morphological changes of the clam feces in example 3 at different soaking times and ambient temperatures;

FIG. 4 is a schematic diagram showing the electrophoresis results of the DNA of the clam feces in example 4 at different soaking times and ambient temperatures;

table 1 shows 2 pairs of clam mitochondrial DNA primers and 2 pairs of clam nuclear genomic DNA primers.

Detailed Description