CN108588065A - A kind of Penaeus Vannmei viral DNA extracting method - Google Patents
A kind of Penaeus Vannmei viral DNA extracting method Download PDFInfo
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- CN108588065A CN108588065A CN201810426885.XA CN201810426885A CN108588065A CN 108588065 A CN108588065 A CN 108588065A CN 201810426885 A CN201810426885 A CN 201810426885A CN 108588065 A CN108588065 A CN 108588065A
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Abstract
The invention discloses a kind of Penaeus Vannmei viral DNA extracting methods, include the following steps：A, sample pre-treatments；B, DNA is extracted；C, DNA is precipitated；D, DNA is purified；E, DNA dissolvings and preservation, the extracting method that the present invention uses is convenient and efficient, at low cost, and extraction accuracy is high, is of great significance to the pathogenesis and prevention of studying Penaeus Vannmei disease.
Description
Technical field
The present invention relates to DNA extractive techniques field, specially a kind of Penaeus Vannmei viral DNA extracting method.
Background technology
Penaeus Vannmei, scientific name east Penaeus Vannmei, also known as Chinese Penaeus Vannmei, spot section shrimp.Arthropoda is soft
First guiding principle, Decapoda, Penaeus Vannmei section, Penaeus Vannmei category.Penaeus Vannmei belongs to individual greatly, is generally called prawn.
General 16-22 centimetres of the female growing individual body length of Penaeus Vannmei, weighs about 50-80 grams, maximum up to 30 lis
Rice, weighs 250 grams;Male is smaller, and body is 13-18 centimetres long, 30-50 grams of weight.Penaeus Vannmei is wide warm eurysalinity aquatic/marine animals.Mang
Ox island Penaeus Vannmei body is in long tubular, and left and right is flat-sided, and body is divided into head, chest and abdomen, is made of 20 body segments.Abdomen compared with
Long, muscular, merogenesis is apparent；Penaeus Vannmei can divide sedentariae (such as Japanese Penaeus Vannmei, wide ditch Penaeus Vannmei, Europe
Continent Penaeus Vannmei, Bohai Sea Penaeus Vannmei etc.) and migration type (such as Chinese Penaeus Vannmei, the lucky Penaeus Vannmei of ink, long Maonan
Penaeus vannamei), preceding one kind is dwelt in littoral shallow sea, and daytime often slips into husky bottom, does not make large-scale movement;Latter class is dwelt in river mouth
Littoral muddiness marine site, often makees large-scale movement and migration.Penaeus Vannmei is mainly food, such as crinosity with benthonic invertebrates
Class, small-sized shell-fish and bivalve etc., also catch zooplankter sometimes.
DNA virus is one kind of biological virus, belongs to level-one virus.DNA virus is widely present in people, vertebrate, elder brother
In polypide and in a variety of continuous cell lines, each virus can only infect a kind of animal (individual exceptions), only a small number of to cause a disease.
The modes of reproduction of DNA virus be replicate, reproductive process be absorption (virion borrow capsid on receptor and host it is thin
Together, otherwise, virion cannot invade host cell to born of the same parents' " adherency ".
Currently, viral DNA extraction step is complicated in Penaeus Vannmei, and it is cumbersome, it is of high cost, and also extraction accuracy is low,
It is therefore desirable to be improved.
Invention content
The purpose of the present invention is to provide a kind of Penaeus Vannmei viral DNA extracting methods, to solve above-mentioned background technology
The problem of middle proposition.
To achieve the above object, the present invention provides the following technical solutions：A kind of Penaeus Vannmei viral DNA extracting method,
Include the following steps：
A, sample pre-treatments；
B, DNA is extracted；
C, DNA is precipitated：It takes 200 μ l supernatants to move in an Amoxcillin EP pipes, 100 microlitres of 10mol/l ammonium acetates, mixing is added
Afterwards, 600 μ l are added, absolute ethyl alcohol mixing, -20 DEG C of placement 20min is pre-chilled.12000rpm, 4 DEG C of centrifugation 15min, abandons supernatant；
D, DNA is purified：Precipitation is washed with 75% ethyl alcohol 2 times, and each 12000rpm centrifuges 1min, carefully discards supernatant liquid, finally sinks
It forms sediment and dries 10min in room temperature；
E, DNA dissolvings and preservation：Precipitation is added 100 microlitres of TE buffer solutions DNA after drying and is used for PCR amplification or preservation
It is spare in -20 DEG C.
Preferably, steps are as follows for sample pre-treatments in the step A：
A, the sample of 50mg-100mg is fitted into EP pipes, 75% alcohol of 1ml is added after being ground with disposable grinding rod,
12000rpm, 4 DEG C of centrifugation 5min, discards supernatant liquid；
B, it is primary to repeat step a；
C, 1ml TE buffer solutions are added in EP pipes, after vibrating mixing, 12000rpm, 4 DEG C of centrifugation 5min discard supernatant liquid；
D, it is primary to repeat step c.
Preferably, the step B includes the following steps：
A, cell cracking：
A, extraction buffer 500 μ l, 85 DEG C of warm bath 10min are added in treated sample;
B, 2.5 microlitres of 20mg/ml Proteinase Ks are added, until 100 micrograms of final concentration/ml, 55 DEG C of water-bath 1h after mixing revolve frequently
It is dynamic;
B, DNA is detached：
A, solution is cooled to room temperature, 500 μ l phenol/chloroform/isoamyl alcohol is added, overturn mixing 2min, in 1200rpm, 4 DEG C from
Heart 5min；
B, take the 300 μ l of upper strata aqueous phase after centrifugation in 1 new EP pipes, be added 200 microlitres of TE buffer solutions and 500 μ l phenol/chloroform/
Isoamyl alcohol mixed liquor mixes and overturns 2min, in 1200rpm, 4 DEG C of centrifugation 2min；
C, the 300 μ l of upper strata aqueous phase after centrifugation are taken, 200 microlitres of TE buffer solutions and 500 μ l chloroforms/isoamyl alcohol is added, overturn mixing
2min, in 1200rpm, 4 DEG C of centrifugation 2min.
Compared with prior art, the beneficial effects of the invention are as follows：
1. the extracting method that the present invention uses is easy to operate, at low cost, extraction accuracy is high, and DNA purity is high；Wherein, the present invention adopts
Sample pre-treatments step can effectively remove the foreign ion in sample, avoid influence of the impurity to extraction accuracy；
It is detached using cell cracking and DNA in the DNA extraction steps of use, extraction efficiency can be further improved.
2. for carrying out the basic research of Penaeus Vannmei DNA virus, the pathogenesis of DNA diseases and preventing that there is weight
It acts on.
Specific implementation mode
The following is a clear and complete description of the technical scheme in the embodiments of the invention, it is clear that described embodiment
Only a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, the common skill in this field
The every other embodiment that art personnel are obtained without making creative work belongs to the model that the present invention protects
It encloses.
The present invention provides the following technical solutions：A kind of Penaeus Vannmei viral DNA extracting method, includes the following steps：
A, sample pre-treatments；
B, DNA is extracted；
C, DNA is precipitated：It takes 200 μ l supernatants to move in an Amoxcillin EP pipes, 100 microlitres of 10mol/l ammonium acetates, mixing is added
Afterwards, 600 μ l are added, absolute ethyl alcohol mixing, -20 DEG C of placement 20min is pre-chilled.12000rpm, 4 DEG C of centrifugation 15min, abandons supernatant；
D, DNA is purified：Precipitation is washed with 75% ethyl alcohol 2 times, and each 12000rpm centrifuges 1min, carefully discards supernatant liquid, finally sinks
It forms sediment and dries 10min in room temperature；
E, DNA dissolvings and preservation：Precipitation is added 100 microlitres of TE buffer solutions DNA after drying and is used for PCR amplification or preservation
It is spare in -20 DEG C.
In the present invention, steps are as follows for sample pre-treatments in step A：
A, the sample of 50mg-100mg is fitted into EP pipes, 75% alcohol of 1ml is added after being ground with disposable grinding rod,
12000rpm, 4 DEG C of centrifugation 5min, discards supernatant liquid；
B, it is primary to repeat step a；
C, 1ml TE buffer solutions are added in EP pipes, after vibrating mixing, 12000rpm, 4 DEG C of centrifugation 5min discard supernatant liquid；
D, it is primary to repeat step c.
In the present invention, step B includes the following steps：
A, cell cracking：
A, extraction buffer 500 μ l, 85 DEG C of warm bath 10min are added in treated sample;
B, 2.5 microlitres of 20mg/ml Proteinase Ks are added, until 100 micrograms of final concentration/ml, 55 DEG C of water-bath 1h after mixing revolve frequently
It is dynamic;
B, DNA is detached：
A, solution is cooled to room temperature, 500 μ l phenol/chloroform/isoamyl alcohol is added, overturn mixing 2min, in 1200rpm, 4 DEG C from
Heart 5min；
B, take the 300 μ l of upper strata aqueous phase after centrifugation in 1 new EP pipes, be added 200 microlitres of TE buffer solutions and 500 μ l phenol/chloroform/
Isoamyl alcohol mixed liquor mixes and overturns 2min, in 1200rpm, 4 DEG C of centrifugation 2min；
C, the 300 μ l of upper strata aqueous phase after centrifugation are taken, 200 microlitres of TE buffer solutions and 500 μ l chloroforms/isoamyl alcohol is added, overturn mixing
2min, in 1200rpm, 4 DEG C of centrifugation 2min.
Viral DNA after extraction is put into high-power microscope, we can clearly observe the shape of the viral DNA
State, embodiment reach the desired effect of the patent.
In conclusion the extracting method that the present invention uses is easy to operate, at low cost, extraction accuracy is high, and DNA purity is high；Its
In, the sample pre-treatments step that the present invention uses can effectively remove the foreign ion in sample, avoid impurity to extraction
The influence of precision；It is detached using cell cracking and DNA in the DNA extraction steps of use, extraction efficiency can be further improved.
It although an embodiment of the present invention has been shown and described, for the ordinary skill in the art, can be with
Understanding without departing from the principles and spirit of the present invention can carry out these embodiments a variety of variations, modification, replace
And modification, the scope of the present invention is defined by the appended.
Claims (3)
1. a kind of Penaeus Vannmei viral DNA extracting method, it is characterised in that：Include the following steps：
A, sample pre-treatments；
B, DNA is extracted；
C, DNA is precipitated：It takes 200 μ l supernatants to move in an Amoxcillin EP pipes, 100 microlitres of 10mol/l ammonium acetates, mixing is added
Afterwards, 600 μ l are added, absolute ethyl alcohol mixing is pre-chilled, -20 DEG C are placed 20min, 12000rpm, 4 DEG C of centrifugation 15min, abandon supernatant；
D, DNA is purified：Precipitation is washed with 75% ethyl alcohol 2 times, and each 12000rpm centrifuges 1min, carefully discards supernatant liquid, finally sinks
It forms sediment and dries 10min in room temperature；
E, DNA dissolvings and preservation：Precipitation is added 100 microlitres of TE buffer solutions DNA after drying and is used for PCR amplification or preservation
It is spare in -20 DEG C.
2. a kind of Penaeus Vannmei viral DNA extracting method according to claim 1, it is characterised in that：In the step A
Steps are as follows for sample pre-treatments：
A, the sample of 50mg-100mg is fitted into EP pipes, 75% alcohol of 1ml is added after being ground with disposable grinding rod,
12000rpm, 4 DEG C of centrifugation 5min, discards supernatant liquid；
B, it is primary to repeat step a；
C, 1ml TE buffer solutions are added in EP pipes, after vibrating mixing, 12000rpm, 4 DEG C of centrifugation 5min discard supernatant liquid；
D, it is primary to repeat step c.
3. a kind of Penaeus Vannmei viral DNA extracting method according to claim 1, it is characterised in that：The step B packets
Include following steps：
A, cell cracking：
A, extraction buffer 500 μ l, 85 DEG C of warm bath 10min are added in treated sample;
B, 2.5 microlitres of 20mg/ml Proteinase Ks are added, until 100 micrograms of final concentration/ml, 55 DEG C of water-bath 1h after mixing revolve frequently
It is dynamic;
B, DNA is detached：
A, solution is cooled to room temperature, 500 μ l phenol/chloroform/isoamyl alcohol is added, overturn mixing 2min, in 1200rpm, 4 DEG C from
Heart 5min；
B, take the 300 μ l of upper strata aqueous phase after centrifugation in 1 new EP pipes, be added 200 microlitres of TE buffer solutions and 500 μ l phenol/chloroform/
Isoamyl alcohol mixed liquor mixes and overturns 2min, in 1200rpm, 4 DEG C of centrifugation 2min；
C, the 300 μ l of upper strata aqueous phase after centrifugation are taken, 200 microlitres of TE buffer solutions and 500 μ l chloroforms/isoamyl alcohol is added, overturn mixing
2min, in 1200rpm, 4 DEG C of centrifugation 2min.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201810426885.XA CN108588065A (en) | 2018-05-07 | 2018-05-07 | A kind of Penaeus Vannmei viral DNA extracting method |
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| CN201810426885.XA CN108588065A (en) | 2018-05-07 | 2018-05-07 | A kind of Penaeus Vannmei viral DNA extracting method |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111394348A (en) * | 2020-03-30 | 2020-07-10 | 南华大学 | Method for extracting and detecting free DNA in sewage |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100999733A (en) * | 2006-12-22 | 2007-07-18 | 苏州大学 | Process for extracting leukasmus rhabdovirus genome DNA of prawn |
| CN101403013A (en) * | 2008-11-18 | 2009-04-08 | 中国水产科学研究院淡水渔业研究中心 | Method for sifting macrobrachium nipponensis microsatellite mark with magnetic bead concentration method |
| CN101748118A (en) * | 2009-09-25 | 2010-06-23 | 中国科学院海洋研究所 | A kind of extracting method of prawn intestinal microbial DNA |
| CN102477423A (en) * | 2010-11-24 | 2012-05-30 | 华中农业大学 | Method for extracting DNA from shrimp shell of procambarus clarkii |
-
2018
- 2018-05-07 CN CN201810426885.XA patent/CN108588065A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100999733A (en) * | 2006-12-22 | 2007-07-18 | 苏州大学 | Process for extracting leukasmus rhabdovirus genome DNA of prawn |
| CN101403013A (en) * | 2008-11-18 | 2009-04-08 | 中国水产科学研究院淡水渔业研究中心 | Method for sifting macrobrachium nipponensis microsatellite mark with magnetic bead concentration method |
| CN101748118A (en) * | 2009-09-25 | 2010-06-23 | 中国科学院海洋研究所 | A kind of extracting method of prawn intestinal microbial DNA |
| CN102477423A (en) * | 2010-11-24 | 2012-05-30 | 华中农业大学 | Method for extracting DNA from shrimp shell of procambarus clarkii |
Non-Patent Citations (1)
| Title |
|---|
| FANG，F.等: "A simple and efficient method for purification of prawn baculovirus DNA", 《JOURNAL OF VIROLOGICAL METHODS》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111394348A (en) * | 2020-03-30 | 2020-07-10 | 南华大学 | Method for extracting and detecting free DNA in sewage |
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Application publication date: 20180928 |
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