CN104946624A - Preparation method of total viral nucleic acid of shellfish - Google Patents
Preparation method of total viral nucleic acid of shellfish Download PDFInfo
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- CN104946624A CN104946624A CN201510201269.0A CN201510201269A CN104946624A CN 104946624 A CN104946624 A CN 104946624A CN 201510201269 A CN201510201269 A CN 201510201269A CN 104946624 A CN104946624 A CN 104946624A
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- nucleic acid
- shellfish
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Abstract
The invention discloses a preparation method of total viral nucleic acid of shellfish. The preparation method comprises the following steps: (1) grinding, namely grinding a shellfish tissue sample into a powder in the environment of liquid nitrogen; (2) homogenizing, namely adding a PBS buffer solution, and then homogenizing into a slurry; (3) obtaining a total viral coarse extract, namely performing centrifugal treatment on the slurry, filtering the obtained supernatant by use of a filter membrane and then performing the centrifugal treatment, abandoning the supernatant, and obtaining the precipitate as the total viral coarse extract; (4) extracting the nucleic acid, namely adding a nuclease solution to the total viral coarse extract to form a suspension, treating the suspension on a shaking table at 37 DEG C for 25-40min, and then extracting the total viral coarse extract; (5) amplifying the nucleic acid, namely amplifying the total viral nucleic acid to increase the nucleic acid concentration to facilitate high-throughput sequencing. The method is simple to operate; the obtained shellfish total viral nucleic acid is high in purity; the nucleic acid is complete, and the nucleic acid concentration meets the requirements of high-throughput sequencing.
Description
Technical field
The preparation method of total virus nucleic acid of the present invention, especially relates to a kind of preparation method of shellfish total virus nucleic acid.
Background technology
The mollusks such as shellfish are the second gates of animal kingdom, and species are very abundant, and distribution widely.Therefore, shellfish biology is an important treasure-house of bio-diversity resources research.The diversity of shellfish determines again the diversity of its body inner virus or symbiotic microorganism further.Carry out deep research to body of shellfish inner virus or microorganism all to have great importance to Safety of Aquatic Products, aquaculture sound development, living aquatic resources utilizes, medicine is researched and developed etc.
But the shellfish virus with complete genomic sequence known at present seldom.OsHV, the AVNV of scallop, AbHV and the AbSV virus of abalone of only having oyster that can find in ncbi database.Due to the limitation of existing investigative technique, existing research is all first be purified from morbidity sample (sample that namely viral level is high) by specific virus by traditional purification process, and then adopts the method such as library construction or pcr amplification to obtain its complete sequence.Such method can only study a kind of virus at every turn, and must obtain from the high infected material of viral level.Viral level in healthy body of shellfish is very low, the virus of q.s cannot be obtained at all, therefore existing method cannot to exist in healthy body of shellfish of a great variety but content is lower virus study simultaneously, this not only seriously constrains the virological development of shellfish, also constrains the management and control of aquaculture industry to shellfish disease.Therefore shellfish virus research is needed badly and is found a kind of research method can identifying all main virus in body of shellfish efficiently simultaneously.
Summary of the invention
The object of the present invention is to provide a kind of preparation method of shellfish total virus nucleic acid.The method is simple to operate, and obtain shellfish total virus nucleic acid purity high, and nucleic acid is complete, nucleic acid concentration meets the requirement of high-flux sequence.
Object of the present invention is achieved through the following technical solutions: a kind of preparation method of shellfish total virus nucleic acid, comprises the following steps:
(1) abrasive dust: under liquid nitrogen environment, by shellfish tissue sample grinds;
(2) homogenate: add PBS damping fluid, then homogenate form slurry in shellfish tissue sample powder;
(3) total virus crude extract is obtained: slurry is through centrifugal treating, and supernatant liquor centrifugal treating after membrane filtration of gained, abandons supernatant liquor, and the precipitation of gained is total virus crude extract;
(4) extract nucleic acid: described total virus crude extract adds nuclease solution, makes suspension, in 37 DEG C of shaking table process 25-40min, then extract total virus nucleic acid;
(5) nucleic acid amplification: described total virus nucleic acid is increased, to improve nucleic acid concentration, is conducive to high-flux sequence.
In step of the present invention (1), shellfish tissue sample comprises whole histoorgans of the abdominal foot, visceral mass, mantle etc. of healthy shellfish.
In step of the present invention (2), the volume mass between PBS damping fluid and shellfish tissue sample is than being 10-12 ﹕ 1.Described PBS damping fluid contains following component: NaCl 135-140mmol/L, KCl 2.5-3.0mmol/L, Na
2hPO
48-12mmol/L, KH
2pO
41-2.5mmol/L, pH7.5.
In step of the present invention (3), the centrifugal treating before filtration at least carries out twice, and centrifugal condition is: 4 DEG C, the centrifugal 25-35min of 10000-12000rpm.Described filter membrane adopts Millex-HV 0.45um filter membrane.The condition of the centrifugal treating after described filtration is: 10 DEG C, the centrifugal 1-1.5h of 35000-45000g.
In step of the present invention (4), nuclease solution contains following component: 100U DNase I, 100ug RNase A, 10-12mM Tris-HCl, 2.0-3.0mM MgCl
2, 0.5-1mM CaCl
2, pH7.5.
In described step (4), HP Viral DNA/RNA Kit is adopted to carry out total virus nucleic acid extraction.
In described step (5), illustra GenomiPhi HY DNA Amplification Kit is adopted to carry out total virus nucleic acid amplification.
Compared with prior art, the present invention has following significant effect:
First the present invention extracts the total virus in shellfish tissue, and carry out purifying, get rid of the interference of host nucleic acids and fragment of tissue as far as possible, then from the total virus sample extraction viral nucleic acid of purifying, recycling amplification technique prepares abundant viral nucleic acid further, make nucleic acid concentration >=20ng/uL, total amount >=3ug, so that the requirement meeting downstream high-flux sequence.Therefore, the present invention can adopt healthy shellfish to be that sample obtains shellfish total virus nucleic acid, utilize high throughput sequencing technologies to carry out disposable qualification with quantitative to tens of kinds of viruses in body of shellfish, fundamentally breach the limitation that traditional method can only utilize a kind of virus of morbidity investigation of materials.
Accompanying drawing explanation
Fig. 1 is the electrophorogram that agarose gel electrophoresis detects total virus nucleic acid integrity.
Loading order: be followed successively by embodiment 1-5 from left to right, λ-Hind III digest DNA Marker
Embodiment
The nucleic acid preparation method of embodiment 1-5 is as follows:
(1) abrasive dust: under liquid nitrogen environment, by 7g shellfish tissue sample grinds.Shellfish tissue sample comprises whole histoorgans of the abdominal foot, visceral mass, mantle etc. of healthy shellfish.
(2) homogenate: add 70ml PBS damping fluid in shellfish tissue sample powder, then fully homogenate, makes slurry.PBS damping fluid is: NaCl 137mmol/L, KCl 2.7mmol/L, Na
2hPO
410mmol/L, KH
2pO
42mmol/L, pH7.5.
(3) total virus crude extract is obtained: 4 DEG C, slurry, 10000rpm centrifugal twice, each centrifugal 30min, gets supernatant liquor.Supernatant liquor is through Millex-HV 0.45um membrane filtration, and filtrate puts into centrifuge tube, 10 DEG C, and the centrifugal 1h of 40000g, abandons supernatant liquor, and the precipitation of gained is total virus crude extract.
(4) extract nucleic acid: total virus crude extract adds nuclease solution, fully suspends, and makes suspension, in 37 DEG C of shaking table process 30min, then use HP Viral DNA/RNA Kit (Omega Bio-Tek company) to extract total virus nucleic acid.Nuclease solution is 100U DNase I, 100ug RNase A, 10mM Tris-HCl, 2.5mM MgCl
2, 0.5mM CaCl
2, pH7.5.
Extract total virus nucleic acid:
A) in viral suspension, add 0.5mL Buffer MPG, put upside down 30-50 time;
B) room temperature places the centrifugal 15min of 1h, 10000g, abandons supernatant;
C) add 200uL water, 25uL OB Protease, fully suspend precipitation;
D) 200uL Buffer BL is added, mixing, 55 DEG C of process 10min;
E) add 250uL ethanol, mixing, room temperature places 5min;
F) added in purification column by solution and (provide in test kit), pillar is enclosed within collection tube, and the centrifugal 1min of 8000g, abandons filtrate and renew collection tube;
G) add 500uL Buffer VHB on post, the centrifugal 1min of 8000g, abandons filtrate and changes collection tube;
H) add 500uL RWB Wash Buffer, the centrifugal 1min of 8000g, abandons filtrate, does not change collection tube;
I) repeating step h) once;
J) change a new 1.5mL centrifuge tube, add 40uL water in pillar central authorities, room temperature places the centrifugal 1min of 5min, 8000g, and filtrate is nucleic acid solution.
(5) nucleic acid amplification: use illustra GenomiPhi HY DNA Amplification Kit (GE Healthcare company) to increase to total virus nucleic acid, to improve nucleic acid concentration, be conducive to high-flux sequence.
A) get 2.5uL viral nucleic acid solution, add 22.5uL sample buffer, 95 DEG C process 3min, after be put in cooled on ice;
22.5uL reaction buffer and 2.5uL enzyme mix (providing in test kit) b) is provided in above-mentioned solution, reacts 4 hours in 30 DEG C, place 10min termination reaction for 65 DEG C.This reaction solution is final viral nucleic acid amplified production.
Use the agarose gel electrophoresis (voltage: 10V/cm) of NanoDrop2000 and 1% to measure the nucleic acid samples concentration of 1ul and purity, result as shown in Table 1 and Table 2.As shown in the results, the total virus nucleic acid concentration >=20ng/uL of the shellfish of embodiment 1-5, total amount >=3ug.For embodiment 5, carry out high-flux sequence identifying virus kind and quantity, result is as shown in table 2.
Table 1:NanoDrop 2000 spectrophotometer detects total virus nucleic acid samples quality
| Sample | Sample message | NanoDrop ng/μl | 260/280 | 260/230 |
| Embodiment 1 | Haliotis discus hannai Ino | 495.4 | 1.69 | 1.96 |
| Embodiment 2 | Babylonia | 532.7 | 1.71 | 1.97 |
| Embodiment 3 | Pacific oyster | 416.9 | 1.64 | 1.97 |
| Embodiment 4 | Haliotis diversicolor 1 | 366.4 | 1.76 | 2.06 |
| Embodiment 5 | Haliotis diversicolor 2 | 353.3 | 1.75 | 2.06 |
Note: in table, last two columns values represent the purity of nucleic acid, and 260/280 between 1.6 ~ 2.0,260/230 to be namely considered as purity about 2.0 qualified.
Table 2: the virus results list that after embodiment 5 sample high-flux sequence, comparison is arrived
Embodiment 6
With embodiment 1-5 unlike:
(1) abrasive dust: under liquid nitrogen environment, by 7g shellfish tissue sample grinds.(2) homogenate: add 77ml PBS damping fluid in shellfish tissue sample powder, then fully homogenate, makes slurry.PBS damping fluid is: NaCl140mmol/L, KCl 3.0mmol/L, Na
2hPO
412mmol/L, KH
2pO
42.5mmol/L, pH7.5.
(3) total virus crude extract is obtained: 4 DEG C, slurry, 12000rpm centrifugal twice, each centrifugal 35min, gets supernatant liquor.Supernatant liquor is through Millex-HV 0.45um membrane filtration, and filtrate puts into centrifuge tube, 10 DEG C, and the centrifugal 1.5h of 45000g, abandons supernatant liquor, and the precipitation of gained is total virus crude extract.
(4) extract nucleic acid: total virus crude extract adds nuclease solution, fully suspends, and makes suspension, in 37 DEG C of shaking table process 30min, then use HP Viral DNA/RNA Kit (Omega Bio-Tek company) to extract total virus nucleic acid.Nuclease solution is 100U DNase I, 100ug RNase A, 12mM Tris-HCl, 3.0mMMgCl
2, 0.5mM CaCl
2, pH7.5.
Embodiment 7
With embodiment 1-5 unlike:
(1) abrasive dust: under liquid nitrogen environment, by 7g shellfish tissue sample grinds.(2) homogenate: add 84ml PBS damping fluid in shellfish tissue sample powder, then fully homogenate, makes slurry.PBS damping fluid is: NaCl135mmol/L, KCl 2.5mmol/L, Na
2hPO
48mmol/L, KH
2pO
41.5mmol/L, pH7.5.
(3) total virus crude extract is obtained: 4 DEG C, slurry, 11000rpm centrifugal twice, each centrifugal 25min, gets supernatant liquor.Supernatant liquor is through Millex-HV 0.45um membrane filtration, and filtrate puts into centrifuge tube, 10 DEG C, and the centrifugal 1h of 35000g, abandons supernatant liquor, and the precipitation of gained is total virus crude extract.
(4) extract nucleic acid: total virus crude extract adds nuclease solution, fully suspends, and makes suspension, in 37 DEG C of shaking table process 30min, then use HP Viral DNA/RNA Kit (Omega Bio-Tek company) to extract total virus nucleic acid.Nuclease solution is 100U DNase I, 100ug RNase A, 11mM Tris-HCl, 2.0mM MgCl
2, 0.5mM CaCl
2, pH7.5.
Embodiments of the present invention are not limited thereto; according to foregoing of the present invention; according to ordinary technical knowledge and the customary means of this area; do not departing under the present invention's above-mentioned basic fundamental thought prerequisite; the present invention can also make the amendment of other various ways, replacement or change, all drops within rights protection scope of the present invention.
Claims (9)
1. a preparation method for shellfish total virus nucleic acid, is characterized in that, comprises the following steps:
(1) abrasive dust: under liquid nitrogen environment, by shellfish tissue sample grinds;
(2) homogenate: add PBS damping fluid, then homogenate form slurry in shellfish tissue sample powder;
(3) total virus crude extract is obtained: slurry is through centrifugal treating, and supernatant liquor centrifugal treating after membrane filtration of gained, abandons supernatant liquor, and the precipitation of gained is total virus crude extract;
(4) extract nucleic acid: described total virus crude extract adds nuclease solution, makes suspension, in 37 DEG C of shaking table process 25-40min, then extract total virus nucleic acid;
(5) nucleic acid amplification: described total virus nucleic acid is increased, to improve nucleic acid concentration, is conducive to high-flux sequence.
2. the preparation method of shellfish total virus nucleic acid according to claim 1, is characterized in that, in described step (2), the volume mass between PBS damping fluid and shellfish tissue sample is than being 10-12 ﹕ 1.
3. the preparation method of shellfish total virus nucleic acid according to claim 1 and 2, is characterized in that, in described step (2), PBS damping fluid contains following component: NaCl 135-140mmol/L, KCl2.5-3.0mmol/L, Na
2hPO
48-12mmol/L, KH
2pO
41-2.5mmol/L, pH7.5.
4. the preparation method of shellfish total virus nucleic acid according to claim 3, is characterized in that, in described step (3), the centrifugal treating before filtration at least carries out twice, and centrifugal condition is: 4 DEG C, the centrifugal 25-35min of 10000-12000rpm.
5. the preparation method of shellfish total virus nucleic acid according to claim 4, is characterized in that, in described step (3), filter membrane adopts Millex-HV 0.45um filter membrane.
6. the preparation method of shellfish total virus nucleic acid according to claim 5, is characterized in that, the condition of the centrifugal treating after described filtration is: 10 DEG C, the centrifugal 1-1.5h of 35000-45000g.
7. the preparation method of shellfish total virus nucleic acid according to claim 6, is characterized in that, in described step (4), nuclease solution contains following component: 100U DNase I, 100ug RNase A, 10-12mM Tris-HCl, 2.0-3.0mM MgCl
2, 0.5-1mM CaCl
2, pH7.5.
8. the preparation method of shellfish total virus nucleic acid according to claim 7, is characterized in that, in described step (4), adopts HP Viral DNA/RNA Kit to carry out nucleic acid extraction.
9. the preparation method of shellfish total virus nucleic acid according to claim 8, is characterized in that, in described step (5), adopts illustra GenomiPhi HY DNA Amplification Kit to carry out nucleic acid amplification.
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Cited By (1)
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Application publication date: 20150930 |