CN103013979A - Preparation method of aeromonas hydrophila nucleic acid standard substance - Google Patents
Preparation method of aeromonas hydrophila nucleic acid standard substance Download PDFInfo
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- CN103013979A CN103013979A CN2012105012237A CN201210501223A CN103013979A CN 103013979 A CN103013979 A CN 103013979A CN 2012105012237 A CN2012105012237 A CN 2012105012237A CN 201210501223 A CN201210501223 A CN 201210501223A CN 103013979 A CN103013979 A CN 103013979A
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Abstract
The invention discloses a preparation method of an aeromonas hydrophila nucleic acid standard substance. The preparation method is characterized in that the aeromonas hydrophila nucleic acid standard substance is obtained through freeze drying of extracted genome nucleic acids of aeromonas hydrophila ATCC35654 and/or ATCC 7966, wherein freeze drying conditions are as follows: starting a freeze drier, and opening a cooling trap when the temperature of a drying room is reduced to minus 35 DEG C; putting a nucleic acid sample prefrozen at a minus 80 DEG C for 2 hours in the drying room when the temperature of the cooling trap is reduced to minus 40 DEG C, and then vacuumizing; and ending freezing after the vacuum degree is reduced to 0.5 Torr; drying the sample at a 15 DEG C, and taking out the sample when the vacuum degree is reduced to 0.1 Torr, and fastening down a penicillin bottle to obtain the genome nucleic acid standard sample, keeping the standard sample out of the sun at a 20 DEG C and storing. The aeromonas hydrophila nucleic acid standard substance is suitable for preventing diseases of aquatic animals, low in preparation cost, long in preservation time and free of pollution and has important practical significance on preventing and treating the aeromonas hydrophila breeding on the lots of aquatic animals.
Description
Technical field
The present invention relates generally to sea-food disease detection technical field, is specifically related to the preparation method of Aeromonas hydrophila nucleic acid standards.
Background technology
China marine site is vast, and superior natural conditions, oceanic resources are very abundant.But the degree of development and use is also very low at present.This also illustrates having a high potential of China's development and use oceanic resources, and marine site, Liaoning is important Living marine resources, fishing resources treasure-house of China, and it abounds with hydrocoles, scallop, mussel, oyster and fish, shrimp and algae etc., drives the marine economy development.Strengthen polygon and bilateral ocean exploitation international co-operation, concentrate intensive development and use marine site and island resource, reasonable disposition advantage marine industries, scientific and efficient exploitation oceanic resources, actively develop scientific research and test, development and utilization, the sea farming stdn is the important means of reasonable development sea farming resource, studies and defines the culturing marine products standard, reasonable development seawater mudflat aquaculture resource, improving cultivation sea-food disease is the key content of current aquaculture.Culturing marine products is a high risk aquaculture, and traditional small family's culturing marine products has brought huge market and technical risk to the raiser.Grow risk and drop to minimumly for cultivation is raised on a household basis, carry out all kinds of aquacultures, comprise that the diseases prevention and treatment task of all kinds of sea-foods such as fish, shrimp, crab, hydrocoles, abalone, shellfish, marine alga is very important.Report that the unwanted bacterias such as vibrio cholerae, floating vibrios, Vibrio parahaemolyticus, vibrio alginolyticus, Vibrio vulnificus are the arch-criminals who causes the sea-food various diseases.
Aeromonas hydrophila (A.hydrophila) is distributed widely in natural various water body, is the primary pathogenic bacterium of multiple hydrocoles, is conditioned pathogen, is altogether ill pathogenic bacteria of typical people-beast-fish.This bacterium is the vibrionaceae Aeromonas, is the Gram-negative tyrothricin, and extreme single flagellum does not have brood cell and pod membrane, and normal two of the pathogenic bacteria that has just separated from focus links to each other.Bacterium colony garden shape, the smooth of the edge, center projections, yellowish pink, canescence or the slightly light pink of cultivating formation at the plain agar plate culture medium are glossy, physically well develop.Aeromonas hydrophila all can breed in 14.0～40.5 ℃ of scopes of water temperature, take 28.0～30.0 ℃ as optimum temperuture.PH value all can be grown in 6～11 scopes; Optimum pH is 7.27; Aeromonas hydrophila can be deposited in the aquatic of saltiness 0 ‰～4 ‰, and optimal salinity is 0.5 ‰.Aeromonas hydrophila can produce the very strong extracellular toxin of toxicity, because of the dissociant of Aeromonas hydrophila more, so will pay special attention to curative effect.
Summary of the invention
By background technology as seen, improve the recall rate of Aeromonas hydrophila (Aeromonas hydrophila), strengthening its accuracy of detection is the important channel that solves multiple aquatic animal disease problem.This research realizes the preparation of Aeromonas hydrophila nucleic acid standards by the following technical solutions: it comprises the steps:
(1) increases bacterium: be inoculated into the common LB substratum from the Aeromonas hydrophila reference culture, ferment, cultivate 12h for 37 ℃ and make tunning; Tunning accessed in the Aeromonas hydrophila substratum again ferments, cultivate 24 ~ 48h for 32 ~ 37 ℃, to the final concentration of bacteria suspension bacterial content be 10
7~ 10
10Cfu/g;
Described Aeromonas hydrophila substratum, its compound method is as follows: Tryptones 10g, yeast extract 5g, sodium-chlor 10g, glucose 5g need not to regulate pH, 121 ℃ of lower sterilization 20min; Cool off for subsequent use;
(2) washing: the bacteria suspension that step (1) is obtained repeats following operation 2 ~ 5 times to clean, and fully mixes centrifugal 5 ~ 10min under 8000 ~ 12000rpm condition, collection thalline with the PBS damping fluid with the volume ratio of 1:10;
(3) adopt the CTAB method to extract bacteria suspension DNA; Use the TE solution of pH8.0 to dissolve the DNA that extracts, obtain genomic nucleic acids solution; Its operation steps is compiled marine organisms Ecological Investigation technical regulation, Maritime Press, 2006,4, p23-26 referring to 908 special offices of National Bureau of Oceanography.Packing nucleic acid samples in cillin bottle;
(4) lyophilize: start Freeze Drying Equipment, when the kiln temperature is down to-35 ℃, open cooling pit; When the cooling pit temperature is down to-40 ℃, will after putting into kiln, the nucleic acid samples of-80 ℃ of pre-freeze 2h vacuumize; Treat that vacuum tightness is down to 0.5Torr and finishes freezing; And with sample in 15 ℃ of dryings, when vacuum tightness is down to 0.1Torr, take out sample, cover tightly cillin bottle and obtain the genomic nucleic acids standard model, lucifuge is stored in-20 ℃.
In above-described all technical schemes, described Aeromonas hydrophila reference culture is Aeromonas hydrophila ATCC35654 and/or Aeromonas hydrophila ATCC 7966;
In above-described all technical schemes, more excellent condition is as follows: the DNA of extraction works as OD with the optical density value at ultraviolet spectrophotometer survey 260nm and 280nm place
260/ OD
280Ratio is better between 1.7～1.9;
Among the present invention, among the said products preparation method, all do not limit other equivalent conditions of method, because it can be determined according to prior art, do not limit the rank of agents useful for same yet, because it affects littlely on the result, and can buy obtain by preparation or commercial sources, the technician can make reference according to adjusting according to listed condition among the embodiment.These believe that about the selection of preparation way and method those skilled in the art can be enlightened fully from prior art, the present invention repeats no more.
Advantage of the present invention is: be applicable to disease control or the detection of hydrocoles; The treating processes of these standard substance is simple, and is consuming time few; Preparation cost is lower than traditional method, and the pulvis shelf time of the present invention is long, is difficult for polluting.The present invention has important practical significance to the control of the aeromonas hydrophila disease of solution hydrocoles cultivation.
Embodiment
Following examples are used for explanation the present invention, but are not used for limiting the scope of the invention.
Embodiment 1
The Aeromonas hydrophila reference culture is selected Aeromonas hydrophila ATCC 35654;
(1) increases bacterium: be inoculated into the LB substratum from the Aeromonas hydrophila reference culture, ferment, cultivate 12h for 37 ℃ and make tunning; Tunning accessed in the Aeromonas hydrophila substratum again ferments, cultivate 48h for 37 ℃, to the final concentration of bacteria suspension bacterial content be 10
9Cfu/g;
Described Aeromonas hydrophila substratum compound method: Tryptones 10g, yeast extract 5g, sodium-chlor 10g, glucose 5g need not to regulate pH, 121 ℃ of lower sterilization 20min;
(2) washing: the bacteria suspension that step (1) is obtained repeats following cleaning operation 3 times, fully mixes with the PBS damping fluid with the volume ratio of 1:10, and centrifugal 5min under the 12000rpm condition collects thalline;
(3) adopt the CTAB method to extract bacteria suspension DNA
Get the TE that thalline adds pH8.0 and suspend, adding 0.5mL concentration is that 100g/L SDS and 30 μ L concentration are the Proteinase K of 30mg/mL, mixing, and 37 ℃ of temperature are bathed 1h; Adding 2mL concentration is 5mol/L NaCl, and mixing adds 1.5mL CTAB-NaCl mixing solutions, mixing, and 65 ℃ of temperature are bathed 20min; Wherein, the CTAB-NaCl mixing solutions is 100g/L CTAB and 0.7mol/L NaCl; Get supernatant, add and the isopyknic phenol-chloroform of supernatant-primary isoamyl alcohol mixed solution, mixing, the centrifugal 10min of 6000g; Wherein, phenol in phenol-chloroform-primary isoamyl alcohol mixed solution: chloroform: the volume ratio of primary isoamyl alcohol is 25:24:1; Get supernatant, add and the isopyknic chloroform of supernatant-primary isoamyl alcohol mixed solution, mixing, the centrifugal 10min of 6000g; Wherein, chloroform in chloroform-primary isoamyl alcohol mixed solution: the volume ratio of primary isoamyl alcohol is 24:1; Get supernatant, add the Virahol of 0.6 times of volume of supernatant, mixing, the centrifugal 10min of 13000g; Get precipitation, clean 2 times with 70% ethanol, drying uses the TE solution of pH8.0 to dissolve the DNA that extracts, and the sample DNA of extraction is got OD with the optical density value at ultraviolet spectrophotometer survey 260nm and 280nm place
260/ OD
280Ratio is at the packing nucleic acid samples in cillin bottle of the sample DNA between 1.8;
(4) lyophilize: start Freeze Drying Equipment, when the kiln temperature is down to-35 ℃, open cooling pit; When the cooling pit temperature is down to-40 ℃, will after putting into kiln, the nucleic acid samples of-80 ℃ of pre-freeze 2h vacuumize; Treat that vacuum tightness is down to 0.5Torr and finishes freezing; And with sample in 15 ℃ of dryings, when vacuum tightness is down to 0.1Torr, take out sample, cover tightly cillin bottle and obtain the genomic nucleic acids standard model, lucifuge is stored in-20 ℃.
Embodiment 2
The Aeromonas hydrophila reference culture is selected Aeromonas hydrophila ATCC 7966;
(1) increases bacterium: be inoculated into the LB substratum from the Aeromonas hydrophila reference culture, ferment, cultivate 12h for 37 ℃ and make tunning; Tunning accessed in the Aeromonas hydrophila substratum again ferments, cultivate 48h for 35 ℃, to the final concentration of bacteria suspension bacterial content be 10
8Cfu/g;
Described Aeromonas hydrophila substratum compound method: Tryptones 10g, yeast extract 5g, sodium-chlor 10g, glucose 5g need not to regulate pH, 121 ℃ of lower sterilization 20min;
(2) washing: the bacteria suspension that step (1) is obtained repeats following cleaning operation 2 times, fully mixes with the PBS damping fluid with the volume ratio of 1:10, and centrifugal 10min abandons supernatant under the 12000rpm condition, collects thalline;
(3) adopt the CTAB method to extract bacteria suspension DNA:
Get the TE that thalline adds pH8.0 and suspend, adding 0.5mL concentration is that 100g/L SDS and 30 μ L concentration are the Proteinase K of 30mg/mL, mixing, and 37 ℃ of temperature are bathed 1h; Adding 2mL concentration is 5mol/LNaCl, and mixing adds 1.5mL CTAB-NaCl mixing solutions, mixing, and 65 ℃ of temperature are bathed 20min; Wherein, the CTAB-NaCl mixing solutions is 100g/L CTAB and 0.7mol/L NaCl; Get supernatant, add and the isopyknic phenol-chloroform of supernatant-primary isoamyl alcohol mixed solution, mixing, the centrifugal 10min of 6000g; Wherein, phenol in phenol-chloroform-primary isoamyl alcohol mixed solution: chloroform: the volume ratio of primary isoamyl alcohol is 25:24:1; Get supernatant, add and the isopyknic chloroform of supernatant-primary isoamyl alcohol mixed solution, mixing, the centrifugal 10min of 6000g; Wherein, chloroform in chloroform-primary isoamyl alcohol mixed solution: the volume ratio of primary isoamyl alcohol is 24:1; Get supernatant, add the Virahol of 0.6 times of volume of supernatant, mixing, the centrifugal 10min of 13000g; Get precipitation, clean 2 times with 70% ethanol, drying uses the TE solution of pH8.0 to dissolve the DNA that extracts, and the sample DNA of extraction is got OD with the optical density value at ultraviolet spectrophotometer survey 260nm and 280nm place
260/ OD
280Ratio is at the packing nucleic acid samples in cillin bottle of the sample DNA between 1.8;
(4) lyophilize: start Freeze Drying Equipment, when the kiln temperature is down to-35 ℃, open cooling pit; When the cooling pit temperature is down to-40 ℃, will after putting into kiln, the nucleic acid samples of-80 ℃ of pre-freeze 2h vacuumize; Treat that vacuum tightness is down to 0.5Torr and finishes freezing; And with sample in 15 ℃ of dryings, when vacuum tightness is down to 0.1Torr, take out sample, cover tightly cillin bottle and obtain the genomic nucleic acids standard model, lucifuge is stored in-20 ℃.
The Aeromonas hydrophila nucleic acid standards of utilizing the embodiment of the invention 1 ~ 2 described method to make is applicable to the disease prevention of hydrocoles; Preparation cost is low, and the shelf time is long, and is pollution-free.Control to the Aeromonas hydrophila that solves hydrocoles in enormous quantities cultivation has important practical significance.
The above only is preferred implementation of the present invention, should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the technology of the present invention principle, can also make some improvement and also should be considered as protection scope of the present invention.
Claims (3)
1. the preparation method of an Aeromonas hydrophila nucleic acid standards is characterized in that, it comprises the steps:
(1) increases bacterium: be inoculated into the LB substratum from the Aeromonas hydrophila reference culture, ferment, cultivate 12h for 37 ℃ and make tunning; Tunning accessed in the Aeromonas hydrophila substratum again ferments, cultivate 24 ~ 48h for 32 ~ 37 ℃, to the final concentration of bacteria suspension bacterial content be 10
7~ 10
10Cfu/g;
Described Aeromonas hydrophila substratum compound method: Tryptones 10g, yeast extract 5g, sodium-chlor 10g, glucose 5g need not to regulate pH, 121 ℃ of lower sterilization 20min;
(2) washing: the bacteria suspension that step (1) is obtained repeats following cleaning operation 2 ~ 5 times, fully mixes with the PBS damping fluid with the volume ratio of 1:10, and centrifugal 5 ~ 10min under 8000～12000rpm condition collects thalline;
(3) adopt the CTAB method to extract bacteria suspension DNA, use the TE solution of pH8.0 to dissolve packing nucleic acid samples in cillin bottle the DNA that extracts;
(4) lyophilize: start Freeze Drying Equipment, when the kiln temperature is down to-35 ℃, open cooling pit; When the cooling pit temperature is down to-40 ℃, will after putting into kiln, the nucleic acid samples of-80 ℃ of pre-freeze 2h vacuumize; Treat that vacuum tightness is down to 0.5Torr and finishes freezing; And with sample in 15 ℃ of dryings, when vacuum tightness is down to 0.1Torr, take out sample, cover tightly cillin bottle and obtain the genomic nucleic acids standard model, lucifuge is stored in-20 ℃.
2. method according to claim 1 is characterized in that, described Aeromonas hydrophila reference culture is Aeromonas hydrophila ATCC 35654 and/or Aeromonas hydrophila ATCC 7966.
3. method according to claim 2 is characterized in that, the sample DNA of extraction is got OD with the optical density value at ultraviolet spectrophotometer survey 260nm and 280nm place
260/ OD
280The sample DNA of ratio between 1.7～1.9 is for subsequent use.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109486959A (en) * | 2018-10-23 | 2019-03-19 | 浙江海洋大学 | The Variations of liver mtDNA copy number in Sepiella maindroni aging course |
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| CN1154410A (en) * | 1995-11-06 | 1997-07-16 | 麦克罗戴克公司 | Method and kit for quantitation and detection of microorganisms |
| CN102321621A (en) * | 2011-08-02 | 2012-01-18 | 宋锋林 | Molecular standard sample for Muscina stabulans and preparation method for molecular standard sample |
Non-Patent Citations (2)
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109486959A (en) * | 2018-10-23 | 2019-03-19 | 浙江海洋大学 | The Variations of liver mtDNA copy number in Sepiella maindroni aging course |
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Application publication date: 20130403 |