CN104450684A - Kit for extracting nucleic acid from bacteria by using paramagnetic particle method and extracting method - Google Patents
Kit for extracting nucleic acid from bacteria by using paramagnetic particle method and extracting method Download PDFInfo
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Abstract
The invention discloses a kit for extracting nucleic acid from bacteria by using a paramagnetic particle method and an extracting method. The kit comprises a bacteria lysate, a magnetic bead binding solution, a magnetic bead scrubbing solution and a nucleic acid eluent. The bacteria lysate comprises sodium dodecyl sulfate, ethylenediaminetetraacetic acid, tri-hydroxymethyl aminomethane and sodium chloride; the magnetic bead binding solution comprises polyethylene glycol-8000 and sodium chloride; the magnetic bead scrubbing solution comprises ethanol; and the nucleic acid eluent comprises tri-hydroxymethyl aminomethane and ethylenediamine tetraacetic acid. The kit comprises the unique bacteria lysate which has a strong lysis function for the bacteria; and the kit can be used for manual extraction and instrument extraction by using a commercially available nucleic acid isolation machine, high-purity and high-concentration bacteria nucleic acid can be extracted.
Description
Technical field
The present invention relates to the biological technical field of nucleic acid extraction, particularly a kind of test kit extracting bacterium amplifying nucleic acid based on paramagnetic particle method.The invention still further relates to the method using paramagnetic particle method to extract bacterium amplifying nucleic acid.
Background technology
Polymerase chain reaction (Polymerase Chain Reaction, PCR) technology is since the eighties emerges, more and more be applied to biology field, the Protocols in Molecular Biology of nucleic acid carries out the pathogenic microorganism examination, build gene pool, one of conventional research means such as the origin of species, diversity evaluation and sibship, phyletic evolution etc.But the extraction of nucleic acid is the prerequisite of round pcr application, and can extract high-quality nucleic acid molecule be key in many nucleic acid molecule biological tests, and sensitivity, the specificity of extracting method also will be directly connected to the success or failure of follow-up test.Alkaline lysis extraction method is to extractive techniques such as column purification, be widely used for the extraction of DNA and RNA of bacterium at present, but still there is shortcomings in these methods, as low in length consuming time, efficiency, and owing to adopting the toxic organic solvent such as phenol, operation has certain risk.
Compare above-mentioned nucleic acid extraction technology, Magnet Treatment is a kind of simple and effective nucleic acid purification method.The magnetic bead surfaces that the method uses is connected to the functional group that can have an effect with DNA specifically, has the characteristic of reversible adsorption DNA.The maximum advantage of paramagnetic particle method purifying nucleic acid is automatization, and the reagent provided can be used for Magnetic tools, this kind of method is without any need for organic solvent, do not need repeatedly centrifugal yet, the steps such as vacuum filtration or post separation, be only by nucleic acid in conjunction with based on magnetic, greatly reduce the harm of test to staff, and reduce the particular requirement to testing installation.Paramagnetic particle method agents useful for same kind and the isoparametric selection of concentration determine the quality and efficiency of extracting nucleic acid, also closely related with cost, therefore constantly explore more convenient and efficient extracting tool and extracting method, engineered every research relevant to nucleic acid extraction is significant.
Summary of the invention
The object of this invention is to provide a kind of test kit of new bacterial nucleic acid rapid extraction and extract the method for nucleic acid, this test kit have simple to operate fast, the feature of high, the low cost of manufacture of use safety, extraction efficiency.Apply this test kit and method, rapid extraction can be realized to the nucleic acid in the various bacteria in the biology detected for round pcr.
Technical scheme of the present invention is:
Paramagnetic particle method extracts a test kit for bacterium amplifying nucleic acid, and comprising component is: bacterial lysate, magnetic bead are in conjunction with liquid, magnetic bead washings and nucleic acid eluents.Described bacterial lysate contains sodium lauryl sulphate, ethylenediamine tetraacetic acid (EDTA), Tutofusin tris and sodium-chlor; Described magnetic bead contains PEG-8 000 and sodium-chlor in conjunction with liquid; Described magnetic bead washings contains ethanol; Described nucleic acid eluents contains hydroxymethyl aminomethane and ethylenediamine tetraacetic acid (EDTA);
Preferably, described magnetic bead is in conjunction with in liquid, and the concentration of PEG-8 000 is 20-30mmol/L, and the concentration of sodium-chlor is 3mol/L;
Preferably, in described magnetic bead washings, the volume by volume concentration of ethanol is 70%-90%;
Preferably, in described nucleic acid eluents, the concentration of Tutofusin tris is for being 5-15mmol/L, and the concentration of described ethylenediamine tetraacetic acid (EDTA) is 1.30-1.50mmol/L;
Preferred further, the concentration of described Tutofusin tris is 10mmol/L, and the concentration of described ethylenediamine tetraacetic acid (EDTA) is 1.35mmol/L;
Preferably, in described bacterial lysate, the concentration of sodium lauryl sulphate is 40-60mmol/L, the concentration of described ethylenediamine tetraacetic acid (EDTA) is 20-40mmol/L, the concentration of described Tutofusin tris is 90-115mmol/L, the concentration of described sodium-chlor is 480-520mmol/L, and surplus is sterilized water; Described bacterial lysate is by adding sodium hydroxide adjust ph to 7.5-8.5.
Paramagnetic particle method extracts an extracting method for bacterium amplifying nucleic acid, comprises the following steps:
Step 1: by bacterium and the centrifugation of nutrient solution mixture, goes supernatant to remove cell culture fluid, leaves microorganism; The rotating speed of centrifugation is 13000r/min, and the time of centrifugation is 2 minutes;
Step 2: add bacterial lysate in thalline, mixing;
Step 3: add magnetic bead and magnetic bead in conjunction with liquid, magnet adsorption removes supernatant;
Step 4: add magnetic bead washings, uses magnet adsorption magnetic bead, removes supernatant;
Step 5: add nucleic acid eluents, uses magnet adsorption magnetic bead, and transfer elutriant, obtains the nucleic acid solution of bacterium.
Preferably, the volume of the bacterial lysate added in above-mentioned steps 2 and the volume ratio of inoculum are 1:1-1:5, the magnetic bead added in step 3 and magnetic bead are 1:10-1:20 in conjunction with the volume ratio of liquid and bacterial lysate, in step 4, the volume ratio of magnetic bead washings and bacterial lysate is 1:0.1-1:0.5, and the volume ratio of step 5 amplifying nucleic acid elutriant and bacterial lysate is 1:10-1:20.
Paramagnetic particle method extracts an extracting method for bacterium amplifying nucleic acid, comprises the following steps:
Step 1: by bacterium and the centrifugation of nutrient solution mixture, goes supernatant to remove cell culture fluid, leaves microorganism; The rotating speed of centrifugation is 13000r/min, and the time of centrifugation is 2 minutes;
Step 2: add bacterial lysate in thalline, after mixing, is transferred to 1 row and 7 rows of 96 hole Sptting plates; Arrange 2 rows and 8 and add magnetic bead and magnetic bead in conjunction with liquid; Magnetic bead washings is added 3 rows, 4 rows, 5 rows and 9 rows, 10 rows, 11 rows; Nucleic acid eluents is added 6 rows and 12 rows;
Step 3: the 96 hole Sptting plates injecting reagent are put into instrument for extracting nucleic acid, obtains bacterial nucleic acid elutriant after automatic end of run.
Preferably, the volume injecting the bacterial lysate of 96 hole Sptting plates in above-mentioned steps 2 is 200-300 μ L, mixes with 200-400 μ L dehydrated alcohol; The magnetic bead injected and magnetic bead are 50-150 μ L in conjunction with the volume of liquid, mix with 300-400 μ L distilled water; The volume of the magnetic bead washings injected is 500-700 μ L; The volume of the nucleic acid eluents injected is 50-300 μ L.
Technical solution of the present invention has the following advantages:
1. the bacterial lysate containing uniqueness in test kit of the present invention, all has stronger splitting action to various bacterium;
2. the invention provides manually and automatic two kinds of methods, utilize test kit to carry out manual extraction, also can carry out instrument extraction in conjunction with now commercially available instrument for extracting nucleic acid, all can extract purity and the higher bacterial nucleic acid of concentration; Use instrument leaching process to be that a step completes automatically, whole leaching process again need not be opened instrument and carry out secondary operation;
3. use test kit of the present invention to extract without the need to heating in calculation process, operation is easier compared with conventional reagents box;
4. the present invention uses less step, reduces the crossed contamination between pipe and the pollution to environment, does not use toxic reagent and organic solvent, greatly reduce the actual bodily harm to experimenter in nucleic acid extraction process.
Accompanying drawing explanation
The stereographic map of the 96 deep hole Sptting plates that Fig. 1 uses when being the inventive method extraction nucleic acid;
Fig. 2 is to the schematic diagram injecting reagent during 96 deep hole Sptting plates are respectively arranged according to the inventive method.
Embodiment
Below in conjunction with accompanying drawing, by embodiment, the present invention will be further described, to understand the present invention better.
Embodiment 1:
1. bacterium amplifying nucleic acid extracts the compound method of test kit and reagent thereof:
1. the preparation of bacterial lysate: first add a small amount of sterilized water in volumetric flask, add sodium lauryl sulphate that concentration is 55mmoL/L, add ethylenediamine tetraacetic acid (EDTA) that concentration is 25mmol/L, add Tutofusin tris that concentration is 100mmol/L, add the sodium-chlor that concentration is 500mmol/L, add sterilized water to volume required, sodium hydroxide adjust ph is used to make the pH value of described bacterial lysate be 8.0, high pressure steam sterilization 10 minutes.
2. magnetic bead is in conjunction with the preparation of liquid: first in volumetric flask, add a small amount of sterilized water, add the PEG-8 000 that concentration is 25mmol/L, concentration is the sodium-chlor of 3mol/L, adds sterilized water to volume required, high pressure steam sterilization 10 minutes.
3. the preparation of magnetic bead washings: 100% ethanol.
4. the preparation of nucleic acid eluents: first add a small amount of sterilized water in volumetric flask, add the Tutofusin tris that concentration is 10mmol/L, add the ethylenediamine tetraacetic acid (EDTA) that concentration is 13.7mmol/L, add sterilized water to volume required, high pressure steam sterilization 10 minutes.
2. the application of test kit:
The application of test kit is the cultivation intestinal bacteria of 16 hours.The concrete operations of application are: cultivate intestinal bacteria, configuration E. coli broth, take Tryptones 10g, yeast extract 5g, sodium-chlor 10g, adds water and is settled to 1L, high pressure steam sterilization 10 minutes, the intestinal bacteria of inoculation logarithmic phase in safety cabinet, after inoculation, shaking table 37 degrees Celsius rocks 16 hours; Getting Escherichia coli bacteria liquid 1ml is added in EP pipe, and 13000r/min is after centrifugal 2 minutes, and abandoning supernatant, adds bacterial lysate 1ml, mixing; Add the magnetic bead 50 μ L of magnetic bead 10ug/ml after 5 minutes, add magnetic bead in conjunction with liquid 50 μ L, mixing; Magnet adsorption magnetic bead after 5 minutes, abandoning supernatant; Add magnetic bead washings 500 μ L, after mixing, magnet adsorption magnetic bead, abandoning supernatant, washs 3 times; Add nucleic acid eluents 100 μ L, mixing; Magnet adsorption magnetic bead after 5 minutes, is transferred to elutriant in new EP pipe, obtains the DNA solution that can be used for pcr amplification.
The traditional method of contrast is: get the cultivation Escherichia coli bacteria liquid 2mL of 16 hours, abandon supernatant after the centrifugal 3min of 5000r/min; Add physiological saline 0.1mL, add 100 μ L5mol/L Proteinase Ks after mixing and fully shake 10min, add chloroform/primary isoamyl alcohol (24:1) 200 μ L and shake the centrifugal 5min of 30s, 10000r/min, get supernatant 100 μ L; Add Virahol 60 μ L to mix gently, the centrifugal 5min of 10000r/min, abandons supernatant; Add the centrifugal 5min of cold dehydrated alcohol 1mL, 10000r/min, abandon dehydrated alcohol (pour out gently, and be inverted in ethanol that filter paper exhausts), add the aseptic ultrapure water mixing of 30 μ L after vacuum-drying and dissolve.
3. the effect detection box of pair test kit application is evaluated:
One-Drop OD-1000 spectrophotometer detects nucleic acid concentration and purity detects above-mentioned elutriant, result is, concentration and the purity of the test kit extraction cultivation colibacillary nucleic acid of 16 hours in employing invention are all higher, illustrate that test kit provided by the invention effectively can extract the nucleic acid in bacterium.
In addition, the bacterial nucleic acid that test kit of the present invention extracts, when without PCR, agarose gel electrophoresis demonstrates obvious DNA band and RNA band, and the mean value of nucleic acid concentration is 220ng/ μ L, and OD260/OD280 mean value is 1.78.Illustrate that test kit of the present invention is not only simple to operate and have higher extraction efficiency.
The traditional method of contrast uses the organic solvents such as chloroform, large to the actual bodily harm of people, compared with the paramagnetic particle method of embodiment 1, the efficiency extracting bacterial nucleic acid is low, concentration and the purity of the nucleic acid of the bacterium liquid extraction of same volume are all on the low side, and consuming time longer, also need use repeatedly centrifugal.
Embodiment 2:
1. bacterium amplifying nucleic acid extracts the compound method of test kit and reagent thereof: with embodiment 1.
2. the application of test kit:
Microbial culture is with embodiment 1;
Getting Escherichia coli bacteria liquid 1ml is added in EP pipe, and 13000r/min is after centrifugal 2 minutes, and abandoning supernatant, adds bacterial lysate 300 μ L, mixing; Aforesaid liquid is added 1 row of 96 deep hole Sptting plates, 7 rows, and add 300 μ L ethanol; Magnetic bead 100 μ L, magnetic bead are added to 2 rows, 8 rows of 96 deep hole Sptting plates in conjunction with liquid 100 μ L, distilled water 400 μ L; Magnetic bead washings 600 μ L is added to the 3-5 row of 96 deep hole Sptting plates, 9-11 row; Nucleic acid eluents 100 μ L is added to 96 deep hole Sptting plates 6 arrange, 12 row; 96 deep hole Sptting plates are put in instrument for extracting nucleic acid, pyrolysis time is set 5 minutes, binding time 5 minutes, washing time 2 minutes, elution time 5 minutes, run instrument for extracting nucleic acid; Liquid rotating in wash-out fluid apertures (6 rows, 12 rows) is moved in new EP pipe, obtains the DNA solution that can be used for pcr amplification.Use One-Drop OD-1000 spectrophotometer to detect nucleic acid concentration and purity, agarose gel electrophoresis detects nucleic acid extraction effect.
The prior art paramagnetic particle method of contrast is generally: get Escherichia coli bacteria liquid 1ml and be added in EP pipe, 13000r/min, after centrifugal 2 minutes, abandons supernatant, adds bacterial lysate 200 μ l, mixing; Aforesaid liquid is added 1 row of 96 deep hole Sptting plates, 7 rows, 96 deep-well plates are put into instrument, after cracking for some time, instrument is opened, 96 deep-well plates are taken out, and then magnetic bead and magnetic bead is added in conjunction with liquid in 1 row, 7 rows, 2-5 row, 8-11 row add four kinds of washingss, and 6 rows, 12 rows add elutriant, and 96 deep hole Sptting plates are put into instrument for extracting nucleic acid, run instrument for extracting nucleic acid, the extraction apparatus automatically run needs heating in elution process; After end, wash-out fluid apertures 6 is arranged, 12 row in liquid rotating move in new EP pipe, obtain the DNA solution that can be used for pcr amplification.
3. the effect detection box of pair test kit application is evaluated:
One-Drop OD-1000 spectrophotometer detects nucleic acid concentration and purity detects above-mentioned elutriant, result is, concentration and the purity of the test kit extraction cultivation colibacillary nucleic acid of 16 hours in employing invention are all higher, illustrate that test kit provided by the invention effectively can apply the nucleic acid in instrument for extracting nucleic acid extraction bacterium.
Compared with the test kit extracting nucleic acid with prior art paramagnetic particle method, the test kit of the present embodiment does not need heating when wash-out; Whole leaching process, without the need to taking out 96 orifice plates, gets final product a step and automatically completes after putting into 96 orifice plates.
Should be understood that above-described embodiment only for technical conceive of the present invention and feature are described, its object is to understand content of the present invention for those skilled in the art and implement according to this, not embodiment is exhaustive, can not limit the scope of the invention with this.All technical schemes according to the present invention's invention are modified or equivalent replacement, and do not depart from aim and the scope of technical solution of the present invention, and it all should be encompassed in the middle of right of the present invention.
Claims (10)
1. paramagnetic particle method extracts a test kit for bacterium amplifying nucleic acid, and the reagent in described test kit contains following component: bacterial lysate, magnetic bead, in conjunction with liquid, magnetic bead washings and nucleic acid eluents, is characterized in that:
Described bacterial lysate contains following component: sodium lauryl sulphate, ethylenediamine tetraacetic acid (EDTA), Tutofusin tris and sodium-chlor;
Described magnetic bead contains following component in conjunction with liquid: PEG-8 000 and sodium-chlor;
Described magnetic bead washings contains following component: ethanol;
Described nucleic acid eluents contains following component: Tutofusin tris and ethylenediamine tetraacetic acid (EDTA).
2. paramagnetic particle method according to claim 1 extracts the test kit of bacterium amplifying nucleic acid, and it is characterized in that: at described magnetic bead in conjunction with in liquid, the concentration of PEG-8 000 is 20-30mmol/L, and the concentration of sodium-chlor is 3mol/L.
3. paramagnetic particle method according to claim 1 extracts the test kit of bacterium amplifying nucleic acid, and it is characterized in that: in described magnetic bead washings, the volume by volume concentration of ethanol is 70%-90%.
4. paramagnetic particle method according to claim 1 extracts the test kit of bacterium amplifying nucleic acid, and it is characterized in that: in described nucleic acid eluents, the concentration of Tutofusin tris is 5-15mmol/L, and the concentration of ethylenediamine tetraacetic acid (EDTA) is 1.30-1.50mmol/L.
5. paramagnetic particle method according to claim 4 extracts the test kit of bacterium amplifying nucleic acid, and it is characterized in that: the concentration of described Tutofusin tris is 10mmol/L, the concentration of described ethylenediamine tetraacetic acid (EDTA) is 1.35mmol/L.
6. the paramagnetic particle method according to any one of claim 1 to 5 extracts the test kit of bacterium amplifying nucleic acid, it is characterized in that: the concentration of described sodium lauryl sulphate is 40-60mmol/L, the concentration of described ethylenediamine tetraacetic acid (EDTA) is 20-40mmol/L, the concentration of described Tutofusin tris is 90-115mmol/L, the concentration of described sodium-chlor is 480-520mmol/L, and surplus is sterilized water; Described bacterial lysate is by adding sodium hydroxide adjust ph to 7.5-8.5.
7. paramagnetic particle method extracts an extracting method for bacterium amplifying nucleic acid, it is characterized in that comprising the following steps:
Step 1: by bacterium and the centrifugation of nutrient solution mixture, goes supernatant to remove cell culture fluid, leaves microorganism; The rotating speed of centrifugation is 13000r/min, and the time of centrifugation is 2min;
Step 2: add bacterial lysate in thalline, mixing;
Step 3: add magnetic bead and magnetic bead in conjunction with liquid, after magnetic bead is combined with nucleic acid, magnet adsorption magnetic bead removes supernatant;
Step 4: add magnetic bead washings, uses magnet adsorption magnetic bead, removes supernatant;
Step 5: add nucleic acid eluents, uses magnet adsorption magnetic bead, and transfer elutriant, obtains the nucleic acid solution of bacterium.
8. paramagnetic particle method according to claim 7 extracts the extracting method of bacterium amplifying nucleic acid, it is characterized in that: the volume of the bacterial lysate added in step 2 and the volume ratio of inoculum are 1:1-1:5, the magnetic bead added in step 3 and magnetic bead are 1:10-1:20 in conjunction with the volume ratio of liquid and bacterial lysate, in step 4, the volume ratio of magnetic bead washings and bacterial lysate is 1:0.1-1:0.5, and the volume ratio of step 5 amplifying nucleic acid elutriant and bacterial lysate is 1:10-1:20.
9. paramagnetic particle method extracts an extracting method for bacterium amplifying nucleic acid, it is characterized in that comprising the following steps:
Step 1: by bacterium and the centrifugation of nutrient solution mixture, goes supernatant to remove cell culture fluid, leaves microorganism; The rotating speed of centrifugation is 13000r/min, and the time of centrifugation is 2min;
Step 2: add bacterial lysate in thalline, after mixing, is transferred to 1 row and 7 rows of 96 hole Sptting plates; Arrange 2 rows and 8 and add magnetic bead and magnetic bead in conjunction with liquid; Magnetic bead washings is added 3 rows, 4 rows, 5 rows and 9 rows, 10 rows, 11 rows; Nucleic acid eluents is added 6 rows and 12 rows;
Step 3: the 96 hole Sptting plates injecting reagent are put into instrument for extracting nucleic acid, obtains bacterial nucleic acid elutriant after automatic end of run.
10. paramagnetic particle method according to claim 9 extracts the extracting method of bacterium amplifying nucleic acid, it is characterized in that: the volume injecting the bacterial lysate of 96 hole Sptting plates in step 2 is 200-300 μ L, mixes with 200-400 μ L dehydrated alcohol; The magnetic bead injected and magnetic bead are 50-150 μ L in conjunction with the volume of liquid, mix with 300-400 μ L distilled water; The volume of the magnetic bead washings injected is 500-700 μ L; The volume of the nucleic acid eluents injected is 50-300 μ L.
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