CN101792803B - Method for preparing hybridization solution and cleaning solution for fluorescence in-situ hybridization - Google Patents
Method for preparing hybridization solution and cleaning solution for fluorescence in-situ hybridization Download PDFInfo
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Abstract
The invention discloses a method for preparing hybridization solution and cleaning solution for fluorescence in-situ hybridization. The method comprises the following steps: preparing the hybridization solution by using solution A, B, C and D, and preparing the cleaning solution by using the solution A, B and C, wherein the solution A is M1 mol/L of NaCl, each 100mL of solution B contains M2g of SDS, the solution C is M3 mol/L of Tris, and the solution D is deionized formamide; the hybridization solution is expected to be prepared by the following components: N1 mol/L of NaCl, N2 percent of SDS, N3 mmol/L of Tris-HCl and X percent of deionized formamide; and the volume ratio of the solution A, B, C and D to the water is (N1/M1): (N2/M2): (N3/1,000M3): (X/100): (1-N1/M1-N2/M2-N3/1,000M3-X/100). The cleaning solution is expected to be prepared by the following components: Y mmol/L of NaCl, S2% of SDS and S3 mmol/L of Tris-HCl; and the volume ratio of the solution A, B and C to the water is (Y/M1): (1,000S2/M2-S3/M3). By using the method, the hybridization solution with different deionized formamide concentration and the cleaning solution corresponding to the hybridization solution can be quickly prepared, and the operation is simple and quick. The hybridization solution and the cleaning solution prepared by the method can be efficiently applied to condition optimization and conventional detection of the fluorescence in-situ hybridization of a probe, and have the advantages of quickness, simple operation, good stability and the like.
Description
Technical field
The present invention relates to a kind of method of preparing the hybridization solution and the washings of fluorescence in situ hybridization.
Background technology
Fluorescence in situ hybridization technique (Fluorescence In Situ Hybridization; FISH) molecular biological particularity and microscopical visuality have been combined; Can monitor in the microbial environment of nature and identify that different mikrobe is individual, simultaneously microflora estimated.Its principle of work is that the nucleic acid fragment sequence that mikrobe has a high conservative of family specificity is detected; In simple terms: according to the nucleic acid fragment sequences Design probe that has the high conservative of family specificity in the mikrobe 16Sr RNA sequence; Through hybridization probe is hybridized mutually with the target sequence of detection then, have the segmental microorganism cells of target nucleic acid through the fluorescence that has on the probe being detected to detect.Facts have proved that in a large number this method has advantages such as directly perceived, accurate, quick, easy to operate to microorganism detection.
In fluorescence in situ hybridization, hybridization temperature combines the melting temperature (Tm) of the molecule that forms relevant with probe and target sequence.Have much for the melting temperature empirical formulas for estimating.For the long oligonucleotide probe of 11～22 bases, the universal experience formula of hybridization temperature is: T
Hyb=Tm-5 ℃.But about the concentration of deionized formamide in the hybridization solution, method can not calculated, and need in experiment, go to test one by one according to different deionized formamide concentration gradients, to confirm to be fit to the best deionized formamide concentration of this probe.
Summary of the invention
The purpose of this invention is to provide a kind of method of preparing the hybridization solution and the washings of fluorescence in situ hybridization.
The invention provides a kind of method of preparing the hybridization solution of following fluorescence in situ hybridization: form by NaCl, SDS, Tris-HCl, deionized formamide and water; The final concentration of NaCl is N1mol/L; The final concentration of SDS is N2% (a quality percentage composition); The final concentration of Tris-HCl is N3mmol/L, and the final concentration of deionized formamide is X% (a quality percentage composition); It is characterized in that: solution A, solution B, solution C, solution D and water are mixed, obtain said hybridization solution; Said solution A is made up of NaCl and water, and the final concentration of NaCl is M1mol/L; Said solution B is made up of SDS and water, and every 100mL solution B contains M2g SDS; Said solution C is made up of Tris and water, and the final concentration of Tris is M3mol/L; Said solution D is a deionized formamide; The volume ratio of said solution A, said solution B, said solution C, said solution D and water=(N1/M1): (N2/M2): (N3/1000M3): (X/100): (1-N1/M1-N2/M2-N3/1000M3-X/100).
In the method for the hybridization solution of said preparation fluorescence in situ hybridization: in the said solution A, the final concentration of NaCl is 3-5mol/L; The said solution B of every 100mL contains 2-10g SDS; In the said solution C, the final concentration of Tris is 0.5-1.5mol/L.
In the method for the hybridization solution of said preparation fluorescence in situ hybridization, the preparation method of said solution A is preferably following: with the water-soluble solution A that obtains of NaCl, the final concentration of NaCl is 4.5mol/L; The preparation method of said solution B is preferably following: with the water-soluble solution B that obtains of SDS, every 100mL solution B contains 2g SDS; The preparation method of said solution C is preferably following: Tris is water-soluble, and using hydrochloric acid to regulate pH is 7.2, obtains solution C, and the final concentration of Tris is 1mol/L.
Work as N1=0.9, N2=0.01, during N3=20, the volume ratio of said solution A, said solution B, said solution C, said solution D and water=40: 1: 4: 2X: (155-2X).Said X is preferably 5,10,15,20,25,30,35,40,45,50,55 or 60.
The present invention also provides a kind of method of preparing the washings of following fluorescence in situ hybridization: be made up of NaCl, SDS, Tris-HCl and water; The final concentration of NaCl is Y mmol/L; The final concentration of SDS is S2% (a quality percentage composition), and the final concentration of Tris-HCl is S3mmol/L; It is characterized in that: solution A, solution B, solution C and water are mixed, obtain said washings; Said solution A is made up of NaCl and water, and the final concentration of NaCl is M1mol/L; Said solution B is made up of SDS and water, and every 100mL solution B contains M2g SDS; Said solution C is made up of Tris and water, and the final concentration of Tris is M3mol/L; The volume ratio of said solution A, said solution B, said solution C and water=(Y/M1): (1000S2/M2): (S3/M3): (1000-Y/M1-1000S2/M2-S3/M3).
In the method for the washings of said preparation fluorescence in situ hybridization: in the said solution A, the final concentration of NaCl is 3-5mol/L; The said solution B of every 100mL contains 2-10g SDS; In the said solution C, the final concentration of Tris is 0.5-1.5mol/L.
In the method for the washings of said preparation fluorescence in situ hybridization, the preparation method of said solution A is preferably following: with the water-soluble solution A that obtains of NaCl, the final concentration of NaCl is 4.5mol/L; The preparation method of said solution B is preferably following: with the water-soluble solution B that obtains of SDS, every 100mL solution B contains 2g SDS; The preparation method of said solution C is preferably following: Tri s is water-soluble, and using hydrochloric acid to regulate pH is 7.2, obtains solution C, and the final concentration of Tris is 1mol/L.
Work as S2=0.01, during S3=20, the volume ratio=2Y of said solution A, said solution B, said solution C and water: 45: 180: (8775-2Y).Said Y is preferably 636,450,318,225,159,112,80,56,40,28,20 or 14.
The present invention also provides a kind of preparation to be used for the method for the test kit of fluorescence in situ hybridization; The said test kit that is used for fluorescence in situ hybridization is made up of hybridization solution and washings; Said hybridization solution is made up of NaCl, SDS, Tris-HCl, deionized formamide and water; The final concentration of NaCl is N1mol/L; The final concentration of SDS is N2% (a quality percentage composition), and the final concentration of Tris-HCl is N3mmol/L, and the final concentration of deionized formamide is X% (a quality percentage composition); Said washings is made up of NaCl, SDS, Tris-HCl and water, and the final concentration of NaCl is Y mmol/L, and the final concentration of SDS is S2% (a quality percentage composition), and the final concentration of Tris-HCl is S3mmol/L;
It is characterized in that: solution A, solution B, solution C, solution D and water are mixed, obtain said hybridization solution; Said solution A, said solution B, said solution C and water are mixed, obtain said washings; Said solution A is made up of NaCl and water, and the final concentration of NaCl is M1mol/L; Said solution B is made up of SDS and water, and every 100mL solution B contains M2g SDS; Said solution C is made up of Tris and water, and the final concentration of Tris is M3mol/L; Said solution D is a deionized formamide; When preparing said hybridization solution, the volume ratio of said solution A, said solution B, said solution C, said solution D and water=(N1/M1): (N2/M2): (N3/1000M3): (X/100): (1-N1/M1-N2/M2-N3/1000M3-X/100); When preparing said washings, the volume ratio of said solution A, said solution B, said solution C and water=(Y/M1): (1000S2/M2): (S3/M3): (1000-Y/M1-1000S2/M2-S3/M3);
When X was 5, Y was 636; When X was 10, Y was 450; When X was 15, Y was 318; When X was 20, Y was 225; When X was 25, Y was 159; When X was 30, Y was 112; When X was 35, Y was 80; When X was 40, Y was 56; When X was 45, Y was 40; When X was 50, Y was 28; When X was 55, Y was 20; When X was 60, Y was 14.
Work as N1=0.9, N2=0.01, during N3=20, when preparing said hybridization solution, the volume ratio of said solution A, said solution B, said solution C, said solution D and water=40: 1: 4: 2X: (155-2X).Work as S2=0.01, during S3=20, when preparing said washings, the volume ratio of said solution A, said solution B, said solution C and water is: 2Y: 45: 180: (8775-2Y).
The present invention also protects a kind of be used to the prepare hybridization solution of fluorescence in situ hybridization and the test kit of washings, comprises solution A, solution B, solution C and solution D;
Said solution A is made up of NaCl and water; In the solution A, the final concentration of NaCl is 3-5mol/L;
Said solution B is made up of SDS and water; Every 100mL solution B contains 2-10g SDS;
Said solution C is made up of Tris and water; In the solution C, the final concentration of Tris is 0.5-1.5mol/L;
Said solution D is a deionized formamide.
Saidly be used to prepare the hybridization solution of fluorescence in situ hybridization and the test kit of washings has and can be made up of solution A, solution B, solution C and solution D; The preparation method of said solution A is following: with the water-soluble solution A that obtains of NaCl; In the solution A, the final concentration of NaCl is 4.5mol/L; The preparation method of said solution B is following: with the water-soluble solution B that obtains of SDS; Every 100mL solution B contains 2g SDS; The preparation method of said solution C is following: Tris is water-soluble, and using hydrochloric acid to regulate pH is 7.2, obtains solution C; In the solution C, the final concentration of Tris is 1mol/L.
The hybridization solution that the different probe that uses in the fluorescence in situ hybridization is required all has the deionized formamide concentration of the best of a correspondence.The invention provides a kind of hybridization solution of quick preparation fluorescence in situ hybridization and the method and the dedicated kit thereof of washings, use method of the present invention or test kit can prepare fast different deionized formamide concentration hybridization solution and with the corresponding washings of this hybridization solution.Test kit preparation method provided by the invention is simple, is easy to industrialization production.Method provided by the invention is simple and quick.But hybridization solution that the present invention preparation obtains and washings efficient application be in the fluorescence in situ hybridization condition optimizing and the fluorescence in situ hybridization conventional sense of probe, has advantages such as quick, simple to operate, good stability.
Description of drawings
Fig. 1 uses the hybridization solution of this test kit preparation and the detected result of washings for the EuB338 probe.
Fig. 2 uses the hybridization solution of this test kit preparation and the detected result of washings for the SRB385 probe.
Fig. 3 uses the hybridization solution of this test kit preparation and the detected result of washings for the ACT465 probe; The orange light that sends for iron-oxidizing bacterium; The I:20% deionized formamide; The II:30% deionized formamide; The III:40% deionized formamide.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique among the following embodiment like no specified otherwise, is ordinary method.Used test materials among the following embodiment like no specified otherwise, is to buy from routine biochemistry reagent shop and obtains.The water that is used for following examples satisfies following condition: resistivity is greater than 18M Ω .cm, and carried out 121 ℃ of high-temperature sterilizations (also can adopt 0.22 μ m membrane filtration degerming).
The preparation of the test kit of embodiment 1, the hybridization solution that is used to prepare fluorescence in situ hybridization and/or washings
One, the preparation of reagent
The preparation method of solution A is following: with the water-soluble solution A that obtains of NaCl; In the solution A, the final concentration of NaCl is 4.5mol/L;
The preparation method of solution B is following: with the water-soluble solution B that obtains of SDS (sodium laurylsulfonate); Every 100mL solution B contains 2g SDS;
The preparation method of solution C is following: Tris (Tutofusin tris) is water-soluble, and use hydrochloric acid to regulate pH and 7.2 obtain solution C (Tris-HCl); In the solution C, the final concentration of Tris is 1mol/L;
Said solution D is a deionized formamide.
Two, the assembling of test kit
Test kit is made up of solution A, solution B, solution C and solution D.
Solution A: colourless transparent liquid; The 100ml/ bottle; 5 bottle/boxes; Normal temperature is preserved;
Solution B: colourless transparent liquid; The 50ml/ bottle; 1 bottle/box; Normal temperature is preserved;
Solution C: colourless transparent liquid; The 50ml/ bottle; 2 bottle/boxes; Normal temperature is preserved;
Solution D: colourless transparent liquid; The 5ml/ bottle; 2 bottle/boxes; Normal temperature is preserved.
Solution A, solution B, solution C and solution D are all through 121 ℃ of high-temperature sterilizations (also can adopt 0.22 μ m membrane filtration degerming).
The preparation and the application of embodiment 2, the hybridization solution that is used for fluorescence in situ hybridization and washings
With bacterium and the sulphate reducing bacteria in the fluorescence in situ hybridization detection Daqing oil field joint station slurry tank water sample.Be used for the probe that bacterial detection uses and be EuB338 (5 '-GCT GCC TCC CGT AGG AGT-3 '), 5 ' end is used the FITC mark; The probe that the detection sulphate reducing bacteria uses is SRB385 (5 ' CGG CGT CGC TGC GTC AGG-3 '), and 5 ' end is used the CY3 mark.Probe EuB338 and SRB385 be synthetic and mark by Jikang Biotechnology Co Ltd, Shanghai.
One, the test kit of application implementation example 1 prepares hybridization solution and washings
According to probe EuB338, confirm that deionized formamide concentration is 20% in this probe hybridization liquid, the concentration of sodium-chlor is 225mmol/L in the washings.Quantity per sample, need confirming the volume of the hybridization solution of preparation is 100 μ l, needing the volume of the washings of preparation is 40ml.
According to probe SRB385, confirm that deionized formamide concentration is 35% in this probe hybridization liquid, the concentration of sodium-chlor is 80mmol/L in the washings.Quantity per sample, need confirming the volume of the hybridization solution of preparation is 100 μ l, needing the volume of the washings of preparation is 40ml.
Utilize the test kit of embodiment 1 to prepare hybridization solution and washings respectively fast to two kinds of probes.
(1) hybridization solution and the washings of preparation probe EuB338.
1, preparing hybrid liquid
According to the deionized formamide concentration of confirming (20%; X=20) and hybridization solution volume (100 μ l), calculate the volume of required solution A, solution B, solution C, solution D and water of preparing hybrid liquid, see table 1.
During table 1 preparation EuB338 hybridization solution, the usage quantity of solution A～D and water in the test kit of embodiment 1
The volume ratio of solution A, solution B, solution C, solution D and water=40: 1: 4: 2X: (155-2X), promptly 20: 0.5: 2: 20: 57.5.
Get the centrifuge tube of 500 μ l specifications; Press table 1 with pipettor, pipette water, solution A, solution B, solution C and solution D successively to centrifuge tube, fully vibrating with the vortex oscillation device then mixes; (final concentration of NaCl is 0.9mol/L to obtain hybridization solution; The final concentration of SDS is 0.01%, and the final concentration of Tris-HCl is 20mmol/L, and the concentration of deionized formamide is 20%).
2, preparation washings
According to the sodium chloride concentration (225mmol/L that confirms; Y=225) and washings volume (40ml), calculate the volume of required solution A, solution B, solution C and water of preparation washings, see table 2.
During table 2 preparation EuB338 washings, the usage quantity of solution A～C and water in the test kit of embodiment 1
Volume ratio=the 2Y of solution A, solution B, solution C and water: 45: 180: (8775-2Y), promptly 2: 0.2: 0.8: 37.
Get the centrifuge tube of 50ml specification; Press table 2 with pipettor; Pipette water, solution A, solution B and solution C successively to centrifuge tube, fully vibrating with the vortex oscillation device then mixes, and (final concentration of NaCl is 225mmol/L to obtain washings; The final concentration of SDS is 0.01%, and the final concentration of Tris-HCl is 20mmol/L).
(2) hybridization solution and the washings of preparation probe SRB385.
1, preparing hybrid liquid
According to the deionized formamide concentration of confirming (35%; X=35) and hybridization solution volume (100 μ l), calculate the volume of required solution A, solution B, solution C, solution D and water of preparing hybrid liquid, see table 3.
During table 3 preparation SRB385 hybridization solution, the usage quantity of solution A～D and water in the test kit of embodiment 1
The volume ratio of solution A, solution B, solution C, solution D and water=40: 1: 4: 2X: (155-2X), promptly 20: 0.5: 2: 35: 42.5.
Get the centrifuge tube of 500 μ l specifications; Press table 3 with pipettor, pipette water, solution A, solution B, solution C and solution D successively to centrifuge tube, fully vibrating with the vortex oscillation device then mixes; (final concentration of NaCl is 0.9mol/L to obtain hybridization solution; The final concentration of SDS is 0.01%, and the final concentration of Tris-HCl is 20mmol/L, and the concentration of deionized formamide is 35%).
2, preparation washings
According to the sodium chloride concentration (80mmol/L that confirms; Y=80) and washings volume (40ml), calculate the volume of required solution A, solution B, solution C and water of preparation washings, see table 4.
During table 4 preparation SRB385 washings, the usage quantity of solution A～C and water in the test kit of embodiment 1
Volume ratio=the Y of solution A, solution B, solution C and water: 45: 180: (8775-2Y), promptly 0.711: 0.2: 0.8: 38.289.
Get the centrifuge tube of 50ml specification; Press table 4 with pipettor; Pipette water, solution A, solution B and solution C successively to centrifuge tube, fully vibrating with the vortex oscillation device then mixes, and (final concentration of NaCl is 80mmol/L to obtain washings; The final concentration of SDS is 0.01%, and the final concentration of Tris-HCl is 20mmol/L).
Adopt the test kit of preparation among the embodiment 1 provided by the invention, in 5 minutes, can make two kinds of hybridization solution and scavenging solutions that probe is required.
Two, the hybridization solution of step 1 preparation and the application of washings
Use the hybridization solution of step 1 preparation and washings is used for detecting the bacterium of Daqing oil field joint station slurry tank water sample and the step of sulphate reducing bacteria is:
1, gets 30ml water sample to be measured, behind 12000rpm, the 4 ℃ of centrifugal 20min, remove supernatant.With the fixing microorganism cells in the water sample of 4% paraformaldehyde solution.After completion is fixing, with 1 * PBS solution (130mM NaCl, 7mMNa
2HPO
4, 3mM NaH
2PO
4, pH 7.2) and washed twice, use 0.5ml1 * PBS solution and 0.5ml absolute ethyl alcohol suspension mixing at last.
2, prepare two slide glasss, the sample of respectively getting after the 1 μ l said fixing spreads upon on two slide glasss, and it is air-dry to leave standstill 1h under the room temperature.Use 50%, 80%, 98% ethanol dehydration then successively.
3, get the hybridization solution whirlpool mixing of the EuB338 for preparing in 12.5 μ l EuB338 probe solutions (concentration is 25 μ g/ml) and the 100 μ l step 1 after, get in the sample smear zone that 9 μ l splash into a slice slide glass; After getting the hybridization solution whirlpool mixing of the SRB385 for preparing in 12.5 μ lSRB385 probe solutions (concentration is 25 μ g/ml) and the 100 μ l step 1, get in the sample smear zone that 9 μ l splash into another sheet slide glass.Under 46 ℃ temperature, hybridize 2h.
The slide glass that 4, will be added with the EuB338 probe is immersed in the washings of the EuB338 for preparing in the 40ml step 1; Another slide glass that is added with the SRB385 probe is immersed in the washings of the SRB385 for preparing in the 40ml step 1.Under 48 ℃ temperature, leave standstill washing 20min.Each cleans slide glass with 1ml zero(ppm) water again, and lucifuge is air-dry.
5, observe the sample on the slide glass with DM6000B fluorescent microscope (come card, Germany).Select the bacterium in the I3 optical filtering piece test sample of this instrument configuration for use, see Fig. 1; Select the sulphate reducing bacteria in the N21 optical filtering piece test sample for use, see Fig. 2.According to the number of fluorescence bright spot in the visual field, in conjunction with area, the amount of smearing of sample and the volume of water sample to be detected that sample on the area in the visual field, the slide glass is smeared, the quantity of bacterium and sulphate reducing bacteria in the calculation sample.
The detected result of present embodiment shows that number of bacteria is 6.78 * 10 in the slurry tank water sample
6Individual/ml, the quantity of sulphate reducing bacteria is 4.36 * 10
5Individual/ml.
The preparation and the application of embodiment 3, the hybridization solution that is used for fluorescence in situ hybridization and washings
With the iron-oxidizing bacterium in the fluorescence in situ hybridization detection Shengli Oil Field combined station re-injection water water sample.The probe that uses is ACT465 (5 '-ACG GUA GAG GAG CAA UC-3 '), and 5 ' end is used the TET mark.Probe ACT465 is synthetic and mark by Jikang Biotechnology Co Ltd, Shanghai.Because probe ACT465 confirms that according to sequence hybridization temperature is 46 ℃ for design voluntarily, also need test and confirm suitable deionized formamide concentration in 46 ℃ of following these probe hybridization liquid.Therefore used the hybridization solution and the corresponding washing liquid of three kinds of deionized formamide concentration (being respectively 20%, 30%, 40%) to investigate crossbreeding effect respectively in the experiment.
One, the test kit of application implementation example 1 prepares hybridization solution and washings
Deionized formamide concentration is 20% in hybridization solution, and the concentration of sodium-chlor is 225mmol/L in the washings; Deionized formamide concentration is 30% in the hybridization solution, and the concentration of sodium-chlor is 112mmol/L in the washings; Deionized formamide concentration is 40% in the hybridization solution, and the concentration of sodium-chlor is 56mmol/L in the washings.Quantity per sample need to confirm the volume of the hybridization solution of preparation to be respectively 100 μ l, needs the volume of the washings of preparation to be respectively 40ml.
Utilize the test kit of embodiment 1 to prepare hybridization solution and washings respectively fast to deionized formamide concentration.
1, preparing hybrid liquid
According to the deionized formamide concentration of confirming (20%, X=20; 30%, X=30; 40%, X=40) with hybridization solution volume (100 μ l), calculate the volume of required solution A, solution B, solution C, solution D and water of preparing hybrid liquid, see table 5.
When table 5 is prepared the hybridization solution of different concns, the usage quantity of solution A～D and water in the test kit of embodiment 1
The volume ratio of solution A, solution B, solution C, solution D and water=40: 1: 4: 2X: (155-2X), promptly be respectively 20: 0.5: 2: 20: 57.5; 20: 0.5: 2: 30: 47.5; 20: 0.5: 2: 40: 37.5.
Get the centrifuge tube of 3 500 μ l specifications, press table 5 with pipettor, pipette water, solution A, solution B, solution C and solution D respectively successively to centrifuge tube, fully vibrating with the vortex oscillation device then mixes, and obtains the hybridization solution of 3 different concns.
2, preparation washings
According to the sodium chloride concentration (225mmol/L, the Y=225 that confirm; 112mmol/L, Y=112; 56mmol/L Y=56) with washings volume (40ml), calculates the volume of required solution A, solution B, solution C and water of preparation washings, sees table 6.
When table 6 is prepared the washings of different concns, the usage quantity of solution A～C and water in the test kit of embodiment 1
Volume ratio=the 2Y of solution A, solution B, solution C and water: 45: 180: (8775-2Y), promptly be respectively 2: 0.2: 0.8: 37; 0.996: 0.2: 0.8: 38.004; 0.498: 0.2: 0.8: 38.502.
Get the centrifuge tube of 3 50ml specifications, press table 6 with pipettor, pipette water, solution A, solution B and solution C successively to centrifuge tube, fully vibrating with the vortex oscillation device then mixes, and obtains the washings of 3 different concns.
Adopt the test kit of preparation among the embodiment 1 provided by the invention, in 10 minutes, can make the hybridization solution and the scavenging solution of 3 kinds of different concns.
Two, the hybridization solution of step 1 preparation and the application of washings
Hybridization solution and washings with the step 1 preparation are used for investigating the suitable deionized formamide concentration of probe ACT465 detection Shengli Oil Field combined station re-injection water iron-oxidizing bacterium.Concrete step is:
1, gets 40ml water sample to be measured, behind 13000rpm, the 4 ℃ of centrifugal 20min, remove supernatant.With the fixing microorganism cells in the water sample of 4% paraformaldehyde solution.After completion is fixing, with 1 * PBS solution (130mM NaCl, 7mMNa
2HPO
4, 3mM NaH
2PO
4, pH 7.2) and washed twice, use 0.2ml1 * PBS solution and 0.2ml absolute ethyl alcohol suspension mixing at last.
2, prepare three slide glasss, the sample of respectively getting after the 3 μ l said fixing spreads upon on two slide glasss, and it is air-dry to leave standstill 2h under the room temperature.Use 50%, 80%, 98% ethanol dehydration then successively.
3, respectively get 12.5 μ l ACT465 probe solutions (concentration is 50 μ g/ml), respectively with 100 μ l step 1 in behind the hybridization solution whirlpool mixing of 3 different concns preparing, respectively get in the sample smear zone that 9 μ l splash into three different slide glasss.Under 46 ℃ temperature, hybridize 2h.
4, after the hybridization, the slide glass that is added with different hybridization solutions is immersed in respectively in the corresponding different washings for preparing in the step 1.Under 48 ℃ temperature, leave standstill washing 20min.Each cleans slide glass with 1ml zero(ppm) water again, and lucifuge is air-dry.
5, observe the sample on the slide glass with DM6000B fluorescent microscope (come card, Germany).Select the iron-oxidizing bacterium in the I3 optical filtering piece test sample of this instrument configuration for use, see Fig. 3.
The detected result of present embodiment shows that the deionized formamide concentration that is fit at 46 ℃ of following probe ACT465 hybridization solutions is 30%.
Claims (9)
1. the method for hybridization solution of the following fluorescence in situ hybridization of preparation: form by NaCl, SDS, Tris-HCl, deionized formamide and water; The final concentration of NaCl is N1mol/L; The final concentration of SDS is N2% (a quality percentage composition); The final concentration of Tris-HCl is N3mmol/L, and the final concentration of deionized formamide is X% (a quality percentage composition); It is characterized in that: solution A, solution B, solution C, solution D and water are mixed, obtain said hybridization solution; Said solution A is made up of NaCl and water, and the final concentration of NaCl is M1mol/L; Said solution B is made up of SDS and water, and every 100mL solution B contains M2g SDS; Said solution C is made up of Tris and water, and the final concentration of Tris is M3mol/L; Said solution D is a deionized formamide; The volume ratio of said solution A, said solution B, said solution C, said solution D and water=(N1/M1): (N2/M2): (N3/1000M3): (X/100): (1-N1/M1-N2/M2-N3/1000M3-X/100);
N1=0.9, N2=0.01, N3=20; The volume ratio of said solution A, said solution B, said solution C, said solution D and water=40: 1: 4: 2X: (155-2X);
X is 5,10,15,20,25,30,35,40,45,50,55 or 60.
2. the method for claim 1, it is characterized in that: in the said solution A, the final concentration of NaCl is 3-5mol/L; The said solution B of every 100mL contains 2-10g SDS; In the said solution C, the final concentration of Tris is 0.5-1.5mol/L.
3. method as claimed in claim 2 is characterized in that: the preparation method of said solution A is following: with the water-soluble solution A that obtains of NaCl, the final concentration of NaCl is 4.5mol/L; The preparation method of said solution B is following: with the water-soluble solution B that obtains of SDS, every 100mL solution B contains 2g SDS; The preparation method of said solution C is following: Tris is water-soluble, and using hydrochloric acid to regulate pH is 7.2, obtains solution C, and the final concentration of Tris is 1mol/L.
4. the method for washings of the following fluorescence in situ hybridization of preparation: be made up of NaCl, SDS, Tris-HCl and water, the final concentration of NaCl is Y mmol/L, and the final concentration of SDS is S2% (a quality percentage composition), and the final concentration of Tris-HCl is S3mmol/L; It is characterized in that: solution A, solution B, solution C and water are mixed, obtain said washings; Said solution A is made up of NaCl and water, and the final concentration of NaCl is M1mol/L; Said solution B is made up of SDS and water, and every 100mL solution B contains M2g SDS; Said solution C is made up of Tris and water, and the final concentration of Tris is M3mol/L; The volume ratio of said solution A, said solution B, said solution C and water=(Y/M1): (1000S2/M2): (S3/M3): (1000-Y/M1-1000S2/M2-S3/M3);
S2=0.01, S3=20; Volume ratio=the 2Y of said solution A, said solution B, said solution C and water: 45: 180: (8775-2Y);
Y is 636,450,318,225,159,112,80,56,40,28,20 or 14.
5. method as claimed in claim 4 is characterized in that: in the said solution A, the final concentration of NaCl is 3-5mol/L; The said solution B of every 100mL contains 2-10g SDS; In the said solution C, the final concentration of Tris is 0.5-1.5mol/L.
6. method as claimed in claim 5 is characterized in that: the preparation method of said solution A is following: with the water-soluble solution A that obtains of NaCl, the final concentration of NaCl is 4.5mol/L; The preparation method of said solution B is following: with the water-soluble solution B that obtains of SDS, every 100mL solution B contains 2g SDS; The preparation method of said solution C is following: Tris is water-soluble, and using hydrochloric acid to regulate pH is 7.2, obtains solution C, and the final concentration of Tris is 1mol/L.
7. method for preparing the test kit that is used for fluorescence in situ hybridization; The said test kit that is used for fluorescence in situ hybridization is made up of hybridization solution and washings; Said hybridization solution is made up of NaCl, SDS, Tris-HCl, deionized formamide and water; The final concentration of NaCl is N1mol/L; The final concentration of SDS is N2% (a quality percentage composition), and the final concentration of Tris-HCl is N3mmol/L, and the final concentration of deionized formamide is X% (a quality percentage composition); Said washings is made up of NaCl, SDS, Tris-HCl and water, and the final concentration of NaCl is Y mmol/L, and the final concentration of SDS is S2% (a quality percentage composition), and the final concentration of Tris-HCl is S3mmol/L;
It is characterized in that: solution A, solution B, solution C, solution D and water are mixed, obtain said hybridization solution; Said solution A, said solution B, said solution C and water are mixed, obtain said washings; Said solution A is made up of NaCl and water, and the final concentration of NaCl is M1mol/L; Said solution B is made up of SDS and water, and every 100mL solution B contains M2g SDS; Said solution C is made up of Tris and water, and the final concentration of Tris is M3mol/L; Said solution D is a deionized formamide; When preparing said hybridization solution, the volume ratio of said solution A, said solution B, said solution C, said solution D and water=(N1/M1): (N2/M2): (N3/1000M3): (X/100): (1-N1/M1-N2/M2-N3/1000M3-X/100); When preparing said washings, the volume ratio of said solution A, said solution B, said solution C and water=(Y/M1): (1000S2/M2): (S3/M3): (1000-Y/M1-1000S2/M2-S3/M3);
When X was 5, Y was 636; When X was 10, Y was 450; When X was 15, Y was 318; When X was 20, Y was 225; When X was 25, Y was 159; When X was 30, Y was 112; When X was 35, Y was 80; When X was 40, Y was 56; When X was 45, Y was 40; When X was 50, Y was 28; When X was 55, Y was 20; When X was 60, Y was 14;
N1=0.9, N2=0.01, N3=20; When preparing said hybridization solution, the volume ratio of said solution A, said solution B, said solution C, said solution D and water=40: 1: 4: 2X: (155-2X); S2=0.01, S3=20; When preparing said washings, the volume ratio=2Y of said solution A, said solution B, said solution C and water: 45: 180: (8775-2Y).
8. the test kit of the washings of hybridization solution that is used to prepare the said fluorescence in situ hybridization of claim 1 and the said fluorescence in situ hybridization of claim 4 comprises solution A, solution B, solution C and solution D;
Said solution A is made up of NaCl and water; In the solution A, the final concentration of NaCl is 3-5mol/L;
Said solution B is made up of SDS and water; Every 100mL solution B contains 2-10g SDS;
Said solution C is made up of Tris and water; In the solution C, the final concentration of Tris is 0.5-1.5mol/L;
Said solution D is a deionized formamide.
9. test kit as claimed in claim 8 is characterized in that: said test kit is made up of solution A, solution B, solution C and solution D;
The preparation method of said solution A is following: with the water-soluble solution A that obtains of NaCl; In the solution A, the final concentration of NaCl is 4.5mol/L;
The preparation method of said solution B is following: with the water-soluble solution B that obtains of SDS; Every 100mL solution B contains 2g SDS;
The preparation method of said solution C is following: Tris is water-soluble, and using hydrochloric acid to regulate pH is 7.2, obtains solution C; In the solution C, the final concentration of Tris is 1mol/L.
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