CN104198357A - Application of flow cytometry method to vaccine production real-time monitoring - Google Patents
Application of flow cytometry method to vaccine production real-time monitoring Download PDFInfo
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- CN104198357A CN104198357A CN201410493364.8A CN201410493364A CN104198357A CN 104198357 A CN104198357 A CN 104198357A CN 201410493364 A CN201410493364 A CN 201410493364A CN 104198357 A CN104198357 A CN 104198357A
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Abstract
The invention discloses the application of a flow cytometry method to vaccine production real-time monitoring. The application is to apply the flow cytometry method to the vaccine production process, and monitor the viable bacteria count in real time during the vaccine production process. The application comprises the steps of dyeing bacterial cell through fluorescent dye, detecting the change of bacteria fluorescence signals through a flow cytometer, and obtaining an FCM diagram with at least two types of the following parameters by measuring the change of the bacteria fluorescence signals: viable bacteria count, dead bacteria count and total bacteria count, obtaining the ratio of the viable bacteria count according to the FCM diagram, obtaining the concentration of target bacteria solution through an absolute counting method, and obtaining the viable bacteria amount and the dead bacteria amount of target bacteria according to the ratio of the viable bacteria count and the concentration of the target bacteria solution. The invention provides a convenient, rapid and reliable bacterial activity monitoring method for the vaccine production process and is convenient and easy, the operation time is only 30 minutes, and the quality of the vaccine production process can be accurately controlled.
Description
Technical field
The present invention relates to the technology application of flow cytometry, the particularly application of flow cytometry method in production of vaccine Real-Time Monitoring.
Background technology
In production of vaccine process, the control of bacterium liquid sweat is the key link of production of vaccine.The many factors such as the nutritional status of temperature, pH value, nutrient solution all affect the sweat of bacterium liquid.Therefore set up easy, quick, reliable bacterial activity detection method the monitoring of production of vaccine is had to practical significance.The method of traditional monitoring production of vaccine mainly contains cultivation counting method and nephometer is counted method, and production of vaccine manufacturer still depends on this two kinds of methods to the control of vaccine quality at present.Cultivation is highly sensitive but length consuming time needs 24h could obtain result above conventionally, can not reach the object of instant control bacterium liquid sweat; Turbidimetry is simple to operate but adopt visual inspection, and detection sensitivity is poor, and can not distinguish viable bacteria and dead bacterium.
Therefore, be badly in need of one and can monitor in real time viable count object method in production of vaccine process.
Summary of the invention
The shortcoming that the object of the invention is to overcome prior art, with not enough, provides the application of a kind of flow cytometry method in production of vaccine Real-Time Monitoring.
Object of the present invention is achieved through the following technical solutions: the application of flow cytometry method in production of vaccine Real-Time Monitoring is that flow cytometry method is applied in production of vaccine process, detects the viable count in production of vaccine process;
Described vaccine comprises bacillus coli vaccine, StaphVAX, swine erysipelas vaccine, streptococcus vaccine, salmonella vaccine, clostridieum welchii vaccine, chicken virus mycoplasma vaccine, pasteurella multocida vaccine and haemophilus parasuis vaccine etc.
The application of described flow cytometry method in production of vaccine Real-Time Monitoring, preferably comprises following steps:
(1) select in fluorescent dye as described below at least two kinds dye object bacteria; Fluorescent dye is the dyestuff that can dye to living bacterial cells, the dyestuff that can dye to total bacteria count and the dyestuff that can dye to dead bacterial cell;
(2) then use the variation of flow cytometer bacterial detection fluorescence signal, by measuring the variation of bacterial fluorescence information, obtain FCM collection of illustrative plates; FCM collection of illustrative plates has at least two kinds in following parameter: viable count, dead bacterium number and total bacteria count; Obtain the ratio of viable count according to FCM collection of illustrative plates;
(3) obtain the concentration of object bacteria bacterium liquid by absolute counting method;
(4) concentration of the object bacteria bacterium liquid that the ratio of the viable count obtaining according to step (2) and step (3) obtain, obtains viable bacteria amount and the dead bacterium amount of object bacteria.
Step (1) is preferably: select fluorescent dye as described below and dye object bacteria; Fluorescent dye is the combination that a kind of dyestuff with dyeing to dead bacterial cell in the dyestuff that can dye to living bacterial cells and the dyestuff that can dye to total bacteria count forms;
The dyestuff that can dye to living bacterial cells described in step (1) is preferably CFDA-SE; Fluorescent dye CFDA-SE can enter cell membrane, after the esterase enzymolysis in somatic cells, is sent fluorescence, is membrane permeability dyestuff; Enzymolysis afterproduct can not pass cell membrane, dyes as nonreversibility, therefore the CFDA-SE living bacterial cells that can only dye;
The dyestuff that can dye to total bacteria count described in step (1) is preferably SYBR Green I; SYBR Green I is a kind of double-stranded DNA combination dye being incorporated in ditch, can combine with all double-stranded DNAs, after being combined, send green fluorescence, and fluorescence strengthens greatly with double-stranded DNA; Its maximum absorption wavelength is about 497nm, and emission wavelength is 520nm to the maximum, SYBR Green I can dye all viable bacterias and dead bacterium;
The dyestuff that can dye to dead bacterial cell described in step (1) is preferably PI; PI (propidium iodide, Propidium Iodide) be a kind of nuclei dyeing color reagent that can dye to DNA, PI is film impermeability dyestuff, can not pass through living cells film, but can to nuclear staining, after embedding double-stranded DNA, discharge red fluorescence through damaged cell membrane;
The concentration that obtains object bacteria bacterium liquid by absolute counting method described in step (3) can be counted or be passed through Flow cytometry by blood counting chamber;
Described is preferably as follows and is realized by Flow cytometry: the magnetic bead of concentration known and described object bacteria bacterium liquid are mixed, detect by flow cytometer, calculate the concentration of object bacteria bacterium liquid by following formula;
Formula is:
(bacterial cell number/magnetic bead number) × (in every pipe, total magnetic bead number/population of samples is long-pending) × extension rate=bacterial concentration;
Bacterial cell number refers to the bacterial cell number that certain hour in-flow cell instrument detects; Magnetic bead number refers to the magnetic bead number that certain hour in-flow cell instrument detects; In every pipe, total magnetic bead number refers to the magnetic bead sum in sample detection pipe; Extension rate refers to the extension rate of bacterium liquid.
The particle diameter of described magnetic bead is preferably 5.5 μ m;
The application of described flow cytometry method in production of vaccine Real-Time Monitoring, more preferably comprises following steps:
I, the blank of setting up bacterial cell and fluorescence intensity compensation contrast, and determine the operating parameter of flow cytometer:
1. by object bacteria activation, be cultured to exponential phase; By a part of bacterium liquid deactivation wherein, the bacterium liquid of deactivation contrasts as dead bacterium; Undressed bacterium liquid is viable bacteria contrast;
2. two kinds of cells that with PBS, 1. step obtained wash respectively, then use PBS resuspended to OD
600be 0.002～0.006, obtain dead bacterium contrast bacterium liquid and viable bacteria contrast bacterium liquid;
3. obtain blank and the fluorescence intensity compensation contrast of bacterial cell:
A, viable bacteria is contrasted to bacterium liquid contrast with dead bacterium the mixed bacteria liquid up flow type cell instrument that bacterium liquid is mixed to get, by adjusting the photoelectric diode voltage of flow cytometer, to regulate the intensity of forward scattering light FSC and side scattered light SSC, establish door with SSC/FSC, determine the position of bacterial cell to be checked in flow cytometer image, in order to eliminate the autofluorescence of bacterium to be checked;
B, in viable bacteria contrast bacterium liquid, add SYBR Green I or the monochromatic fluorescent dye of CFDA-SE, vibration mixes, lucifuge reaction; Then up flow type cell instrument detects, with the SSC/FSC door of setting in steps A, the PMT voltage of adjusting SYBR Green I or CFDA-SE carries out fluorescence compensation, for removing from detected fluorescence signal the interference fluorescence signal causing because fluorescein emission spectrum is overlapping, can be used for getting rid of due to the impact of factor on testing result such as quality, concentration and the colouring methods of dyestuff simultaneously, obtain the compensation contrast figure of SYBR Green I or CFDA-SE, determine the voltage of SYBR Green I or CFDA-SE fluorescence intensity; In dead bacterium contrast bacterium liquid, add the monochromatic fluorescent dye of PI, vibration mixes, lucifuge reaction; Then up flow type cell instrument detects, and with the SSC/FSC door of setting in steps A, the PMT voltage of adjusting PI carries out fluorescence compensation, obtains PI compensation contrast figure, determines the voltage of PI fluorescence intensity; Contrast the composite fluorescent dye that adds a kind of and PI in SYBR Green I and CFDA-SE to form in the mixed bacteria liquid that bacterium liquid is mixed to get with dead bacterium at viable bacteria contrast bacterium liquid, vibration mixes, and lucifuge is reacted; Then up flow type cell instrument detects, and with the SSC/FSC door of setting in steps A, the PMT voltage of adjusting composite fluorescent dye carries out Two Colour Fluorescence compensation, obtains composite fluorescent dye compensation contrast figure, determines the voltage of composite fluorescent dye fluorescence intensity;
The monitoring of II, bacterium liquid sweat
1. in the different phase of production of vaccine, get zymocyte liquid sample;
2. PBS washing zymocyte liquid sample, then uses the resuspended zymophyte of PBS to OD
600be 0.002～0.006; By two kinds of fluorescent dyes on the bacterium liquid mark obtaining, the fluorescent dye using is identical with step I, and the voltage of use is the 3. final definite voltage of B of step I; Flow cytometer detects, and after picture door hives off, obtains each group of ratios;
3. by the magnetic bead of concentration known and zymocyte liquid sample mix, detect by flow cytometer, calculate the concentration of zymocyte liquid by following formula;
Formula is:
(bacterial cell number/magnetic bead number) × (in every pipe, total magnetic bead number/population of samples is long-pending) × extension rate=bacterial concentration;
4. according to the concentration of each group of ratios and zymocyte liquid, the viable bacteria in zymophyte and dead bacterium are carried out quantitatively.
In the present invention, the washing of bacterium is first centrifugal bacterium liquid, gets precipitation, then add PBS resuspended, centrifugal, abandon supernatant, complete once washing;
The step of the deactivation of step I described in is 1. preferably: by centrifugal bacterium liquid, get precipitation; Working concentration is to boil 5～10min after the ethanolic solution of mass volume ratio 70% disperses precipitation standing at least 1 hour or adds PBS to be uniformly dispersed;
The PBS of step I described in is 2. the PBS of pH6.2～7.4,0.01～0.1mol/L;
The number of times of the washing of step I described in is 2. preferably 2 times;
The consumption of the SYBR Green I of step I described in is 3. preferably that 0.1～20 μ M calculates by the final concentration in bacterium liquid;
The consumption of the CFDA-SE of step I described in is 3. preferably that 0.1～20 μ M calculates by the final concentration in bacterium liquid;
The consumption of the PI of step I described in is 3. preferably 0.1～10 μ g.mL by the final concentration in bacterium liquid
-1calculate;
The time of the lucifuge reaction of step I described in is 3. 5min at least; Be preferably as follows: when used dyestuff is during for SYBR Green I or CFDA-SE, the time of lucifuge reaction is 10min; When used dyestuff is during for PI, the time of lucifuge reaction is 5min;
The temperature of described lucifuge reaction is preferably 20～30 DEG C;
The compensation contrast figure of step I described in 3. in the time making, preferably first single microbiological contamination cell, more two microbiological contamination cell;
The volume of the zymocyte liquid sample of Step II described in is 1. preferably 0.2mL～1mL;
The particle diameter of the magnetic bead of Step II described in is 3. preferably 5.5 μ m.
The present invention has following advantage and effect with respect to prior art:
(1) the present invention provides a kind of easy, quick, reliable bacterial activity monitoring method for production of vaccine process.
(2) the present invention is simple and easy to do, and the running time only needs 30min, far fewer than traditional cultivation.Can detect one by one bacterium, without amount reproduction, can accurately detect at short notice a large amount of bacteriums, therefore the bacterium long to growth cycle, the method has superiority more significantly.
(3) the present invention can accurately control the quality of production of vaccine process.
Brief description of the drawings
Fig. 1 is the SYBR Green I of chicken virus mycoplasma and the scatter diagram of PI dyeing, wherein: figure A, B and E are living bacterial cells; Figure C, D and F are dead bacterial cell; Figure G is viable bacteria and dead bacterium mixed bacteria liquid; Figure H is the bacterium liquid blank that is unstained; In the time of double staining, Q2 district is dead bacterial cell district, and Q3 is living bacterial cells district; In the time singly dying PI, Q1 district is dead bacterial cell district; In the time singly dying SYBR Green I, dead bacterial cell and living bacterial cells are all shown as the positive, WeiQ3 district.
Fig. 2 is the scatter diagram of flow cytometry method monitoring chicken virus mycoplasma production of vaccine bacterium liquid sweat.
Fig. 3 is chicken virus mycoplasma bacterial cell absolute counting scatter diagram, and wherein: P1 district is bacterial cell district, P2 district is magnetic bead district.
Fig. 4 is the scatter diagram of clostridieum welchii living bacterial cells and dead bacterial cell CFDA-SE and PI dyeing contrast, wherein: figure A, B and E are living bacterial cells; Figure C, D and F are dead bacterial cell; Figure G is viable bacteria and dead bacterium mixed bacteria liquid; Figure H is the bacterium liquid blank that is unstained; Q3 district is living bacterial cells district, and Q1 is dead bacterial cell district.
Fig. 5 is the scatter diagram of flow cytometry method monitoring clostridieum welchii production of vaccine bacterium liquid sweat.
Fig. 6 is clostridieum welchii bacterial cell absolute counting scatter diagram, and wherein: P1 district is bacterial cell district, P2 district is magnetic bead district.
Embodiment
Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited to this.
Embodiment 1
(1) set up the blank of chicken virus mycoplasma bacterial cell and fluorescence intensity compensation contrast
1. chicken virus mycoplasma F-36 strain (China Veterinery Drug Inspection Office) is activated through improvement FreyShi nutrient culture media, more than 37 DEG C of cultivation 80h, inoculation Medium of shaking flask fermentation (improvement FreyShi nutrient culture media), 37 DEG C are cultured to bacterial growth logarithmic phase, get 2 pipe fermentation liquors, each 1mL; By centrifugal fermentation liquor (the centrifugal 10min of 9000rpm), abandon supernatant, obtain bacterial cell.The ethanolic solution that adds therein 1mL mass volume ratio 70% in a pipe bacterial cell, mixes rear placement 1 hour, or adds 1mL PBS, boils 10min, and object is for deactivation, obtains dead bacterium contrast; Another is managed to undressed bacterial cell contrasts as viable bacteria simultaneously.
2. use PBS (pH7.4,0.01mol/L) that step two kinds of cells are 1. washed respectively 2 times, then resuspended to OD respectively with PBS
600be 0.002.
A, contrast 1:1 by volume with viable bacteria contrast and dead bacterium and be mixed to get mixed bacteria liquid, as blank.By adjusting the photoelectric diode voltage of flow cytometer, to regulate the intensity of forward scattering light FSC and side scattered light SSC, establish door with SSC/FSC, determine the position of bacterial cell to be checked in flow cytometer image, in order to eliminate the autofluorescence of bacterium to be checked, the operating voltage that obtains FSC and SSC is 412 volts and 363 volts, and result is as shown in H in Fig. 1;
B, mono-the dying of SYBR Green I, by way of compensation contrast.Step is: (be OD at bacterium liquid
600be 0.002 bacterium liquid) in add SYBR Green I dyeing liquor, the final concentration of SYBR Green I dyeing liquor is 0.1 μ M, vibration mixes, room temperature lucifuge reaction 10min, then up flow type cell instrument detects, establish door with the SSC/FSC setting in steps A, the PMT voltage of adjusting SYBR Green I carries out fluorescence compensation, for removing from detected fluorescence signal the interference fluorescence signal causing because fluorescein emission spectrum is overlapping, can be used for getting rid of the quality due to dyestuff simultaneously, the impact of the factor such as concentration and colouring method on testing result, obtain SYBR Green I compensation contrast figure, the voltage of determining SYBR Green I fluorescence intensity is 414 volts, result is as shown in the A in Fig. 1 (viable bacteria contrast) and C (dead bacterium contrast),
C, PI are mono-to be dyed, by way of compensation contrast.Step is: in bacterium liquid, adding PI dyeing liquor, the final concentration of PI dyeing liquor is 0.1 μ g.mL
-1, vibration mixes, room temperature lucifuge reaction 5min; Then up flow type cell instrument detects, establish door with the SSC/FSC setting in steps A, the PMT voltage of adjusting PI carries out fluorescence compensation, obtain PI compensation contrast figure, the voltage of determining PI fluorescence intensity is 533 volts, and result is as shown in the B in Fig. 1 (viable bacteria contrast) and D (dead bacterium contrast);
D, SYBR Green I and PI are two to be dyed, contrast by way of compensation.Step is: in bacterium liquid, add PI dyeing liquor and SYBR Green I dyeing liquor, the final concentration of SYBR Green I dyeing liquor is 0.1 μ M, and the final concentration of PI dyeing liquor is 0.1 μ g.mL
-1, vibration mixes, room temperature lucifuge reaction 10min; Then up flow type cell instrument detects, establish door with the FSC/SSC setting in steps A, the PMT voltage of adjusting SYBR Green I and PI carries out Two Colour Fluorescence compensation, obtain SYBR Green I and PI compensation contrast figure, the voltage of determining SYBR Green I and PI fluorescence intensity is 414 and 533 volts, and result is as shown in the E in Fig. 1 (viable bacteria contrast), F (dead bacterium contrast) and G (viable bacteria contrast and dead bacterium contrast the mixed bacteria liquid that 1:1 is mixed to get by volume).
Set blank and the fluorescence intensity compensation contrast of chicken virus mycoplasma bacterial cell, using this as the impact of getting rid of autofluorescence, simultaneously for arranging and adjust voltage, and remove the interference fluorescence signal that causes because fluorescein emission spectrum is overlapping etc.When flow cytometer detects, each bacterial cell is by four kinds of parameter-definitions: two non-fluorescence signals and two fluorescence signals.Wherein two non-fluorescence signals are respectively lateral scattering light signal (for measuring cell granulations degree) and forward-scattering signal (for measuring cell size), and the combination of the two can be used for telling different cell masses from mixed cellularity group.Two fluorescence signals are respectively that energy that SYBR Green I and PI red fluorescence dyestuff receive laser is excited and the light signal launched; The excitation wavelength of SYBR Green I is 488nm, and emission wavelength is 525nm, is detected as green fluorescence.The excitation wavelength of PI is 488nm, and emission wavelength is 625nm, is detected as red fluorescence.
(2) get each 2 pipes of the fermentation liquor of different phase in chicken virus mycoplasma F-36 strain vaccine production run, every pipe 1mL (the centrifugal 10min of 9000rpm), a pipe detects for flow cytometer, and a pipe uses culture-based method to cultivate.
(3) flow cytometer detects:
1. PBS washs after 2 times, and re-suspended cell concentration is OD
600=0.002, with SYBR Green I and PI double staining (2. D of the same step of operating process (1)), the magnitude of voltage of operating parameter: FSC is 412 volts, the magnitude of voltage of SSC is 363 volts, the magnitude of voltage of SYBR Green I and PI is 414 volts and 533 volts, flow cytometer detects (as shown in Figure 2), after picture door hives off, obtains each group of ratios.
2. by the magnetic bead of concentration known (particle diameter be 5.5 μ m) mix with bacterium liquid after, detect (as shown in Figure 3) by flow cytometer, calculate the concentration of fermentation liquor by following formula;
(bacterial cell number/magnetic bead number) × (in every pipe, total magnetic bead number/population of samples is long-pending) × extension rate=bacterial concentration;
Bacterial cell number refers to the bacterial cell number that certain hour in-flow cell instrument detects; Magnetic bead number refers to the magnetic bead number that certain hour in-flow cell instrument detects; In every pipe, total magnetic bead number refers to the magnetic bead sum in sample detection pipe; Extension rate refers to the extension rate of bacterium liquid.Example magnetic bead adds up to 54829, fully dissolve with 20 μ L PBS, add 480 μ L bacterium liquid, after mixing, up flow type cell instrument detects, magnetic bead number is 1000, bacterial cell number 2261, and bacterial concentration is (2261/1000) * (54829/ (500*0.001)) * (500/480)==2.5*10
5ccu (change color unit) mL
-1.
3. SYBR Green I dyeing obtains total bacterium amount, and PI dyeing obtains dead bacterium amount.1. the fermentation liquor concentration that 2. the each group of ratios that obtain by step and step obtain, can obtain dead bacterium number, thereby calculate viable count, obtains quantitative result.Computing formula is: dead bacterium ratio × concentration=dead bacterium number; Viable bacteria ratio × concentration=viable count.
(4) classic flat-plate method of counting: the 1 pipe fermentation liquor taking out in step (2) is cooked to 10 times of gradient dilutions to 10
-6, respectively by 10
-4, 10
-5, 10
-6the bacterium liquid of three gradients is coated with improvement FreyShi culture medium flat plate, and each gradient is coated with 3 flat boards, puts in 37 DEG C of incubators and cultivates after 3-7 days, and counting is dull and stereotyped, averages, and obtaining viable bacteria concentration is 3 × 10
5ccumL
-1, consistent with flow cytometry methods and results, but about long 3-7 days consuming time, can not be used for the real-time monitoring of production of vaccine.Tradition turbidimetry: bacterium liquid in step (2) is done after 10 times of gradient dilutions, and with Maxwell opacity tube comparison, the order of magnitude that obtains bacterial concentration by visual inspection is 10
5～10
6ccumL
-1, operation is very fast, but can not distinguish viable bacteria and dead bacterium, and by visual inspection, can not obtain objective, experimental result accurately, thereby causes the instability of vaccine quality.
Flow cytometry method provided by the invention, the running time is in 30 minutes, and data are accurately objective, and strong operability can effectively overcome the drawback in current production of vaccine process.
Embodiment 2
(1) set up the fluorescence intensity blank of clostridieum welchii bacterial cell and compensate contrast
1. by clostridieum welchii (ATCC13124, purchased from American type culture collection strain library), inoculation seed culture medium liquid sulfur glycollate (FT) nutrient culture media, cultivate 24h activation for 37 DEG C, inoculation Medium of shaking flask fermentation (FT nutrient culture media), cultivate 24h to bacterial growth logarithmic phase, get 2 pipe fermentation liquors, each 1mL for 37 DEG C; By centrifugal fermentation liquor (the centrifugal 10min of 5000rpm), abandon supernatant, obtain bacterial cell.In a pipe bacterial cell, add therein 1mL PBS (pH6.2,0.01mol/L), boil 5min, object is for deactivation, obtains dead bacterium contrast; Another is managed to undressed bacterial cell contrasts as viable bacteria simultaneously.
2. with PBS, step two kinds of cells are 1. washed respectively 2 times, then use PBS resuspended to OD
600be 0.004.
A, contrast 1:1 by volume with viable bacteria contrast and dead bacterium and be mixed to get mixed bacteria liquid, as blank.By adjusting the photoelectric diode voltage of flow cytometer, to regulate the intensity of forward scattering light FSC and side scattered light SSC, establish door with SSC/FSC, determine the position of bacterial cell to be checked in flow cytometer image, in order to eliminate the autofluorescence of bacterium to be checked, the operating voltage that obtains FSC and SSC is 476 volts and 500 volts, and result is as shown in H in Fig. 4;
B, CFDA-SE are mono-to be dyed, by way of compensation contrast.Step is: in bacterium liquid, add CFDA-SE dyeing liquor, the final concentration of CFDA-SE dyeing liquor is 0.1 μ M, vibration mixes, room temperature lucifuge reaction 10min, then up flow type cell instrument detects, establish door with the SSC/FSC setting in steps A, the PMT voltage of adjusting CFDA-SE carries out fluorescence compensation, for removing from detected fluorescence signal the interference fluorescence signal causing because fluorescein emission spectrum is overlapping, can be used for getting rid of the quality due to dyestuff simultaneously, the impact of the factor such as concentration and colouring method on testing result, obtain CFDA-SE compensation contrast figure, the voltage of determining CFDA fluorescence intensity is 450 volts, result is as shown in the A in Fig. 4 (viable bacteria contrast) and C (dead bacterium contrast),
C, PI are mono-to be dyed, by way of compensation contrast.Step is: in bacterium liquid, adding PI dyeing liquor, the final concentration of PI dyeing liquor is 0.1 μ g.mL
-1, vibration mixes, room temperature lucifuge reaction 5min; Then up flow type cell instrument detects, establish door with the SSC/FSC setting in steps A, the PMT voltage of adjusting PI carries out fluorescence compensation, obtain PI compensation contrast figure, the voltage of determining PI fluorescence intensity is 545 volts, and result is as shown in the B in Fig. 4 (viable bacteria contrast) and D (dead bacterium contrast);
D, CFDA-SE and PI are two to be dyed, contrast by way of compensation.Step is: in bacterium liquid, add PI dyeing liquor and CFDA dyeing liquor, the final concentration of CFDA dyeing liquor is 0.1 μ M, and the final concentration of PI dyeing liquor is 0.1 μ g.mL
-1, vibration mixes, room temperature lucifuge reaction 10min; Then up flow type cell instrument detects, establish door with the FSC/SSC setting in A step, the PMT voltage of adjusting CFDA-SE and PI carries out Two Colour Fluorescence compensation, obtain CFDA-SE and PI compensation contrast figure, the voltage of determining CFDA-SE and PI fluorescence intensity is 450 and 545 volts, and result is as shown in the E in Fig. 4 (viable bacteria contrast), F (dead bacterium contrast) and G (viable bacteria contrast and dead bacterium contrast the mixed bacteria liquid that 1:1 is mixed to get by volume).
Set blank and the fluorescence intensity compensation contrast of clostridieum welchii bacterial cell, using this as the impact of getting rid of autofluorescence, simultaneously for arranging and adjust voltage, and remove the interference fluorescence signal that causes because fluorescein emission spectrum is overlapping etc.When flow cytometer detects, each bacterial cell is by four kinds of parameter-definitions: two non-fluorescence signals and two fluorescence signals.Wherein two non-fluorescence signals are respectively lateral scattering light signal (for measuring cell granulations degree) and forward-scattering signal (for measuring cell size), and the combination of the two can be used for telling different cell masses from mixed cellularity group.Two fluorescence signals are respectively that energy that CFDA-SE and PI red fluorescence dyestuff receive laser is excited and the light signal launched; The excitation wavelength of CFDA-SE is 488nm, and emission wavelength is 525nm, is detected as green fluorescence.The excitation wavelength of PI is 488nm, and emission wavelength is 625nm, is detected as red fluorescence.
(2) get each 2 pipes of fermentation liquor of different phase in clostridieum welchii production of vaccine process, every pipe 1mL, a pipe detects for flow cytometer, and a pipe uses culture-based method to cultivate.
(3) flow cytometer detects:
1. PBS washs after 2 times, and re-suspended cell concentration is OD
600=0.004, with CFDA-SE and PI double staining (2. D of the same step of operating process (1)), operating parameter is that the magnitude of voltage of FSC is 476 volts, the magnitude of voltage of SSC is 500 volts, the magnitude of voltage of CFDA-SE and PI is 450 volts and 545 volts, flow cytometer detects, and after picture door hives off, obtains each group of ratios (as shown in Figure 5).
2. by the magnetic bead of concentration known (particle diameter be 5.5 μ m) mix with bacterium liquid after, detect by flow cytometer, the formula providing by embodiment 1 calculates the concentration (as shown in Figure 6) of fermentation liquor;
3. CFDA-SE dyeing obtains viable bacteria amount, and PI dyeing obtains dead bacterium amount.1. the bacterial concentration that 2. the each group of ratios that obtain by step and step obtain, can obtain viable count and dead bacterium number, obtains quantitative result.Technology formula is: viable bacteria ratio * concentration=viable count.
(4) classic flat-plate method of counting: the 1 pipe fermentation liquor taking out in step (2) is cooked to 10 times of gradient dilutions to 10
-6, respectively by 10
-4, 10
-5, 10
-6the bacterium liquid of three gradients is coated with FT nutrient agar panel, and each gradient is coated with 3 plates, and (36 ± 1) DEG C anaerobism is cultivated 24 hours, and counting is dull and stereotyped, and averaging and obtaining viable bacteria concentration is 3 × 10
5ccumL
-1, consistent with flow cytometry methods and results, complex operation, length consuming time.Tradition turbidimetry: the fermentation liquor in step (2) is done after 10 times of gradient dilutions, and with Maxwell opacity tube comparison, the order of magnitude that obtains bacterial concentration by visual inspection is 10
5～10
6ccumL
-1, operation is very fast, but can not distinguish viable bacteria and dead bacterium, and by visual inspection, can not obtain objective, experimental result accurately, thereby causes the instability of vaccine quality.
Flow cytometry method provided by the invention, the running time is in 30 minutes, and data are accurately objective, and strong operability can effectively overcome the drawback in current production of vaccine process.
Above-described embodiment is preferably embodiment of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under Spirit Essence of the present invention and principle, substitutes, combination, simplify; all should be equivalent substitute mode, within being included in protection scope of the present invention.
Claims (10)
1. the application of flow cytometry method in production of vaccine Real-Time Monitoring, is characterized in that: be that flow cytometry method is applied in production of vaccine process, detect the viable count in production of vaccine process;
Described vaccine comprises bacillus coli vaccine, StaphVAX, swine erysipelas vaccine, streptococcus vaccine, salmonella vaccine, clostridieum welchii vaccine, chicken virus mycoplasma vaccine, pasteurella multocida vaccine and haemophilus parasuis vaccine.
2. the application of flow cytometry method in production of vaccine Real-Time Monitoring according to claim 1, is characterized in that comprising following steps:
(1) select in fluorescent dye as described below at least two kinds dye object bacteria; Fluorescent dye is the dyestuff that can dye to living bacterial cells, the dyestuff that can dye to total bacteria count and the dyestuff that can dye to dead bacterial cell;
(2) then use the variation of flow cytometer bacterial detection fluorescence signal, by measuring the variation of bacterial fluorescence information, obtain FCM collection of illustrative plates; FCM collection of illustrative plates has at least two kinds in following parameter: viable count, dead bacterium number and total bacteria count; Obtain the ratio of viable count according to FCM collection of illustrative plates;
(3) obtain the concentration of object bacteria bacterium liquid by absolute counting method;
(4) concentration of the object bacteria bacterium liquid that the ratio of the viable count obtaining according to step (2) and step (3) obtain, obtains viable bacteria amount and the dead bacterium amount of object bacteria.
3. the application of flow cytometry method in production of vaccine Real-Time Monitoring according to claim 2, is characterized in that: step (1) is: select fluorescent dye as described below and dye object bacteria; Fluorescent dye is the combination that a kind of dyestuff with dyeing to dead bacterial cell in the dyestuff that can dye to living bacterial cells and the dyestuff that can dye to total bacteria count forms.
4. according to the application of flow cytometry method in production of vaccine Real-Time Monitoring described in claim 2 or 3, it is characterized in that:
The dyestuff that can dye to living bacterial cells described in step (1) is CFDA-SE; ;
The dyestuff that can dye to total bacteria count described in step (1) is SYBR Green I;
The dyestuff that can dye to dead bacterial cell described in step (1) is PI.
5. the application of flow cytometry method in production of vaccine Real-Time Monitoring according to claim 2, is characterized in that: the concentration that obtains object bacteria bacterium liquid by absolute counting method described in step (3) is counted by blood counting chamber or passed through Flow cytometry.
6. the application of flow cytometry method in production of vaccine Real-Time Monitoring according to claim 5, it is characterized in that: described is to realize as follows by Flow cytometry: the magnetic bead of concentration known and described object bacteria bacterium liquid are mixed, detect by flow cytometer, calculate the concentration of object bacteria bacterium liquid by following formula;
Formula is:
(bacterial cell number/magnetic bead number) * (in every pipe, total magnetic bead number/population of samples is long-pending) * extension rate=bacterial concentration;
Bacterial cell number refers to the bacterial cell number that certain hour in-flow cell instrument detects; Magnetic bead number refers to the magnetic bead number that certain hour in-flow cell instrument detects; In every pipe, total magnetic bead number refers to the magnetic bead sum in sample detection pipe; Extension rate refers to the extension rate of bacterium liquid.
7. according to the application of flow cytometry method in production of vaccine Real-Time Monitoring described in any one in claim 2,3,5 and 6, it is characterized in that comprising following steps:
I, the blank of setting up bacterial cell and fluorescence intensity compensation contrast, and determine the operating parameter of flow cytometer:
1. by object bacteria activation, be cultured to exponential phase; By a part of bacterium liquid deactivation wherein, the bacterium liquid of deactivation contrasts as dead bacterium; Undressed bacterium liquid is viable bacteria contrast;
2. two kinds of cells that with PBS, 1. step obtained wash respectively, then use PBS resuspended to OD
600be 0.002～0.006, obtain dead bacterium contrast bacterium liquid and viable bacteria contrast bacterium liquid;
3. obtain blank and the fluorescence intensity compensation contrast of bacterial cell:
A, viable bacteria is contrasted to bacterium liquid contrast with dead bacterium the mixed bacteria liquid up flow type cell instrument that bacterium liquid is mixed to get, by adjusting the photoelectric diode voltage of flow cytometer, to regulate the intensity of forward scattering light FSC and side scattered light SSC, establish door with SSC/FSC, determine the position of bacterial cell to be checked in flow cytometer image, in order to eliminate the autofluorescence of bacterium to be checked;
B, in viable bacteria contrast bacterium liquid, add SYBR Green I or the monochromatic fluorescent dye of CFDA-SE, vibration mixes, lucifuge reaction; Then up flow type cell instrument detects, with the SSC/FSC door of setting in steps A, the PMT voltage of adjusting SYBR Green I or CFDA-SE carries out fluorescence compensation, for removing from detected fluorescence signal the interference fluorescence signal causing because fluorescein emission spectrum is overlapping, the impact on testing result for quality, concentration and the colouring method of getting rid of dyestuff simultaneously, obtain the compensation contrast figure of SYBR Green I or CFDA-SE, determine the voltage of SYBR Green I or CFDA-SE fluorescence intensity; In dead bacterium contrast bacterium liquid, add the monochromatic fluorescent dye of PI, vibration mixes, lucifuge reaction; Then up flow type cell instrument detects, and with the SSC/FSC door of setting in steps A, the PMT voltage of adjusting PI carries out fluorescence compensation, obtains PI compensation contrast figure, determines the voltage of PI fluorescence intensity; Contrast the composite fluorescent dye that adds a kind of and PI in SYBR Green I and CFDA-SE to form in the mixed bacteria liquid that bacterium liquid is mixed to get with dead bacterium at viable bacteria contrast bacterium liquid, vibration mixes, and lucifuge is reacted; Then up flow type cell instrument detects, and with the SSC/FSC door of setting in steps A, the PMT voltage of adjusting composite fluorescent dye carries out Two Colour Fluorescence compensation, obtains composite fluorescent dye compensation contrast figure, determines the voltage of composite fluorescent dye fluorescence intensity;
The monitoring of II, bacterium liquid sweat
1. in the different phase of production of vaccine, get zymocyte liquid sample;
2. PBS washing zymocyte liquid sample, then uses the resuspended zymophyte of PBS to OD
600be 0.002～0.006; By two kinds of fluorescent dyes on the bacterium liquid mark obtaining, the fluorescent dye using is identical with step I, and the voltage of use is the 3. final definite voltage of B of step I; Flow cytometer detects, and after picture door hives off, obtains each group of ratios;
3. by the magnetic bead of concentration known and zymocyte liquid sample mix, detect by flow cytometer, calculate the concentration of zymocyte liquid by following formula;
Formula is:
(bacterial cell number/magnetic bead number) * (in every pipe, total magnetic bead number/population of samples is long-pending) * extension rate=bacterial concentration;
4. according to the concentration of each group of ratios and zymocyte liquid, the viable bacteria in zymophyte and dead bacterium are carried out quantitatively.
8. the application of flow cytometry method in production of vaccine Real-Time Monitoring according to claim 7, is characterized in that:
The step of the deactivation of step I described in is 1.: by centrifugal bacterium liquid, get precipitation; Working concentration is to boil 5～10min after the ethanolic solution of mass volume ratio 70% disperses precipitation standing at least 1 hour or adds PBS to be uniformly dispersed;
The PBS of step I described in is 2. the PBS of pH6.2～7.4,0.01～0.1mol/L;
The time of the lucifuge reaction of step I described in is 3. 5min at least;
The particle diameter of the magnetic bead of Step II described in is 3. 5.5 μ m.
9. the application of flow cytometry method in production of vaccine Real-Time Monitoring according to claim 7, is characterized in that:
The consumption of the SYBR Green I of step I described in is 3. that 0.1～20 μ M calculates by the final concentration in bacterium liquid;
The consumption of the CFDA-SE of step I described in is 3. that 0.1～20 μ M calculates by the final concentration in bacterium liquid;
The consumption of the PI of step I described in is 3. 0.1～10 μ g.mL by the final concentration in bacterium liquid
-1calculate.
10. the application of flow cytometry method in production of vaccine Real-Time Monitoring according to claim 9, is characterized in that:
The lucifuge reaction time of described SYBR Green I is 10min;
The lucifuge reaction time of described CFDA-SE is 10min;
The time of the lucifuge reaction of described PI is 5min.
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