CN104830950B - A method of quickly judging mycobacterium paratuberculosis existing state in milk and dairy produce using BLU-V PMA technologies - Google Patents
A method of quickly judging mycobacterium paratuberculosis existing state in milk and dairy produce using BLU-V PMA technologies Download PDFInfo
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Abstract
The invention discloses a kind of methods quickly judging mycobacterium paratuberculosis existing state in milk and dairy produce using BLU-V PMA technologies.It has been announced according to GenBankMap's（Accession number：AF286339）ISMAV2 genome sequences choose the conserved sequence of the gene, design TaqMan fluorescence probes and primer.The advantages of fluorescent quantitative PCR technique is combined in PMA technologies using BLU-V systems as light source applications, has been successfully established in a kind of quick detection milk and dairy produceMAPThe method of viable bacteria.This method is easy to operate, quick and more accurate.
Description
Technical field
A kind of application BLU-V PMA technologies of the present invention quickly judge mycobacterium paratuberculosis existing state in milk and dairy produce
Method, belong to technical field of microbial detection.
Background technology
Johne's disease（Paratuberculosis）It is by mycobacterium paratuberculosis, i.e. mycobacterium avium perituberculosis subspecies
（Mycobacteriumaviumsubsp.Paratuberculosis,MAP）It is caused a kind of chronic based on ruminant
Consume sexually transmitted disease, also referred to as Johne, s disease.The disease be difficult purification, and with the Chron of the mankind（Crohn,s）Disease exists latent
Contact, some researches show that this serious disease of people may be since the pasteurization of milk can not kill this completely
Bacterium, and find that it is the positive that perituberculosis bacterium is all detected in almost 44% dairy produce in studying.Not with China's national life level
Disconnected to improve, milk demand increases in positive exponent, is badly in need of a large amount of high-quality, high yield cow, promotes import kind ox abruptly increase in recent years,
There is an urgent need to establish the fast and accurately johne's disease diagnostic method of early stage, with prevention, purification and the disease is eradicated, protects China
The healthy and rapid development of animal husbandry.
The common methods of detection mycobacterium paratuberculosis have bacteriodiagnosis, immunology diagnosis, molecular biology to examine at present
It is disconnected.Wherein smear for microscopic examination method cannot identify whether the pathogen in the infected's body survives, and bacterial cultivation, which exists, to be difficult to cultivate
And time-consuming at least 4-6 weeks, drug sensitive test also takes long problem.Complement fixation test（CF）Although to there is clinical symptoms
Ill domestic animal recall rate it is higher, but for latent infection case when it is ineffective.Agar gel diffusion test（AGID）Method sensibility is low,
It should not be used as screening and identifying subclinical infection case.Enzyme-linked immunosorbent assay（ELISA）Compare it is sensitive, hold promise as often
Diagnosis.In recent years, Nogva et al. distinguished viable bacteria and dead bacterium using EMA-PCR technologies.But EMA has centainly
Toxicity, if the cell membrane that will be embedded in living cells with cytosis time slightly length is combined with its DNA, to influence, detection knot
Fruit.Single folded aziridine（propidium mono azide,PMA）It can be combined with DNA by the dead bacterium of cell membrane breakage,
So that dead bacterium DNA is no longer expanded in PCR, and then detects the signal of viable bacteria.The profits such as Tomas Garc a-Cayuela [53]
Quantitative detection is carried out to four kinds of lactic acid bacterias in dairy products with PMA-qPCR methods, PMA-PCR technologies are most to apply at present
The living stems technology of foreground.Current non-someone carries out perituberculosis branch bar using BLU-V PMA combination fluorescent quantitative PCR techniques
The quick judgement of the viability of bacterium.
However, light source type is also a key factor for influencing PMA processing, to PMA processing in previous experiment
When, used light source is all the halogen lamp of 600W, although its cost is reasonable, illuminance is abundant, it reaches peak brightness
Time it is short, and influenced by air dielectric and sample turbidity, the treatment effect of PMA can all reduced, experiment is caused to occur
The result of false positive and false negative.
Invention content
Overcome the deficiencies in the prior art of the present invention, the technical problem to be solved is that using BLU-V systems as light source applications
In PMA technologies, in conjunction with the advantages of fluorescent quantitative PCR technique, it has been successfully established in a kind of quick detection milk and dairy produceMAPIt is living
The method of bacterium.
In order to solve the above technical problems, the technical solution adopted in the present invention is：A kind of application BLU-V PMA technologies are quick
The method for judging mycobacterium paratuberculosis existing state in milk and dairy produce, using BLU-V systems as light source applications in PMA technologies
In；
The described mycobacterium paratuberculosis in PMA technology determinations milk and dairy produce using BLU-V systems as light source applications
The method of existing state includes the following steps：
A takes sample to be tested, fluid sample directly to centrifuge, and solid sample adds distillation water dissolution centrifugation, 14500r to centrifuge 5min；
It after centrifugation, discards supernatant, EBBuffer is added in precipitation, mixing obtains bacteria suspension；
B is into the bacterium solution of step a plus list folds aziridine, makes the final concentration of 10 μ g/mL of single folded aziridine, mixes well,
And it sets and is not added with the bacteria suspension of PMA as a contrast；EP pipes are placed in avoid light box, 10min is protected from light；EP pipes are taken out, are put it into
In BLU-V systems (light intensity of instrument is 100), 20min is acted on, PMA and the DNA double chain of dead bacterium is made to be combined；It is taken out every 2min
EP pipes vibrate suspension once in vortex oscillation instrument, to increase Percentage bound；
After step c b, EP pipes are taken out, 14500r centrifuges 5min；After centrifugation, discard supernatant（Pay attention to abandoning and fill
Point）；It is eventually adding the TE of 400 μ L, mixing is resuspended again；It is follow-up to carry out conventional DNA extractions；
D carries out the object bacteria in fluorescence quantitative PCR detection sample using the DNA extracted in step c as template, and upstream and downstream is drawn
Object and probe sequence are respectively
Upstream：5'-AACGAGGGGACGATGAGG-3’
Downstream：5'-CTGTGATGTAACGCGATTCG-3’
5 '-FAM-CACGGCGTTGCTGATGTCCACC-TAMRA of probe
Quantitative fluorescent PCR reaction system is 10 × PCRBuffer500mM, and Kcl, 100mMTris-cl (pH8.3) are at room temperature
Each 1.0 μ of each 3.2 μm of ol2.0 μ L, upstream and downstream primer 10pmol of 2.5 μ L, Mgcl225mM1.5 μ L, dATP, dCTP, dTTP, dGTP
L, TaqDNA enzyme 5U/ μ L2.5 μ L, probe 3pmol1.0 μ L, template DNA 2.0 μ L, ddH2O11.5 μ L, 25.0 μ of overall reaction system
L。
A kind of application BLU-V PMA technologies quickly judge mycobacterium paratuberculosis existing state in milk and dairy produce
Method, quantitative fluorescent PCR reaction condition：93 DEG C of denaturation 15s；56 DEG C of annealing 15s, 68 DEG C of extension 30s, 40 recycle.
A kind of application BLU-V PMA technologies quickly judge mycobacterium paratuberculosis existing state in milk and dairy produce
Method, when determining the Ct value < 25 of TaqMan fluorescence quantitative PCR detections, result judgement be the positive；With without PMA processing group phases
Than, PMA treated Ct values than being greater than 6 or more without the Ct values at PMA, judge not containing viable bacteria in milk sample.
Compared with prior art the invention has the advantages that.
Compared with all halogen lamp of 600W of used light source when previous PMA is handled, and it is used in the present invention
BLU-V systems are a kind of laser thermostabilization equipment that can be continuously generated stable light stimulus amount, i.e., the equipment has specific wave
Long and continual and steady laser irradiation intensity, detection sample can be placed in confined space simultaneously to 12 parts by PMA by it
The sample of reason carries out photochemical excitation reaction.The influence of air dielectric and sample turbidity is not only avoided, and improves sample inspection
The efficiency of survey.
The present invention uses BLU-V systems, in conjunction with the advantages of PMA and fluorescent quantitative PCR technique, has been successfully established a kind of quick
It detects in milk and dairy produceMAPThe method of viable bacteria.This method is easy to operate, quick and more accurate.And make milk and milk
Whether product meets an important indicator of bio-safety, to prevention, purification and removes the disease, protects our people's life security
Have great importance with the healthy and rapid development of animal husbandry.
Description of the drawings
Fig. 1 is the fluorescent quantitation AFLP system in embodiment 1；
The corresponding fluorescent quantitation ct values of different light application time processing in Fig. 2 embodiments 2；
The corresponding fluorescent quantitation ct values of different quality concentration PMA processing in Fig. 3 embodiments 2；
Fig. 4 embodiments 3 are handled through PMA and the MAP reference culture P18 fluorescent quantitation AFLP systems without PMA processing；
Fig. 5 embodiments 3 are handled through PMA and the MAP reference culture P18 fluorescent quantitation Ct values without PMA processing are reported；
Fig. 6 embodiments 3 are handled through PMA and the MAP reference culture P10 fluorescent quantitation AFLP systems without PMA processing；
Fig. 7 embodiments 3 are handled through PMA and the MAP reference culture P10 fluorescent quantitation Ct values without PMA processing are reported.
Specific implementation mode
Below in conjunction with specific embodiment, the invention will be further described.
Embodiment 1
Primer specificity is verified
Extract Map（P10 and P18）, staphylococcus aureus, salmonella, Escherichia coli, 6 kinds of standards of Mycobacterium graminis
Strain gene group DNA carries out TaqMan fluorescent quantitative PCRs, detects the specificity of the method for the present invention.（Perituberculosis branch bar
Bacterium（M.paratuberculosis）Reference culture：P18（The U.S.）, P10（Japan）（All it is ox type perituberculosis reference culture）,
It is provided by Jilin veterinary institute.Staphylococcus aureus, salmonella, Escherichia coli, Mycobacterium graminis are come in and gone out by Shanxi
Border inspection and quarantine bureau technique center laboratory preserves.）
The preparation of 1.DNA
50 μ L bacterium solutions are taken, are added in 400 μ L TE, mixing.Extraction DNA profiling is as follows：
（1）80 DEG C of 20 min of heating water bath, are cooled to room temperature, are allowed to inactivate；
（2）Add 50 μ L lysozymes, 37 DEG C of 1 h of gas bath shake culture；
（3）Add 75 μ L SDS/ Proteinase Ks, mixing, 65 DEG C of 10 min of heating water bath；
（4）The CTAB/Nacl for adding 65 DEG C of 5 min of preheating of Nacl and 100 μ L of 100 μ L, 5 mol/L, turns upside down
Mixing, until liquid becomes white（It is creamy）, 65 DEG C of 10 min of heating water bath；
（5）Add 750 μ L chloroforms: isoamyl alcohol（24:1）, mixing, 12000 r/min, 5 min of room temperature centrifugation；
（6）Take upper strata aqueous phase（Supernatant）In Yu Xinguan, add isometric chloroform: isoamyl alcohol（24:1）, mixing,
12000 r/min, room temperature centrifuge 5 min；
（7）It takes upper strata aqueous phase in new pipe, is carefully added into the isopropanol of 0.6 volume, precipitate nucleic acids, small hand mixing, -20
DEG C place 30 min, 12000 r/min, room temperature centrifuge 15 min；
（8）Supernatant to be abandoned, 70% ethyl alcohol of 500 μ L precoolings, 12000 r/min is added, room temperature centrifuges 5 min,；
（9）Liquid carefully is siphoned away, dries 10 min or so at room temperature, with 50 μ L TE dissolvings, 4 DEG C of preservations.
2.TaqMan quantitative fluorescent PCRs reaction system and cycling condition
Quantitative fluorescent PCR reaction system is 10 × PCRBuffer500mM, and Kcl, 100mMTris-cl (pH8.3) are at room temperature
Each 1.0 μ of each 3.2 μm of ol2.0 μ L, upstream and downstream primer 10pmol of 2.5 μ L, Mgcl225mM1.5 μ L, dATP, dCTP, dTTP, dGTP
L, TaqDNA enzyme 5U/ μ L2.5 μ L, probe 3pmol1.0 μ L, template DNA 2.0 μ L, ddH2O11.5 μ L, 25.0 μ of overall reaction system
L。
Quantitative fluorescent PCR reaction condition：93 DEG C of denaturation 15s；56 DEG C of annealing 15s, 68 DEG C of extension 30s, 40 recycle.
The results are shown in Figure 1, only uses Map（P10 and P18）Template DNA can just obtain specific amplification curve, and
The template DNA of staphylococcus aureus, salmonella, Escherichia coli, Mycobacterium graminis cannot obtain specific amplification curve, table
The specificity of bright this method is good.
Embodiment 2
PMA acts on the optimization of MAP reference culture optimum reaction conditions
1. MAP reference cultures（P10 and P18）The preparation of membrane damage bacterium
P10 the and P18 bacterium solutions of 1 mL are taken respectively（It is as possible to take fungus block more）In the 2 mL EP pipes for being placed with 50 magnetic beads, and
It is sealed with sealed membrane, is put into instrument for extracting nucleic acid（Every 5 s effects are primary）, bacterium solution is made fully to be suspended；After suspension, it is placed in 80 DEG C of water
20 min are inactivated in bath.
2. PMA is to MAP reference cultures（P10 and P18）Processing
By the PMA of 0.5 mg be dissolved in 1 mL without the PMA stostes in RNA enzyme water, being made into 0.5 mg/mL, -20 DEG C of guarantors
It deposits.The bacterium solution inactivated is placed in a centrifuge, 14500 r centrifuge 5 min；After centrifugation, discard supernatant；Then it is added
The EB Buffer of 500 μ L are in remaining thalline, mixing.
The optimization of 3 .PMA mass concentrations
Inactivated in bacteria suspension to 0.5 mL and a certain amount of PMA be added, make its final concentration be respectively 0.0,0.5,1.0,3.0,
5.0,10.0,20.0 μ g/mL, and set and be not added with the inactivation bacteria suspension of PMA as a contrast.After handling 10 min in avoid light box, use
BLU-V systems（Light intensity is 100）20 min of photo-irradiation treatment takes out EP pipes every 2 min and vibrates suspension one in vortex oscillation instrument
It is secondary, after, EP pipes are taken out, 14500 r centrifuge 5 min, discard supernatant, and the TE of 400 μ L is added, carries out conventional DNA and carries
It takes, best PMA mass concentrations is determined by TaqMan quantitative fluorescent PCRs.
The optimization of 4 .PMA photo-irradiation treatment times
PMA is added in 0.5 mL inactivates bacteria suspension, makes its final concentration of 10 μ g/mL.After being protected from light 10 min of processing, use
BLU-V systems (light intensity 100) are to 0,5,10,15,20,25,30 min of bacterium solution difference photo-irradiation treatment.EP is taken out every 2 min
Pipe vibrates suspension once in vortex oscillation instrument, after, EP pipes are taken out, 14500 r centrifuge 5 min, discard supernatant, and are added
The TE of 400 μ L carries out conventional DNA extractions, best PMA mass concentrations is determined by TaqMan quantitative fluorescent PCRs.
As seen from Figure 2, Ct values constantly increase with the extension of light application time, when light application time is more than 20 min
When, the amplitude of Ct values variation is smaller and smaller, illustrates when light application time is more than 20 min, and PMA can be tied with dead bacterium DNA completely
It closes, and the PMA to dissociate in milk sample is made to be passivated completely, therefore, this experiment is using 20 min as optimum illumination processing time.
As can be known from Fig. 3, viable bacteria（Unused PMA processing）Fluorescent quantitation reaction Ct values be basically unchanged, illustrate that PMA does not influence
The amplification of viable bacteria, and with the increase of PMA mass concentrations, hot inactivated bacteria（Dead bacterium）The Ct values of TaqMan quantitative fluorescent PCRs are also shown
It writes and increases, when the mass concentration of PMA increases to 10 μ g/mL, Ct values start to keep stablizing, gradual with the increased amplitude of concentration
Reduce, therefore, in order to make the PMA to dissociate in bacterium solution be passivated completely, this experiment is with 10 μ g/mL for best PMA reaction densities.
Embodiment 3
Best PMA reaction conditions stimulate the secretion of milk and dairy produce in viable bacteria quantitative detection
1. in artificial contamination's MAP milk samples（P10 and P18）The preparation of membrane damage bacterium
P10 the and P18 bacterium solutions of 1 mL are taken respectively（It is as possible to take fungus block more）In the 2 mL EP pipes for being placed with 50 magnetic beads, and
It is sealed with sealed membrane, is put into instrument for extracting nucleic acid（Every 5 s effects are primary）, it is in bacteria suspension so that fungus block is fully suspended；Sterile working takes
Be not detected the negative milk sample of MAP using fluorescent PCR, regular-PCR and electrophoresis, i.e. Yoghourt respectively takes 1 mL, milk powder respectively take 0.5 g in
In 2 mL EP pipes of sterilizing；500 μ L bacteria suspensions are respectively added into acquired milk sample, are mixed well in vortex oscillation instrument；
After mixing, it is placed in 80 DEG C of water-baths and inactivates 20 min.By the processing, make the cell wall of strain journey different from cell membrane generation
The damage of degree prepares pasteurized milk to reach artificial（One is to 62 ~ 65 DEG C, keep Milk During Heating 30 minutes.Second
Milk During Heating to 75 ~ 90 DEG C, is kept the temperature 15 ~ 16 seconds by method）Purpose.
2. PMA is in artificial contamination's MAP milk samples（P10 and P18）The processing of membrane damage bacterium
The milk sample inactivated is placed in a centrifuge, 14500 r centrifuge 5 min；After centrifugation, discard supernatant；Then
The EB Buffer of 500 μ L are added in remaining thalline, mixing；In the bacterium solution with EB Buffer mixings, 10 μ g/mL are added
PMA is mixed well, and is set and be not added with the bacterium solution of PMA as a contrast；EP pipes are placed in avoid light box, 10 min are protected from light；Take out EP
Pipe puts it into BLU-V systems (light intensity of instrument is 100), acts on 20 min, PMA and the DNA double chain of dead bacterium is made to be combined；
EP pipes are taken out every 2 min and vibrate suspension in vortex oscillation instrument once, to increase Percentage bound；After, EP pipes are taken out,
14500 r centrifuge 5 min；After centrifugation, discard supernatant（Pay attention to abandoning fully）；It is eventually adding the TE of 400 μ L, is weighed again
Outstanding mixing；It is follow-up to carry out conventional DNA extractions.
The extraction of 3.DNA templates is the same as embodiment 1
4.TaqMan quantitative fluorescent PCRs reaction system and cycling condition are the same as embodiment 1
PMA acts on MAP reference cultures（P10 and P18）TaqMan PCR results such as Fig. 4,5,6,7 afterwards.From Fig. 4 and Fig. 6
As it can be seen that P10 and P18 has obtained good amplification curve by TaqMan quantitative fluorescent PCRs；In Fig. 5 and Fig. 7, by PMA processing
MAP reference cultures afterwards（P10 and P18）The Ct values of bacterial strain of the Ct values than being handled without PMA be greater than 6 or more, illustrate MAP
Viable bacteria and dead bacterium obtained good differentiation, and PMA is obtained in the preprocessing process to MAP with the dead bacterium cell of the bacterium
Effective combination.
The present invention can be summarized with others without prejudice to the concrete form of the spirit or essential characteristics of the present invention.Therefore, nothing
By from the point of view of which point, the embodiment above of the invention can only all be considered the description of the invention and cannot limit invention,
Claims indicate the scope of the present invention, and above-mentioned explanation does not point out the scope of the present invention, therefore, with the present invention
The comparable meaning and scope of claims in any variation, be all considered as being included within the scope of the claims.
Claims (3)
1. a kind of method quickly judging mycobacterium paratuberculosis existing state in milk and dairy produce using BLU-V PMA technologies,
It is characterized in that using BLU-V systems as light source applications in PMA technologies；
The described mycobacterium paratuberculosis in PMA technology determinations milk and dairy produce using BLU-V systems as light source applications is survived
The method of state includes the following steps：
A takes sample to be tested, fluid sample directly to centrifuge, and solid sample adds distillation water dissolution centrifugation, 14500r to centrifuge 5min；Centrifugation
After, it discards supernatant, EB Buffer is added in precipitation, mixing obtains bacteria suspension；
B is into the bacterium solution of step a plus list folds aziridine, makes the final concentration of 10 μ g/mL of single folded aziridine, mixes well, and set
It is not added with the bacteria suspension of PMA as a contrast；EP pipes are placed in avoid light box, 10min is protected from light；EP pipes are taken out, BLU-V systems are put it into
In system, the light intensity of instrument is 100, acts on 20min, PMA and the DNA double chain of dead bacterium is made to be combined；EP pipes are taken out in whirlpool every 2min
Oscillation is suspended primary on the shaker of whirlpool, to increase Percentage bound；
After step c b, EP pipes are taken out, 14500r centrifuges 5min；After centrifugation, discard supernatant；It is eventually adding 400 μ L's
Mixing is resuspended in TE again；It is follow-up to carry out conventional DNA extractions；
D using the DNA extracted in step c as template, carry out fluorescence quantitative PCR detection sample in object bacteria, upstream and downstream primer and
Probe sequence is respectively
Upstream：5'-AACGAGGGGACGATGAGG-3’
Downstream：5'-CTGTGATGTAACGCGATTCG-3’
5 '-FAM-CACGGCGTTGCTGATGTCCACC-TAMRA of probe
Quantitative fluorescent PCR reaction system is 10 × PCR Buffer:500mM KCl, 100mMTris-cl, pH8.3 are at room temperature
2.5 μ L, MgCl2Each 3.2 μm of ol of 1.5 μ L, dATP, dCTP, dTTP, dGTP of 25mM, 2.0 μ L, upstream and downstream primer 10pmol are each
1.0 μ L, TaqDNA enzyme 5U/ μ 1.0 μ L of L, 2.5 μ L, probe 3pmol, template DNA 2.0 μ L, ddH211.5 μ L of O, overall reaction body
It is 25.0 μ L.
2. a kind of application BLU-V PMA technologies according to claim 1 quickly judge perituberculosis branch in milk and dairy produce
The method of bacillus existing state, it is characterised in that quantitative fluorescent PCR reaction condition：93 DEG C of denaturation 15s；56 DEG C of 15s that anneal, 68 DEG C
Extend 30s, 40 cycles.
3. a kind of application BLU-V PMA technologies according to claim 1 quickly judge perituberculosis branch in milk and dairy produce
The method of bacillus existing state, which is characterized in that when determining the Ct value < 25 of TaqMan fluorescence quantitative PCR detections, result judgement
For the positive；Compared with without PMA processing groups, PMA treated Ct values are sentenced than being greater than 6 or more without the Ct values at PMA
Determine not containing viable bacteria in milk sample.
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| CN101168783A (en) * | 2007-11-06 | 2008-04-30 | 广东出入境检验检疫局检验检疫技术中心 | Paratuberculosis fluorescence PCR rapid diagnosis kit |
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| CN101168783A (en) * | 2007-11-06 | 2008-04-30 | 广东出入境检验检疫局检验检疫技术中心 | Paratuberculosis fluorescence PCR rapid diagnosis kit |
Non-Patent Citations (3)
| Title |
|---|
| 奶及奶制品中副结核分枝杆菌存活状态快速鉴定方法的建立;梁慧霞;《中国优秀硕士学位论文全文数据库 农业科技辑》;20160215;D050-220 * |
| 牛副结核分枝杆菌实时荧光定量PCR检测方法的建立;王素华等;《中国畜牧兽医》;20121231;第39卷(第10期);22-26 * |
| 病原体分子检测与食品安全解决方案;李娜;《新发传染病防治热点研讨会论文集》;20141231;第66页 * |
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