CN110343744A - A kind of brucella viable count method based on PMA-qPCR technology - Google Patents
A kind of brucella viable count method based on PMA-qPCR technology Download PDFInfo
- Publication number
- CN110343744A CN110343744A CN201910801583.0A CN201910801583A CN110343744A CN 110343744 A CN110343744 A CN 110343744A CN 201910801583 A CN201910801583 A CN 201910801583A CN 110343744 A CN110343744 A CN 110343744A
- Authority
- CN
- China
- Prior art keywords
- brucella
- pma
- qpcr
- sample
- detection
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention provides a kind of brucella viable count methods based on PMA-qPCR technology, belong to microorganism count technical field.The present invention optimizes concentration for the treatment of and the time for exposure of PMA processing brucella S2, detects brucella BCSP31 gene, the method for establishing PMA-qPCR quantitative detection brucella viable bacteria in conjunction with qPCR method.Sensitivity using PMA-qPCR detection brucella is 100 times of regular-PCR, accurate quantitative analysis can be carried out to brucella viable count, and PMA processing does not interfere qPCR reaction.Counting brucella viable count with PMA-qPCR method has the characteristics that quick, easy, specificity is preferable.Compared with plate technique method, the measurement of brucella viable count can not only be rapidly completed, but also the quantitative and qualitative detection of brucella can be completed at the same time.
Description
Technical field
The invention belongs to microorganism count technical fields, and in particular to a kind of brucella based on PMA-qPCR technology is living
Bacterium method of counting.
Background technique
Brucellosis (referred to as " cloth disease ", Brucellosis) is a kind of Amphixenosis as caused by brucella, can
Infect the mankind, a variety of domestic animals and wild animal.Infected animal shows night sweat, arthralgia, pregnant female miscarriage, male animal testis
The clinical symptoms such as inflammation, lead to huge economic loss and serious public health problem, are classified as Category B notifiable disease by China.
Conventional cloth disease detects mainly by the methods of bacteriology and serology, and time-consuming for these methods, has certain vacation
It is positive.Currently, Protocols in Molecular Biology is widely used to pathogenic bacteria detection the features such as quick with its, accurate.With cloth Lu Shi
Bacterium disease research is goed deep into, and discussion brucella survival condition, quantity and equilibrium relation of host in host is more and more important,
With the presence or absence of brucella living and its quantity in especially accurate analysis machine body, to the effect of the infection of assessment cloth disease or vaccine immunity
It is even more important.The method of brucella viable count is mainly based on plate count in previous measurement host, but this method it is time-consuming,
Heavy workload, error are larger.Therefore it is badly in need of researching and developing a kind of measurement side of quick, accurate, easy brucella viable count intracellular
Method, in favor of further studying brucella pathogenic mechanism and its effective prevention and control.
The third ingot of nitrine bromination (propidium monoazide, PMA) be it is a kind of can be with the dyestuff in conjunction with DNA molecular, the dye
Material can enter the ditch of DNA molecular, make that PMA decomposition generates a kind of nitrence substance and DNA molecular occurs under the action of light
Covalent cross-linking reaction, this covalent cross-linked thing can inhibit the PCR amplification of DNA.Viable bacteria has complete cell membrane, and film is damaged thin
Bacterium is due to not complete membrane structure, so being difficult to survive.Damaged cell film is penetrated in conjunction with dead bacterium DNA using PMA, inhibits its expansion
Increase, to realize the detection to viable bacteria.The researchs such as NockerA have shown that PMA excessive concentration has toxicity to living cells, to influence
The DNA cloning of living cells, while the PMA to dissociate in the sample not in conjunction with DNA can detect qPCR and impact, it can be through overexposure
Light makes it inactivate and be passivated, so needing to optimize PMA working concentration and time for exposure.
PMA-qPCR detection technique is usually used in the living stems of various microorganism at present, such as CN107354228A
Patent discloses the method for detecting VBNC vibrio parahemolyticus in freezing matrix using PMA-qPCR method, and CN108504753A is disclosed
A kind of method detecting staphylococcus aureus live bacteria in milk, CN109321666A disclose SDS-PMA-qPCR method detection cream
The method of middle Streptococcusagalactiae viable bacteria has the characteristics that detection sensitivity height and specificity are good using PMA-qPCR detection technique,
Operating procedure is simplified simultaneously, detection time is shortened, reduces testing cost.But different types of microorganism live bacteria is examined
Survey in PMA processing method and PCR context of detection there are species variation, there is presently no using PMA-qPCR detection technique to cloth Shandong
The report that Salmonella viable bacteria is detected.
Summary of the invention
In view of this, the purpose of the present invention is to provide a kind of brucella count plate sides based on PMA-qPCR technology
Method realizes viable bacteria accurate counting.
The present invention provides a kind of brucella viable count methods based on PMA-qPCR technology, comprising the following steps:
1) sample suspension is made in sample；
2) PMA is added in Xiang Suoshu sample suspension obtains final concentration of 15~19 μ g/mL, 10~25min of exposure-processed
To the sample suspension of PMA processing；
3) microbe genome DNA in the sample suspension of the PMA processing is extracted；
4) using the microbe genome DNA as template, it is fixed that real-time fluorescence is carried out for brucella BCSP31 gene
Measure PCR detection；
5) the CT value by real-time fluorescence quantitative PCR detection substitutes into standard curve regression equation, obtains in sample suspension
Brucella viable bacteria concentration.
Preferably, in step 2) PMA final concentration of 15~17 μ g/mL.
Preferably, the time of exposure-processed is 10~15min in step 2).
Preferably, the real-time fluorescence quantitative PCR detection of brucella BCSP31 gene includes with amplimer in step 4)
The upstream primer as shown in sequence table SEQ ID No.1 and the downstream primer as shown in sequence table SEQ ID No.2.
Preferably, the response procedures of the fluorescence quantitative PCR detection of brucella BCSP31 gene are 94 DEG C pre- in step 4)
It is denaturalized 2min；Then 40 recycle: 94 DEG C of denaturation 45s, 55 DEG C of annealing 30s, 72 DEG C extend and acquire fluorescence signal 32s.
Preferably, the reaction system of the fluorescence quantitative PCR detection of brucella BCSP31 gene is 20 μ L in step 4):
Fast StartUniversal SYBR GreenMaster (Rox) 10 μ L, 2 μ L of template, upstream and downstream primer each 1 μ L, ddH2O is mended
It is charged to 20 μ L.
Preferably, step 5) standard curve regression equation is as follows: y=﹣ 3.492x+36.303, wherein y represents CT value, x
Represent the logarithm of gene copy Particle density；R2=0.999.
Preferably, sample described in step 1) includes non-human or animal body source sample；
Preferably, described non-human or animal body source sample includes bacterium solution containing brucella, culture cell, fermentation
Liquid, water environment sample, soil sample or food sample.
The present invention provides a kind of brucella viable count method based on PMA-qPCR technology, by PMA-qPCR and
Common qPCR tests discovery to the contrasting detection of various concentration brucella S2, when bacterial concentration is 103~108CFU/mL range
Interior, brucella S2 is handled through PMA and untreated CT value is coincide substantially, all has good linear relationship (R2Respectively
0.986 and 0.991), sensitivity 103CFU/mL, and its sensitivity is detected 10 by regular-PCR5CFU/mL, it is seen that PMA-
The sensitivity of qPCR detection brucella S2 is 100 times of regular-PCR, and accurate quantitative analysis can be carried out to brucella viable count, and
QPCR reaction is not interfered, more accurately count results can be obtained.In addition, by PMA-qPCR method and colony counting method
Living stems result to be compared, discovery analyzes pure bacterium solution with two methods, and the gained bacterial concentration order of magnitude matches, and
Without the significant meaning of statistical difference.Viable count intracellular is detected attacking bacterium cell A and B group, the detection of PMA-qPCR method
Attack the intracellular viable count of bacterium and be significantly higher than colony counting method viable count, may can not be counted due to colony counting method it is intracellular it is non-can
The work bacterial population of cultivation conditions.In addition, the viable bacteria of known concentration/dead bacterium is mixed by different proportion, PMA-qPCR is carried out
Detection, while being compared so that the sample of PMA processing is not added, the results showed that PMA-qPCR on living stems without influence, and can be to work
Bacterium carries out accurate quantitative analysis.
Meanwhile using method of counting provided by the invention, detected using brucella BCSP31 gene as specific gene,
Brucella and Escherichia coli, salmonella, yersinia enterocolitica, vibrio parahaemolytious can be subjected to specific area
Point；With brucella bacterial strain (the brucella S2 low virulent strain, 2308 plants and A19 plants of B. abortus, sheep kind in source not of the same race
16M plants and M5 plants of brucella, sheep epididymis kind brucella are as brucella representative strains) it is test object, as a result, it has been found that
With PMA-qPCR method energy specific amplification brucella bacterial strain not of the same race, it is not of the same race to illustrate that method provided by the invention is suitable for
The counting of brucella bacterial strain.
Detailed description of the invention
Fig. 1 is the optimization of PMA treatment conditions, and wherein Fig. 1-A is influence of the different exposure time to amplification；Fig. 1-B is difference
The PMA of concentration acts on dead bacterium；Fig. 1-C is that the PMA of various concentration acts on viable bacteria；
Fig. 2 is the sensitivity that PMA-qPCR detects brucella S2；Fig. 2-A is that PMA-qPCR and common qPCR detection is different
The standard curve of the brucella S2 of concentration；Fig. 2-B is the sensitivity that regular-PCR detects brucella S2, and wherein M is indicated:
DL2000 DNAMarker；1~10 indicates: 108~10-1CFU/mL；
Fig. 3 be PMA-qPCR detection method specificity verification, wherein Fig. 3-A:PMA-qPCR specific amplification pig kind S2,
B. abortus 2308, B. abortus A19, brucella melitensis M5, brucella melitensis 16M, sheep epididymis kind cloth
Shandong Salmonella；Fig. 3-B:PMA-qPCR detection method expands pig kind brucella S2, B. abortus 2308, B. abortus
A19, brucella melitensis M5, brucella melitensis 16M, sheep epididymis kind brucella solubility curve；Fig. 3-C: regular-PCR
Confirm the specificity of PMA-qPCR method；Wherein M.DL5000 DNAMarker；1. pig kind brucella S2；2. ox kind cloth Lu Shi
Bacterium 2308；3. B. abortus A19；4. brucella melitensis M5；5. brucella melitensis 16M；6. sheep epididymis kind cloth Shandong
Salmonella；The specificity that Fig. 3-D:PMA-qPCR method detects different microorganisms, wherein M.DL2000 DNAMarker；1. pig kind
Brucella S2；2. Escherichia coli；3. salmonella；4, yersinia enterocolitica；5, vibrio parahaemolytious；6, nothing
RNase water；
Fig. 4 is that PMA-qPCR and qPCR detects the S2 viable bacteria of different proportion；
Fig. 5 is that brucella S2 expands electrophoretogram, wherein M.2000DNAMarker；1. brucella S2 genome；
Fig. 6 is the standard curve that qPCR detects recombinant plasmid pMD-18T-BCSP31；
Fig. 7 is PMA-qPCR method and colony counting method to living stems (unit: CFU/mL).
Specific embodiment
The present invention provides a kind of brucella viable count methods based on PMA-qPCR technology, comprising the following steps:
1) sample suspension is made in sample；
2) PMA is added in Xiang Suoshu sample suspension obtains final concentration of 15~19 μ g/mL, 10~25min of exposure-processed
To the sample suspension of PMA processing；
3) microbe genome DNA in the sample suspension of the PMA processing is extracted；
4) using the microbe genome DNA as template, it is fixed that real-time fluorescence is carried out for brucella BCSP31 gene
Measure PCR detection；
5) the CT value by real-time fluorescence quantitative PCR detection substitutes into standard curve regression equation, obtains in sample suspension
Brucella viable bacteria concentration.
Sample suspension is made in sample by the present invention.The sample includes non-human or animal body source sample, to avoid
The method of counting is related to the diagnostic method of disease.Described non-human or animal body source sample is preferably included containing brucella
Bacterium solution, culture cell, fermentation liquid, water environment sample, soil sample or food sample, mainly use in terms of scientific research, belong to
In the method for non-diagnostic purpose.Sample suspension is made to guarantee that the brucella contained in sample is in free shape in the sample
State is handled convenient for subsequent PMA.When sample is the solid samples such as soil or food, it is preferred to use PBS buffer solution dispersion；Work as sample
The brucella contained when product are bacterium solution, fermentation liquid, water environment sample, in sample has been in free state, then can directly into
Row subsequent operation；When sample is culture cell, first the bland cell lysate of cell routine is cracked, brucella intracellular
Subsequent operation can directly be carried out.
After obtaining sample suspension, PMA is added into the sample suspension by the present invention makes final concentration of 15~19 μ g/
ML, 10~25min of exposure-processed obtain the sample suspension of PMA processing.
In the present invention, the final concentration of PMA is preferably 15~17 μ g/mL.The time of exposure-processed is preferably 10~
15min.Light source used in the exposure-processed is preferably capable distributing the light source of strong light.In embodiments of the present invention, the exposure
Light source used in light processing is 650W halogen lamp.Intensity of illumination is preferably light of the 650W halogen lamp at 15-30cm above sample
According to intensity.After PMA is added into the sample suspension, PMA penetrates the dead bacterium of the brucella in sample suspension or impaired thin
The cell membrane of born of the same parents forms covalent bond with DNA under the action of strong light, to inhibit dead cell DNA to expand during qPCR
Increase.When brucella is viable bacteria, PMA can not penetrate the cell membrane of viable bacteria, subsequent brucella cannot be kept special in conjunction with DNA
Specific gene can go on smoothly qPCR amplification, therefore viable bacteria concentration in sample is calculated by qPCR result.
After obtaining the sample suspension of PMA processing, the present invention extracts microorganism base in the sample suspension of the PMA processing
Because of a group DNA.The present invention is to the method for microbe genome DNA in the sample suspension for extracting the PMA processing without spy
Different limitation, using microbial DNA extracting method known in the art.In embodiments of the present invention, described in the extraction
The method of microbe genome DNA is carried out using bacterial genomes DNA extraction kit in the sample suspension of PMA processing.
After obtaining microbe genome DNA, the present invention is using the microbe genome DNA as template, for brucella
BCSP31 gene carries out real-time fluorescence quantitative PCR detection.
In the present invention, the real-time fluorescence quantitative PCR detection amplimer of the brucella BCSP31 gene is preferred
The upstream primer as shown in sequence table SEQ ID No.1 (5 '-AAACATCAAATCGGTCGC-3 ') and such as sequence table SEQ ID
Downstream primer shown in No.2 (5 '-CCGCCCACAAAGAAATAG-3 ').The expanding fragment length of the amplimer is
200bp.The reaction system of the fluorescence quantitative PCR detection of brucella BCSP31 gene is 20 μ L:Fast StartUniversal
10 μ L of SYBR GreenMaster (Rox), 2 μ L of template, upstream and downstream primer each 1 μ L, ddH2O is supplemented to 20 μ L.The Fast
StartUniversal SYBR Green Master (Rox) is purchased from Roche company.The fluorescence of brucella BCSP31 gene is fixed
The response procedures for measuring PCR detection are 94 DEG C of initial denaturation 2min；Then 40 circulations: 94 DEG C of denaturation 45s, 55 DEG C of annealing 30s, 72 DEG C
Extend and acquires fluorescence signal 32s.The present invention is not particularly limited the real-time fluorescence quantitative PCR detection method, using this
Real-time fluorescence quantitative PCR detection method known to field.
In the present invention, in order to realize quantitative detection brucella viable bacteria, preparation and reorganization plasmid pMD-18T-BCSP31 is marked
Quasi- product and establish standard curve.The preparation method of the standard items is the BCSP31 gene expanded using above-mentioned amplimer
Segment is inserted into pMD-18T, and recombinant plasmid is formed.The method for building up of the standard curve copies the measurement of above-mentioned preparation and reorganization plasmid
After shellfish Particle density, 10 times of doubling dilutions carry out qPCR amplification according to the method described above, to copy the logarithm of Particle density as abscissa,
It is that ordinate draws standard curve to reach the average number cycles (threshold cycle, CT) of threshold value.Standard curve recurrence side
Journey is as follows: y=﹣ 3.492x+36.303, and wherein y represents CT value, and x represents the logarithm of gene copy Particle density；R2=0.999.
According to formula: E=10-1/k- 1 (E is amplification efficiency, and k is the slope of standard curve), calculates its amplification efficiency.Amplification efficiency is
93.36%.
After obtaining the CT value of real-time fluorescence quantitative PCR detection, the CT value that the present invention detects real-time fluorescence quantitative PCR is substituted into
In standard curve regression equation, brucella viable bacteria concentration in sample suspension is obtained.(1) calculates sample according to the following formula
Viable bacteria concentration.
Sample viable bacteria concentration=(volume of suspension is made in brucella viable bacteria concentration × sample in sample suspension) ÷
Solid sample quality or liquid sample volume formula (1)
Below with reference to embodiment to a kind of brucella count plate side based on PMA-qPCR technology provided by the invention
Method is described in detail, but they cannot be interpreted as limiting the scope of the present invention.
Experimental material and reagent source introduction: experimental strain, cell and genome pig kind brucella S2 low virulent strain, large intestine
Bacillus type strain, salmonella, Caused by Yersinia enterocolitica, vibrio parahaemolytious, mouse macrophage RAW 264.7, ox kind cloth
2308 plants of Shandong Salmonella and A19 pnca gene group, 16M plants of brucella melitensis and M5 pnca gene group, sheep epididymis kind brucella base
Because group is saved by bacterial disease research department, Amphixenosis research institute, Jilin University.
Key instrument and reagent gel imager are purchased from UVP company；650W halogen lamp is purchased from OSRAM company；ABI
7500real-time PCR system is purchased from American AB I company；The small purification of bacterial genomes DNA extraction kit, plasmid
Kit is purchased from Tiangeng Bioisystech Co., Ltd；The third ingot of nitrine bromination (PMA) is purchased from Biotium company；Fast Start
Universal SYBR Green Master (Rox) is purchased from Roche company；Brucella broth culture medium is purchased from Qingdao day aquatic organism
Technology Co., Ltd.；TritonX-100 is purchased from Shanghai Sheng Gong bioengineering limited liability company.Other raw materials of the present invention
And reagent is all made of commodity purchasing approach purchase known in the art.
Embodiment 1
1, brucella outer membrane protein gene BCSP31 (GenBank accession number: M20404.1) sequence published according to NCBI
Column design 1 pair of specific primer, upstream primer sequence are as follows: 5 '-with PremierPrimer 5
AAACATCAAATCGGTCGC-3 ' (SEQ ID No.1), downstream primer sequence are as follows: 5 '-CCGCCCACAAAGAAATAG-3 '
(SEQ ID No.2), expanding fragment length 200bp, by Changchun, Ku Mei Biotechnology Co., Ltd is synthesized.
2, PMA treatment conditions
Following 3 groups of processing methods are set:
(1) 7 parts of brucella low virulent strain each 500 μ L of S2 Thermal killed bacterium solution are taken, wherein 1 part of sample that PMA is not added compares,
Remaining 6 parts are separately added into PMA and make its final concentration of 15 μ g/mL, with 650W halogen lamp respectively to sample exposure different time, warp
QPCR detection, determines optimum exposure time.
(2) 7 parts of brucella low virulent strain each 500 μ L of S2 Thermal killed bacterium solution are taken, PMA is separately added into, keeps final concentration different, presses
It is handled according to optimum exposure time, is detected through qPCR, determine that PMA completely inhibits the optium concentration of dead bacterium DNA cloning.
(3) 6 parts of brucella low virulent strain each 500 μ L of S2 viable bacteria bacterium solution are taken, PMA is separately added into, keep final concentration different, according to
Optimum exposure time is handled, and is detected through qPCR, and determining PMA not influences the optium concentration of viable bacteria DNA cloning.
3, brucella low virulent strain S2 is inoculated in 5mLTSB culture medium by the extraction of brucella low virulent strain S2 genome,
37 DEG C are extracted genome after shaking table culture 12 hours, according to bacterial genomes DNA extraction kit specification, are examined using microplate reader
Its concentration is surveyed, A is made260/A280Between 1.8~2.0, -20 DEG C save backup value.
4, PMA-qPCR experimental design is using the genomic DNA for the processed brucella S2 bacterium solution of PMA extracted as mould
Plate carries out PMA-qPCR amplification, and reaction system is 20 μ L:Fast Start Universal SYBR Green Master (Rox)
10 μ L, 2 μ L of template, upstream and downstream primer each 1 μ L, ddH2O 6μL；Response procedures are as follows: 94 DEG C of initial denaturation 2min；Then 40 are followed
Ring: 94 DEG C of denaturation 45s, 55 DEG C of annealing 30s, 72 DEG C extend and acquire fluorescence signal 32s；Each concentration is repeated 3 times.
5, statistical analysis is examined, the difference as P < 0.05 using the data obtained in the statistical analysis experiment of SPSS 19.0 through t
Significantly, indifference is significant when P > 0.05.
The optimum exposure time of PMA, optimum concentration the result is shown in Figure 1.Different exposure time as the result is shown (Fig. 1-A), with
The extension of time for exposure, CT value constantly increase, upon exposure between when being greater than or equal to 10min each exposure time point CT value without
Significant difference (P > 0.05)；(Fig. 1-B) as the result is shown that PMA acts on dead bacterium, with the increase of PMA concentration, CT value constantly increases
Greatly, but when concentration is more than or equal to 15 μ g/ml, CT value is greater than 35 or without CT value, shows that PMA completely inhibits dead bacterium
DNA；(Fig. 1-C) as the result is shown that PMA acts on viable bacteria, when PMA concentration is in 0~15 μ g/ml range, the CT value of each concentration
Increase as PMA concentration >=20 μ g/ml with concentration without significant difference (P > 0.05), CT value obviously increases, and shows higher concentration
PMA influence detection to viable bacteria.So determine PMA effective exposure time be 10-25min, optimum exposure time 10min,
15~19 μ g/ml of effective working concentration, optimum concentration are 15 μ g/ml.
Embodiment 2
The sensitivity analysis of PMA-qPCR detection method
With the brucella S2 bacterium solution of the PMA processing known concentration of 15 μ g/ml of final concentration, time for exposure 10min is extracted
Its 10 times of doubling dilutions are carried out PMA-qPCR detection, while regular-PCR compares by genomic DNA, analysis method it is sensitive
Property.
(Fig. 2-A) is found by comparative experiments of the PMA-qPCR and common qPCR to the brucella S2 of various concentration, when
Bacterial concentration is 103~108Within the scope of CFU/mL, brucella S2 is handled through PMA and untreated CT value is coincide substantially, all
With good linear relationship (R2Respectively 0.986 and 0.991), sensitivity 103CFU/mL, and pass through regular-PCR (Fig. 2-
B its sensitivity) is detected 105CFU/mL, it is seen that the sensitivity of PMA-qPCR detection brucella S2 is 100 times of regular-PCR,
More sensitive accurate quantitative analysis can be carried out to brucella viable count, and qPCR reaction is not interfered.
Embodiment 3
The specificity analysis of PMA-qPCR detection method
With Escherichia coli, salmonella, yersinia enterocolitica, vibrio parahaemolytious, compare without RNase water,
With pig kind brucella S2 low virulent strain, 2308 plants and A19 plants of B. abortus, 16M plants of brucella melitensis and M5 plants, sheep
Epididymis kind brucella carries out PMA-qPCR experiment and regular-PCR experiment, analysis as the representative of source brucella not of the same race
The specificity of method.
Specificity experiments result is template as control (Fig. 3-A) using each non-brucella and without RNase water, as the result is shown
Amplification is negative, and carries out melting curve analysis (Fig. 3-B) to reaction product, when Tm value is melted at 87.87~88.36 DEG C in single
Xie Feng, primer free dimer and non-specific amplification, illustrate that primer specificity is good, while regular-PCR also confirms that PMA-qPCR
Method specificity is good (Fig. 3-C).
Embodiment 4
The brucella S2 of the repeatability analysis PMA optimal treatment condition processing known concentration of PMA-qPCR detection method
Bacterium solution extracts genomic DNA, and gradient is diluted, and selects 105、106、107The DNA of three dilutions is criticized as template
Between and batch in PMA-qPCR experiment, calculate the coefficient of variation (CV), analysis method repeatability.
Selection 105、106、107The DNA of three dilutions is carried out between criticizing and batch interior experiment, analytical variance coefficient (CV), according to
The visible coefficient of variation of experimental result (table 1, table 2) illustrates qPCR detection stability, reproducible between 0.63~2.04%.
The coefficient of variation of 1 batch of interior experiment of table
The coefficient of variation tested between 2 batches, table
Embodiment 5
PMA-qPCR is to the detection of the live/dead bacterium of different proportion by two kinds of bacterium solutions of the viable bacteria of known concentration and dead bacterium solution by difference
Ratio mixing, setting viable bacteria accounting are 0,10,30,50,70,100%, extract genomic DNA after respectively taking 1mL that PMA processing is added
PMA-qPCR reaction is carried out, while comparing group so that the sample of PMA processing is not added.
PMA-qPCR is as follows to the testing result of the live/dead bacterium of different proportion: by the viable bacteria of known concentration/dead bacterium by not year-on-year
Example is mixed, and carries out PMA-qPCR detection, while comparing so that the sample of PMA processing is not added.Work as viable bacteria as seen from Figure 4
When content is 0%, CT value is not detected, and illustrates that dead bacterium DNA is inhibited by PMA completely, not can be carried out amplification.With viable bacteria ratio
Increasing, CT value gradually decreases, but is above without the processed CT value of PMA, when viable bacteria content is 100%, PMA-qPCR inspection
The CT value of survey with without the processed CT value of PMA there is no the significant meaning of statistical difference (P > 0.05), while can be seen by table 3
Out, as viable bacteria ratio increases, △ CT value is also gradually reduced, compared with common qPCR result processed without PMA, result
Closer to the theoretical viable count in sample.Illustrate PMA-qPCR on living stems without influence, and can to viable bacteria carry out accurate quantitative analysis.
3 different proportion S2 viable bacteria PMA-qPCR result of table
Note: the CT value of △ CT:PMA-qPCR-control group CT value absolute value；- indicate that PMA-qPCR testing result is yin
Property.
Embodiment 6
The brucella S2 of the preparation of recombinant plasmid pMD-18T-BCSP31 standard items and the foundation of standard curve to extract
Strain gene group DNA is template, PCR amplification target fragment.Reaction system is 50 μ L:TaKaRa EX Taq (5U/ μ L) 0.25 μ
L, 10 × Ex TaqBuffer (Mg2+Free) 5 μ L, dNTP 4 μ L, 1 μ L of template, upstream and downstream primer each 1 μ L, ddH2O complements to 50
μL.Reaction condition: 94 DEG C of initial denaturation 2min；Then 35 recycle: denaturation: 94 DEG C of 45s, annealing: 55 DEG C of 30s extend: 72 DEG C
1min；Terminal extends: 72 DEG C of 10min；4 DEG C of preservations.It is detected through agarose gel electrophoresis, and recovery purifying PCR product, by product
It is connected to pMD-18T carrier, preparation and reorganization plasmid is inverted to obtain clone bacterium, and sequencing and bacterium solution PCR identification are carried out to it, will
It identifies that correct bacterium solution extracts plasmid, measures its concentration, and be converted into the respective copies number of standard items: copy number as follows
=concentration (ng/ μ L) × Avogadro constant number (NA) × 10-9/ (the base number of 660 × recombinant plasmid).
10 times of doubling dilutions of standard plasmid are subjected to qPCR amplification, reaction system is 20 μ L:Fast Start
Universal SYBR Green Master (Rox) 10 μ L, 2 μ L of template, upstream and downstream primer each 1 μ L, ddH2O 6μL；Reaction interval
Sequence is 94 DEG C of initial denaturation 2min；Then 40 recycle: denaturation: 94 DEG C of 45s, annealing: 55 DEG C of 30s extend: 72 DEG C of 32s；It is each dense
Degree is repeated 3 times.To copy the logarithm of Particle density as abscissa, to reach the average number cycles (threshold of threshold value
Cycle, CT) it is ordinate drafting standard curve, and according to formula: E=10-1/k- 1 (E is amplification efficiency, and k is standard curve
Slope), calculate its amplification efficiency.
After the preparation of plasmid standard is analyzed through 1% agarose gel electrophoresis, brucella target gene PCR product is big
Small is 200bp, is consistent with expected results (Fig. 5)；Sequencing result similarly shows that recombinant plasmid pMD-18T-BCSP31 is built into
Function.Recombinant plasmid detectable concentration is 398.408ng/ μ L；Show that copy number is about 6.28 × 10 through formula scales10Copy/μ L.
By 10 times of doubling dilutions of standard plasmid, copy number concentration gradient is 6.28 × 10 for the foundation of standard curve0~
6.28×108It is detected using 7500 real-time fluorescence quantitative PCR instrument of ABI, amplification terminates to draw standard curve (Fig. 6), returns
Equation are as follows: y=﹣ 3.492x+36.303, wherein y represents CT value, and x represents the logarithm of gene copy Particle density；R2=0.999,
By standard curve it can be seen that the linear relationship between CT value and the logarithm for copying Particle density is good, amplification is calculated to obtain according to formula
Efficiency is 93.36%.
Embodiment 7
Two methods of PMA-qPCR method and colony counting method comparative analysis number of viable application are to pure culture brucella liquid
Carry out Comparative and Quantitative Analysis experiment.Then, then to analog sample test and analyze: taking 12 porocyte culture plates, every hole inoculation 2
×105264.7 cell of/mL mouse macrophage RAW changes culture medium culture 1h without double antibody after cultivating 12h, carries out attacking bacterium reality
It tests.It is divided into A and two groups of B, (wherein PMA-qPCR analysis is done in 3 holes to every group of 6 repeating holes, and in addition plate count point is done in 3 holes
Analysis), brucella S2 plants of concentration of A group infection is 1.28 × 1012CFU/mL, B group infection bacteria concentration be 0.64 ×
1012CFU/mL.It after attacking bacterium 1h, changes containing dual anti-culture medium culture 2h, collects simultaneously lytic cell.Take the 100 μ L lysates in wherein 3 holes
Gradient dilution is carried out, TSA solid medium is applied to, overnight incubation counts clump count；The lysate in other 3 hole is taken to be collected by centrifugation
Intracellular thallus, sterile saline are washed twice, with most suitable PMA concentration, after most suitable time for exposure effect, carry out qPCR detection,
It brings CT value obtained by each group into standard curve, calculates to obtain bacterial concentration, the intracellular infection bacterium number that two methods obtain is united
Meter.
PMA-qPCR method and colony counting method analyze pure bacterium solution living stems with two methods, and gained bacterium solution is dense
Degree magnitude matches, and statistical difference is not significant.Bacterium cell A and B group viable count intracellular of then attacking against each other is detected, and two
Viable count obtained by kind method has certain statistical difference conspicuousness (Fig. 7), but viable count in an order of magnitude and is presented one
The viable count of cause trend, the measurement of PMA-qPCR method is higher than the viable count that colony counting method measures, this may be due to plate meter
Number method can only measure educable number of viable at normal temperatures and pressures, and Intracellular bacterial is due to the shadow by intracellular environment
Ring, part viable bacteria be likely to be at it is non-can cultivation conditions, therefore colony counting method can not reflect it is non-can cultivation conditions number of viable.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Sequence table
<110>Jilin University
<120>a kind of brucella viable count method based on PMA-qPCR technology
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
aaacatcaaa tcggtcgc 18
<210> 2
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
ccgcccacaa agaaatag 18
Claims (9)
1. a kind of brucella viable count method based on PMA-qPCR technology, which comprises the following steps:
1) sample suspension is made in sample；
2) PMA is added in Xiang Suoshu sample suspension obtains final concentration of 15~19 μ g/mL, 10~25min of exposure-processed
The sample suspension of PMA processing；
3) microbe genome DNA in the sample suspension of the PMA processing is extracted；
4) using the microbe genome DNA as template, real-time fluorescence quantitative PCR is carried out for brucella BCSP31 gene
Detection；
5) the CT value by real-time fluorescence quantitative PCR detection substitutes into standard curve regression equation, obtains cloth Shandong in sample suspension
Salmonella viable bacteria concentration.
2. method of counting according to claim 1, which is characterized in that final concentration of 15~17 μ g/ of PMA in step 2)
mL。
3. method of counting according to claim 1 or 2, which is characterized in that in step 2) time of exposure-processed be 10~
15min。
4. method of counting according to claim 1, which is characterized in that brucella BCSP31 gene is real-time in step 4)
Fluorescence quantitative PCR detection amplimer includes the upstream primer as shown in sequence table SEQ ID No.1 and such as sequence table SEQ
Downstream primer shown in ID No.2.
5. method of counting according to claim 1, which is characterized in that the fluorescence of brucella BCSP31 gene in step 4)
The response procedures of quantitative PCR detection are 94 DEG C of initial denaturation 2min；Then 40 circulations: 94 DEG C of denaturation 45s, 55 DEG C of annealing 30s, 72
DEG C extends and acquire fluorescence signal 32s.
6. according to claim 1, method of counting described in 4 and 5 any one, which is characterized in that brucella in step 4)
The reaction system of the fluorescence quantitative PCR detection of BCSP31 gene is 20 μ L:Fast Start Universal SYBR
10 μ L of GreenMaster (Rox), 2 μ L of template, each 1 μ L, dd H of upstream and downstream primer2O is supplemented to 20 μ L.
7. method of counting according to claim 1, which is characterized in that step 5) standard curve regression equation is as follows: y
=﹣ 3.492x+36.303, wherein y represents CT value, and x represents the logarithm of gene copy Particle density；R2=0.999.
8. according to claim 1, method of counting described in 2,4,5 and 7 any one, which is characterized in that sample described in step 1)
Product include non-human or animal body source sample.
9. method of counting according to claim 8, which is characterized in that described non-human or animal body source sample includes containing
Bacterium solution, culture cell, fermentation liquid, water environment sample, soil sample or the food sample of brucella.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910801583.0A CN110343744A (en) | 2019-08-28 | 2019-08-28 | A kind of brucella viable count method based on PMA-qPCR technology |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910801583.0A CN110343744A (en) | 2019-08-28 | 2019-08-28 | A kind of brucella viable count method based on PMA-qPCR technology |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN110343744A true CN110343744A (en) | 2019-10-18 |
Family
ID=68181310
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910801583.0A Pending CN110343744A (en) | 2019-08-28 | 2019-08-28 | A kind of brucella viable count method based on PMA-qPCR technology |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110343744A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111500752A (en) * | 2020-04-23 | 2020-08-07 | 江苏省农业科学院 | PMA-qPCR detection method of vibrio parahaemolyticus |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102146466A (en) * | 2011-02-15 | 2011-08-10 | 浙江国际旅行卫生保健中心 | Reagent for detecting brucella and complex probe fluorescence quantitative PCR (polymerase chain reaction) brucella detection method |
| CN107354228A (en) * | 2017-09-06 | 2017-11-17 | 上海出入境检验检疫局动植物与食品检验检疫技术中心 | A kind of method and reagent of detection VBNC vibrio parahemolyticus |
| CN108504753A (en) * | 2018-03-30 | 2018-09-07 | 中国农业科学院北京畜牧兽医研究所 | A method of detection staphylococcus aureus live bacteria in milk |
| CN109321666A (en) * | 2018-09-06 | 2019-02-12 | 新疆农业科学院农业质量标准与检测技术研究所 | The method of Streptococcusagalactiae viable bacteria in SDS-PMA-qPCR method detection cream |
-
2019
- 2019-08-28 CN CN201910801583.0A patent/CN110343744A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102146466A (en) * | 2011-02-15 | 2011-08-10 | 浙江国际旅行卫生保健中心 | Reagent for detecting brucella and complex probe fluorescence quantitative PCR (polymerase chain reaction) brucella detection method |
| CN107354228A (en) * | 2017-09-06 | 2017-11-17 | 上海出入境检验检疫局动植物与食品检验检疫技术中心 | A kind of method and reagent of detection VBNC vibrio parahemolyticus |
| CN108504753A (en) * | 2018-03-30 | 2018-09-07 | 中国农业科学院北京畜牧兽医研究所 | A method of detection staphylococcus aureus live bacteria in milk |
| CN109321666A (en) * | 2018-09-06 | 2019-02-12 | 新疆农业科学院农业质量标准与检测技术研究所 | The method of Streptococcusagalactiae viable bacteria in SDS-PMA-qPCR method detection cream |
Non-Patent Citations (1)
| Title |
|---|
| 石丽瑞等: "布鲁氏菌实时定量荧光PCR检测体系的建立及应用", 《中国动物检疫》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111500752A (en) * | 2020-04-23 | 2020-08-07 | 江苏省农业科学院 | PMA-qPCR detection method of vibrio parahaemolyticus |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Nucera et al. | Comparison of API 20E and invA PCR for identification of Salmonella enterica isolates from swine production units | |
| Pal et al. | Rapid detection and differentiation of Erysipelothrix spp. by a novel multiplex real‐time PCR assay | |
| Wen et al. | A rapid novel visualized loop-mediated isothermal amplification method for Salmonella detection targeting at fimW gene | |
| CN113249499B (en) | Salmonella typhi detection kit, and preparation method and application thereof | |
| Zhou et al. | Multiple PCR assay based on the cigR gene for detection of Salmonella spp. and Salmonella Pullorum/Gallinarum identification | |
| CN111876502B (en) | Method for identifying Brucella S2 vaccine strain by dual real-time fluorescent quantitative PCR and kit used by same | |
| CN104212901A (en) | Composition for detecting drug resistance of staphylococcus aureus | |
| CN107475374A (en) | The kit and detection method of Vibrio vulnificus in a kind of accurate quantification detection food | |
| CN114774563B (en) | Detection reagent for brucellosis in dog and application | |
| Murugaiyan et al. | MALDI spectra database for rapid discrimination and subtyping of Mycobacterium kansasii | |
| CN114891902A (en) | Primer-probe combination for rapidly detecting five virulent pathogenic bacteria based on liquid drop digital PCR and application method thereof | |
| CN104263842B (en) | A kind of fluorescent quantitative PCR detection method of source of fish streptococcus agalactiae | |
| CN114438214A (en) | Colorectal cancer tumor marker and detection method and device thereof | |
| CN105525023A (en) | Fluorescent quantitative PCR detection kit for clostridium difficile toxin A/B and detection method | |
| CN109337995B (en) | PCR detection method and kit for eubacterium terrae and subspecies thereof | |
| US20180330044A1 (en) | Methods Associated With A Database That Stores A Plurality Of Reference Genomes | |
| CN104328174B (en) | A kind of detect the LAMP primer of Mus Klebsiella pneumonia, test kit and method | |
| CN110343744A (en) | A kind of brucella viable count method based on PMA-qPCR technology | |
| Ojha et al. | Examination of animal and zoonotic pathogens using microarrays | |
| Müştak et al. | Detection and differentiation of Salmonella Enteritidis and Salmonella Typhimurium by multiplex quantitative PCR from different poultry matrices | |
| Ganji et al. | Comparison of the Detection Limits of the Culture and PCR Methods for the Detection of Clostridium difficile, Clostridium perfringens, Campylobacter jejuni, and Yersinia enterocolitica in Human Stool | |
| CN112795673B (en) | CRISPR (clustered regularly interspaced short palindromic repeats) detection method for Cronobacter in food and kit thereof | |
| CN108866216A (en) | It is a kind of for identifying the primer combination of probe of mycobacterium abscessus and Marseille mycobacteria | |
| AU2019100071A4 (en) | Pcr detection kit for rapidly identifying salmonella of specific serotypes | |
| Ferrandis-Vila et al. | Using unique ORFan genes as strain-specific identifiers for Escherichia coli |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20191018 |