CN104651482A - Method for detecting living bacteria body of vibrio parahemolyticus in sample - Google Patents
Method for detecting living bacteria body of vibrio parahemolyticus in sample Download PDFInfo
- Publication number
- CN104651482A CN104651482A CN201310583134.6A CN201310583134A CN104651482A CN 104651482 A CN104651482 A CN 104651482A CN 201310583134 A CN201310583134 A CN 201310583134A CN 104651482 A CN104651482 A CN 104651482A
- Authority
- CN
- China
- Prior art keywords
- vibrio parahemolyticus
- sample
- biomolecular
- viable bacteria
- bacteria body
- 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.)
- Granted
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
- C12Q1/6804—Nucleic acid analysis using immunogens
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- Immunology (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Pathology (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 invention discloses a method for detecting living bacteria bodies of vibrio parahemolyticus in a sample. The method includes following steps: (1) coupling an antibody capable of being specifically combined with the living bacteria bodies of vibrio parahemolyticus onto magnetic balls to obtain immune magnetic balls, wherein the antibody capable of being specifically combined with the living bacteria bodies of vibrio parahemolyticus cannot be combined with dead bacteria bodies; (2) mixing the immune magnetic balls with a liquid sample to obtain a mixture, wherein the mixing condition is described as that when the living bacteria bodies of vibrio parahemolyticus exists in the sample, the immune magnetic balls can specifically adsorb the living bacteria bodies of vibrio parahemolyticus; (3) placing the mixture in a separation magnetic field for immobilizing the immune magnetic balls and eluting substances being not adsorbed on the immune magnetic balls to obtain eluted immune magnetic balls; (4) performing DNA extraction to substances adsorbed onto the immune magnetic balls to obtain a template sample; (5) performing PCR amplification to the template sample with a specific primer aiming to the living bacteria bodies of vibrio parahemolyticus, and determining the content of the living bacteria bodies of vibrio parahemolyticus in the sample according to the content of targeted fragments in a PCR amplification product.
Description
Technical field
The present invention relates to biomedicine field, particularly, relate to a kind of method detecting Vibrio parahemolyticus viable bacteria body in sample.
Background technology
Pathogenic bacterium (Pathogenic bacteria) can cause the microorganism of disease to be called pathogenic micro-organism or pathogenic bacterium.Pathogenic micro-organism comprises bacterium, virus, spirochete, Rickettsiae, chlamydozoan, mycoplasma, fungi and actinomycetes etc.General said pathogenic bacterium refer to the bacterium in pathogenic micro-organism.Pathogenic and its virulence of bacterium, invade quantity and portal entry relevant.Although most bacterium is harmless even useful, a large portion can be caused a disease.Conditioned pathogen is only caused a disease under given conditions, bacterium can be allowed to enter blood if any wound, or when immunizing power reduces.Such as, streptococcus aureus and suis are also normal microfloras, often may reside in skin surface, nasal cavity and do not cause disease, but potentially can cause skin infections, as pneumonia (pneumonia), meningitis (meningitis) and septicemia (sepsis) etc.
Vibrio parahemolyticus (Vibrio Parahemolyticus) is a kind of halophilism bacterium.Vibrio parahaemolytisus poisoning is caused by the food of feed containing this bacterium, and this bacterium, mainly from sea-food, as inkfish, ocean fish, extra large shrimp, sea crab, jellyfish, and contains the higher pickling food of salinity, as salted vegetables, butcher's meat etc.This bacterium survival ability is strong, rag and chopping block can be survived more than 1 month, can survive 47 days in seawater.Clinically with Acute onset, stomachache, vomiting, diarrhoea and watery stool for cardinal symptom.This disease is many betides coastland in summer and autumn, often causes collective to fall ill.
At present, the method detecting pathogenic bacterium can only detect the total number of the pathogenic bacterium in sample, and the quantity of the Vibrio parahemolyticus viable bacteria body can not distinguished in sample and dead thalline is respectively how many.
Summary of the invention
In order to the dead thalline quantity in testing sample and the difference of Vibrio parahemolyticus viable bacteria body quantity are come, the invention provides a kind of method detecting Vibrio parahemolyticus viable bacteria body in sample, the method comprises: the antibody coupling that (1) can be combined with Vibrio parahemolyticus viable bacteria body specifically, on magnetic ball, obtains biomolecular; The described antibody that can be combined with Vibrio parahemolyticus viable bacteria body specifically can not be combined with dead thalline; (2) biomolecular is mixed with the testing sample of liquid form, obtain mixed material; The condition of mixing makes when there is Vibrio parahemolyticus viable bacteria body in testing sample, and biomolecular can adsorb Vibrio parahemolyticus viable bacteria body specifically; (3) described mixed material is placed in sorting magnetic field with fixing biomolecular, and wash-out is not adsorbed on the material on biomolecular, obtains the biomolecular after wash-out; (4) material be adsorbed on biomolecular is carried out the operation of extracting DNA, obtain template sample; (5) use the Auele Specific Primer for described Vibrio parahemolyticus viable bacteria body to carry out pcr amplification to template sample, and judge the content of the Vibrio parahemolyticus viable bacteria body in testing sample according to the content of the object fragment in pcr amplification product
By technique scheme, the dead thalline quantity in testing sample and the difference of Vibrio parahemolyticus viable bacteria body quantity can come by the present invention effectively.
Other features and advantages of the present invention are described in detail in embodiment part subsequently.
Embodiment
Below the specific embodiment of the present invention is described in detail.Should be understood that, embodiment described herein, only for instruction and explanation of the present invention, is not limited to the present invention.
In the present invention, when not doing contrary explanation, the term " biomolecular " of use to refer to by linked reaction by antibodies on the surface of magnetic microsphere, the immune magnetic microsphere of formation.Term " PCR " refers to polymerase chain reaction.Term " primer " comprises at least one upstream primer and at least one downstream primer.The liquid volume used in the present invention is the numerical value at 20 DEG C.
The invention provides a kind of method detecting Vibrio parahemolyticus viable bacteria body in sample, the method comprises:
(1) antibody coupling that can be combined with Vibrio parahemolyticus viable bacteria body specifically, on magnetic ball, obtains biomolecular; The described antibody that can be combined with Vibrio parahemolyticus viable bacteria body specifically can not be combined with dead thalline;
(2) biomolecular is mixed with the testing sample of liquid form, obtain mixed material; The condition of mixing makes when there is Vibrio parahemolyticus viable bacteria body in testing sample, and biomolecular can adsorb Vibrio parahemolyticus viable bacteria body specifically;
(3) described mixed material is placed in sorting magnetic field with fixing biomolecular, and wash-out is not adsorbed on the material on biomolecular, obtains the biomolecular after wash-out;
(4) material be adsorbed on biomolecular is carried out the operation of extracting DNA, obtain template sample;
(5) use the Auele Specific Primer for described Vibrio parahemolyticus viable bacteria body to carry out pcr amplification to template sample, and judge the content of the Vibrio parahemolyticus viable bacteria body in testing sample according to the content of the object fragment in pcr amplification product.
Wherein,, as the particularly preferred a kind of embodiment of the present invention, the described antibody that can be combined with Vibrio parahemolyticus viable bacteria body is specifically be numbered the intelligent of VP-mab-01 to weed biological monoclonal antibody, this antibody can be commercially available from Hui Yun biotech firm, and article number is VP-mab-01.
Wherein, the magnetic ball relative to every milligram, the consumption of described antibody can be 0.05-2mg.
Wherein, the testing sample of the liquid form relative to every milliliter, with the weighing scale of magnetic ball, the consumption of described biomolecular can be 10-25 μ g.
Wherein, in step (3), wash-out liquid used can be phosphoric acid buffer, and described phosphoric acid buffer contains the Sodium phosphate dibasic of 0.144-0.162mol/L and the SODIUM PHOSPHATE, MONOBASIC of 0.038-0.056mol/L.
Wherein, as the particularly preferred a kind of embodiment of the present invention, for Vibrio parahemolyticus viable bacteria body, pcr amplification primer used comprises forward primer as shown in SEQ ID NO:1 (5 '-AAGCGGATTATGCAGAAGCA-3 ') and the reverse primer as shown in SEQ ID NO:2 (5 '-GCTACTTTCTAGCATTTTCTCTGC-3 ').
Wherein, described magnetic ball can have the superparamagnetism Fe of amino or carboxyl for finishing
3o
4polystyrene complex microsphere.
According to method of the present invention, wherein, testing sample can be food and/or medicine.Particularly, testing sample can be obtained according to the method in standard GB/T 4789.1-2010 " national food safety standard food microbiological analysis general provisions ".
Below will be described the present invention by embodiment.In following examples, described phosphoric acid buffer is the aqueous solution of the SODIUM PHOSPHATE, MONOBASIC of Sodium phosphate dibasic containing 0.153mol/L and 0.047mol/L.To be commercially available from Hui Yun biotech firm, article number is that the antibody of VP-mab-01 is as antibody 1.
Preparation embodiment 1
This is prepared embodiment 1 and uses antibody 1 to prepare method of the present invention biomolecular used.
The finishing activated by 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimide (EDC) and N-hydroxy-succinamide (NHS) there is the superparamagnetism Fe of carboxyl
3o
4polystyrene complex microsphere is (purchased from Aorun Weina New Material Science and Technology Co., Ltd., Shanghai, PM3-020 type polymkeric substance magnetic ball, concentration is 10mg/mL, particle diameter is 180nm, finishing has carboxyl functional group) use as magnetic ball, get 2mg magnetic ball, in dispersion phosphate buffer solution, in dispersion liquid, the concentration of magnetic ball is 2mg/mL.Get 0.2mg specific antibody (being dissolved in the phosphoric acid buffer of 1mL) to mix with 2mg magnetic ball, in shaking table (200r/min), keep 3h under room temperature, then cleaned by Magneto separate, remove the specific antibody not being coupled to magnetic ball surface.Then be 1%(w/v by concentration) bovine serum albumin solution (being dissolved in phosphoric acid buffer), at 37 DEG C, close 30min, then cleaned by Magneto separate, wash away unnecessary bovine serum albumin solution, namely obtain biomolecular.
This prepares the biomolecular 1 that embodiment obtains using antibody 1 to prepare.
Embodiment 1
By Vibrio parahemolyticus (Vibrio Parahemolyticus) (purchased from ATCC, article No. 17802) at LB substratum (Tryptones 10g/L, yeast extract 5g/L, sodium-chlor 10g/L) in be cultured to logarithmic phase, with LB substratum adjustment cell concentration to 10
3individual/L, as the positive containing Vibrio parahemolyticus viable bacteria body.
Above-mentioned positive is obtained after high pressure deactivation (121 DEG C, 15min) not containing Vibrio parahemolyticus viable bacteria body, but containing 10
3the negative sample of the dead thalline of individual/L.
Positive and negative sample are carried out equal-volume mix, namely obtain containing 5 × 10
2the dead thalline of individual/L and 5 × 10
2the biased sample of the viable bacteria body of individual/L.
Respectively by the positive of 1mL, negative sample and biased sample and biomolecular 1(with the weighing scale of magnetic ball, 15mg) mix, obtain mixed material, 30min is hatched at 29 DEG C, then in sorting magnetic field, biomolecular is fixed, wash-out is not adsorbed on the material on biomolecular, the material obtaining the biomolecular after wash-out He elute.
Use the bacterial genomes DNA extraction kit (DP302) purchased from Tian Gen biochemical technology company limited, according to the working method of its specification sheets, the operation of extracting DNA is carried out respectively to the biomolecular after wash-out and the material eluted, obtain respectively being enriched in the DNA sample in the material on immunomagnetic beads and the DNA sample in the remaining material of enrichment, using them as template sample.Forward primer as shown in SEQID NO:1 (5 '-AAGCGGATTATGCAGAAGCA-3 ') and the reverse primer as shown in SEQID NO:2 (5 '-GCTACTTTCTAGCATTTTCTCTGC-3 ') is used to carry out pcr amplification (real-time quantitative PCR to template sample, positive Ct value is proofreaded outside using), result is as shown in table 1.
According to the method specified in GB/T4789.7-2008 (inspection of microbiological test of food hygiene Vibrio parahemolyticus), respectively the material on immunomagnetic beads and the remaining material of enrichment are carried out to the viable bacteria body number detection of colony counting method, result is as shown in table 1.
Table 1
Visible according to the data of table 1, the change of the viable bacteria body number that PCR quantifiable signal value and colony counting method obtain is completely the same.
Visible by the result of table 1 in embodiment 1, the dead thalline quantity of Vibrio parahemolyticus in testing sample and the difference of viable bacteria body quantity can come by the present invention effectively, because method of the present invention does not need to carry out yeast culture to testing sample, thus greatly accelerate and simplify the detection of the pathogenic bacterium in sample.
More than describe the preferred embodiment of the present invention in detail; but the present invention is not limited to the detail in above-mentioned embodiment, within the scope of technical conceive of the present invention; can carry out multiple simple variant to technical scheme of the present invention, these simple variant all belong to protection scope of the present invention.
It should be noted that in addition, each concrete technical characteristic described in above-mentioned embodiment, in reconcilable situation, can be combined by any suitable mode, in order to avoid unnecessary repetition, the present invention illustrates no longer separately to various possible array mode.
In addition, also can carry out arbitrary combination between various different embodiment of the present invention, as long as it is without prejudice to thought of the present invention, it should be considered as content disclosed in this invention equally.
Claims (7)
1. detect a method for Vibrio parahemolyticus viable bacteria body in sample, the method comprises:
(1) antibody coupling that can be combined with Vibrio parahemolyticus viable bacteria body specifically, on magnetic ball, obtains biomolecular; The described antibody that can be combined with Vibrio parahemolyticus viable bacteria body specifically can not be combined with dead thalline;
(2) biomolecular is mixed with the testing sample of liquid form, obtain mixed material; The condition of mixing makes when there is Vibrio parahemolyticus viable bacteria body in testing sample, and biomolecular can adsorb Vibrio parahemolyticus viable bacteria body specifically;
(3) described mixed material is placed in sorting magnetic field with fixing biomolecular, and wash-out is not adsorbed on the material on biomolecular, obtains the biomolecular after wash-out;
(4) material be adsorbed on biomolecular is carried out the operation of extracting DNA, obtain template sample;
(5) use the Auele Specific Primer for described Vibrio parahemolyticus viable bacteria body to carry out pcr amplification to template sample, and judge the content of the Vibrio parahemolyticus viable bacteria body in testing sample according to the content of the object fragment in pcr amplification product.
2. method according to claim 1, wherein, the described antibody that can be combined with Vibrio parahemolyticus viable bacteria body is specifically be numbered the intelligent of VP-mab-01 to weed biological monoclonal antibody.
3. method according to claim 1 and 2, wherein, the magnetic ball relative to every milligram, the consumption of described antibody is 0.05-2mg.
4. method according to claim 1 and 2, wherein, the testing sample of the liquid form relative to every milliliter, with the weighing scale of magnetic ball, the consumption of described biomolecular is 10-25 μ g.
5. method according to claim 1 and 2, wherein, in step (3), wash-out liquid used is phosphoric acid buffer, and described phosphoric acid buffer contains the Sodium phosphate dibasic of 0.144-0.162mol/L and the SODIUM PHOSPHATE, MONOBASIC of 0.038-0.056mol/L.
6. method according to claim 1, wherein, pcr amplification primer used is for comprising the forward primer as shown in SEQ ID NO:1 and the reverse primer as shown in SEQ ID NO:2.
7. method according to claim 1, wherein, described magnetic ball is the superparamagnetism Fe that finishing has amino or carboxyl
3o
4polystyrene complex microsphere.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310583134.6A CN104651482B (en) | 2013-11-19 | 2013-11-19 | A kind of detect the method for vibrio parahaemolyticus viable bacteria body in sample |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310583134.6A CN104651482B (en) | 2013-11-19 | 2013-11-19 | A kind of detect the method for vibrio parahaemolyticus viable bacteria body in sample |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104651482A true CN104651482A (en) | 2015-05-27 |
| CN104651482B CN104651482B (en) | 2016-09-14 |
Family
ID=53243121
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310583134.6A Active CN104651482B (en) | 2013-11-19 | 2013-11-19 | A kind of detect the method for vibrio parahaemolyticus viable bacteria body in sample |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104651482B (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101158635A (en) * | 2007-11-19 | 2008-04-09 | 上海师范大学 | Method using quantum dot to detect bacterium |
| CN102586157A (en) * | 2012-03-12 | 2012-07-18 | 上海海洋大学 | Method for enriching and capturing vibrio patahaemolyticus with high throughput |
| CN102586452A (en) * | 2012-03-12 | 2012-07-18 | 上海海洋大学 | Vibrio parahemolyticus detection kit and detection method thereof |
| CN105300963A (en) * | 2015-10-22 | 2016-02-03 | 宁波大学 | Preparing method and application of sandwich type electrochemical luminescence immunosensor for detecting marine pathogenic bacteria |
-
2013
- 2013-11-19 CN CN201310583134.6A patent/CN104651482B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101158635A (en) * | 2007-11-19 | 2008-04-09 | 上海师范大学 | Method using quantum dot to detect bacterium |
| CN102586157A (en) * | 2012-03-12 | 2012-07-18 | 上海海洋大学 | Method for enriching and capturing vibrio patahaemolyticus with high throughput |
| CN102586452A (en) * | 2012-03-12 | 2012-07-18 | 上海海洋大学 | Vibrio parahemolyticus detection kit and detection method thereof |
| CN105300963A (en) * | 2015-10-22 | 2016-02-03 | 宁波大学 | Preparing method and application of sandwich type electrochemical luminescence immunosensor for detecting marine pathogenic bacteria |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104651482B (en) | 2016-09-14 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| AU2021236442B9 (en) | Methods and systems for rapid detection of microorganisms using recombinant bacteriophage | |
| Yang et al. | Rapid detection of Listeria monocytogenes by nanoparticle-based immunomagnetic separation and real-time PCR | |
| Fu et al. | Rapid detection of Escherichia coli O157: H7 by immunomagnetic separation and real-time PCR | |
| US20200140919A1 (en) | Methods and Systems for Rapid Detection of Microorganisms Using Infectious Agents | |
| Huang et al. | Development of a rapid Salmonella detection method via phage-conjugated magnetic bead separation coupled with real-time PCR quantification | |
| CN105112519A (en) | CRISPR-based Escherichia coli O157:H7 strain detection reagent box and detection method | |
| CN102943113B (en) | Loop-mediated isothermal amplification detection primer groups of Escherichia coli 0157, detection method and reagent kit | |
| McKindles et al. | Dissolved microcystin release coincident with lysis of a bloom dominated by Microcystis spp. in western Lake Erie attributed to a novel cyanophage | |
| CN101613745B (en) | Staphylococcus aureus multiple PCR-MIX rapid detection kit and detection method thereof | |
| CN102586157A (en) | Method for enriching and capturing vibrio patahaemolyticus with high throughput | |
| CN103468823A (en) | Listeria monocytogenes LAMP detection primer and detection method, kit and preparation method | |
| CN102321762A (en) | Method for rapid detection of listeria monocytogenes with high sensitivity and kit thereof | |
| Donati et al. | The gut microbiota of healthy and Flavobacterium psychrophilum-infected rainbow trout fry is shaped by antibiotics and phage therapies | |
| CN103160603B (en) | LAMP (loop-mediated isothermal amplification) detection kit of vibrio parahaemolyticus and detection method thereof | |
| CN104212887A (en) | Method for rapidly detecting salmonella | |
| Liedig et al. | Blood supplementation enhances Bartonella henselae growth and molecular detection of bacterial DNA in liquid culture | |
| Kumar et al. | Rapid detection of Salmonella typhi in foods by combination of immunomagnetic separation and polymerase chain reaction | |
| Almand et al. | Capture and concentration of viral and bacterial foodborne pathogens using apolipoprotein H | |
| Bai et al. | Exploring magnetic capture to improve the detection of human norovirus in strawberries | |
| CN104651482A (en) | Method for detecting living bacteria body of vibrio parahemolyticus in sample | |
| Wu et al. | Current and emerging innovations for detection of food-borne Salmonella | |
| O'Connell et al. | Alternatives to antibiotics in veterinary medicine: considerations for the management of Johne's disease | |
| CN104651479A (en) | Method for detecting living bacteria body in sample | |
| CN105203578B (en) | A kind of method for detecting microorganism | |
| CN103160602B (en) | Real-time fluorescent PCR (polymerase chain reaction) detection kit of vibrio parahaemolyticus and detection method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |