CN102586157A - Method for enriching and capturing vibrio patahaemolyticus with high throughput - Google Patents
Method for enriching and capturing vibrio patahaemolyticus with high throughput Download PDFInfo
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- CN102586157A CN102586157A CN2012100632144A CN201210063214A CN102586157A CN 102586157 A CN102586157 A CN 102586157A CN 2012100632144 A CN2012100632144 A CN 2012100632144A CN 201210063214 A CN201210063214 A CN 201210063214A CN 102586157 A CN102586157 A CN 102586157A
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Abstract
The invention relates to a method for enriching and capturing vibrio patahaemolyticus with high throughput. The method is characterized by comprising the following steps: coupling a polyclonal antibody of the vibrio patahaemolyticus to a magnetic bead, whose surface is coated with carboxyl, by using an EDC/NHS activation method so as to obtain an immunomagnetic bead of the vibrio patahaemolyticus; suspending the immunomagnetic bead coupled with polyclonal antibody; adding the immunomagnetic bead to a reaction tube; adding a reaction solution and a sample solution to the reaction tube; adding a washing solution to an elution tube; and carrying out immune enriching on the vibrio patahaemolyticus by using a system.
Description
Technical field
The invention belongs to field of biological detection, the method for be specifically related to a kind of high-throughput enrichment, catching Vibrio parahemolyticus.
Background technology
Vibrio parahemolyticus is a kind of important food-borne pathogens, and the people is in case edible by the fishery products of this fungi pollution, is prone to cause diarrhoea, enterospasm, feels sick, typical gastrointestinal symptoms such as vomiting, heating.Therefore, be the important content of fishery products safety detection to the detection of Vibrio parahemolyticus, as importing and exporting the project that fishery products detect, in tap water, food inspection and foodborne illness source, infect in the work such as pathogeny investigation, all need this bacterium is detected.The traditional detection of Vibrio parahemolyticus need be passed through and increased that bacterium, selectivity are cultivated, Physiology and biochemistry is identified and process such as serological reaction, and complex operation, length consuming time, recall rate are low, is unfavorable for that diagnosis in time and epidemiology traces to the source.Along with development of biology, correlation techniques such as PCR, dna probe, ELISA have been applied to the detection of Vibrio parahemolyticus in recent years, but the complex component in food samples and the clinical sample tends to disturb the accuracy of Vibrio parahemolyticus detection; And under low contamination levels, usually need to cultivate reaching detectability, so also prolong greatly detection time through increasing bacterium.
(Immonumagnetic Separation, IMS) technology is that a kind of pair cell separates the immunological method of catching with enrichment with mikrobe to the separation of immunity magnetic.It possesses fast enriching and catches the purpose target and reduce other impurity interferential advantage.Immunomagnetic beads is formed through the coupling of covalency group by carrier microballoons and immune aglucon.Object bacteria somatocyte surface antigen forms somatic cells and immunomagnetic beads mixture with the antibodies that is connected with magnetic bead, and it is mobile that orientation can take place under the action of a magnetic field this mixture, accumulate in around the magnetic field, thereby with other separating substances.IMS can effectively separate the purpose cell, and can effectively remove the supressor in food samples or the clinical sample, is effective sample-pretreating method.This technology and PCR, technical batterys such as ELISA are with can effectively improving recall rate and sensitivity.At present, utilize IMS to adopt manually-operated method to carry out to the separation of purpose bacterium, operating process is loaded down with trivial details, easy contaminated samples, and reaction time consumption is long, and the treatment capacity of single sample is limited, and can not realize the batch processing of sample.
Summary of the invention
The technical issues that need to address of the present invention are to be: a kind of immunomagnetic beads is provided, and this immunomagnetic beads is applicable to that
system's high-throughput enrichment catches Vibrio parahemolyticus.
This high-throughput enrichment, the method for catching Vibrio parahemolyticus; Adopt the polyclonal antibody of Vibrio parahemolyticus; Utilize the EDC/NHS activation method that the polyclonal antibody of said Vibrio parahemolyticus is coupled at pan coating and on the magnetic bead of carboxyl, obtain the immunomagnetic beads of preparation Vibrio parahemolyticus; The immunomagnetic beads that resuspended coupling resists more; It is joined in the reaction tubes; Add reaction solution and sample liquid again; In the wash-out pipe, add washings,
system of employing carries out immunity enrichment.
The preparation method of immunomagnetic beads:
Magnetic bead activation: wash magnetic bead 1-3 time with 2-(N-morpholino) ethyl sulfonic acid (pH 5), add the carbodiimide solution of 50mg/ml and the N-hydroxy-succinamide solution of 50mg/ml then, room temperature reaction 20-40 minute, obtain the activatory magnetic bead;
Magnetic bead and how anti-coupling: join in the activatory magnetic bead incubated at room temperature 2-4 hour with Vibrio parahaemolyticus is how anti-; Add the PBS solution that contains 1% (w/v) bovine serum albumin, capping 20-40 minute; As preserving liquid, being made into final concentration is the immunomagnetic beads solution of 15mg/ml with the PBS that contains 0.02% (w/v) sodiumazide NaN3,0.1% (w/v) bovine serum albumin BSA in adding.
Among the present invention also the technical issues that need to address provide the scheme that a cover is applicable to the operation of
system, be used for to the Vibrio parahemolyticus high-throughput enrichment and catch.
The scheme that solves the problems of the technologies described above is:
method that Vibrio parahemolyticus is caught in system's high-throughput enrichment of application, its reactions step is following:
(1) getting concentration is that the immunomagnetic beads solution of 15mg/ml is no less than 50 μ l repetitive scrubbing 1-3 time in the PBS lavation buffer solution, adds the resuspended immunomagnetic beads of 100 μ l PBS again.
(2) the reaction suit is installed: place sample hose with the 50ml reaction solution with like the said 100 μ l immunomagnetic beadses of step (1), in the wash-out pipe, add the elutriant of 35ml.
(3) open
instrument; To react suit and be installed in the reaction support, and carry out enrichment and catch.Preferably: the consumption of immunomagnetic beads is 15mg/ml in the step (1), 100 μ l; Reaction solution is the Tris-HCl of pH 6.4 in the step (2), and elutriant is PBS+0.05%Tween-20.
After the application aforesaid method is caught Vibrio parahemolyticus, can adopt the method for selectivity cultivation or PCR to detect enrichment capture ability and the specificity of this method to Vibrio parahemolyticus.
Method of the present invention is compared with prior art; Set up the high-throughput enrichment catching method to Vibrio parahemolyticus based on
system; Easy, the consuming time weak point of operating process, high specificity, highly sensitive; And can unite use with many detection techniques, improve recall rate and sensitivity effectively.This is invented required immunomagnetic beads and prepares simple and easyly, and can preserve the several months at 4 ℃, and stability preferably arranged; The processing volume ratio usual way of
system's single sample enlarges 25-50 doubly, effectively the analytic sample carrier state; And this system can realize high-throughput processing sample, saves the labor force greatly.This method can be widely used in food and medicine supervision and administration organizations at different levels, prevention and control of diseases laboratory etc. the enrichment of Vibrio parahemolyticus in the samples such as fishery products and clinical patient gi tract vomitus, blood, ight soil is caught and subsequent detection.It can be sensitivity, fast, the early diagnosis Vibrio parahemolyticus pollutes and infection state provides reliable experimental evidence.
Embodiment
Embodiment 1
One, the many anti-preparations of Vibrio parahemolyticus:
Get the fresh bacterium liquid of Vibrio parahemolyticus ATCC33846, centrifugal collection thalline, under the uv lamp after the inactivation treatment, immune new zealand white rabbit (available from western pul Bi Kai company), indirect ELISA detects serum, waits to tire＞1: 4000, stops immunity, gets blood after 10 days.Adopt ammonium sulfate multistep precipitator method purified rabbit antiserum(antisera), and adopt SDS-PAGE to detect sero-fast molecular weight and purity.
Two, the preparation of immunomagnetic beads:
The concrete operations of carboxyl magnetic bead coupling Vibrio parahemolyticus polyclonal antibody are following:
Get the 1ml magnetic bead in the centrifuge tube of 2ml, with twice of 2-(N-morpholino) the ethyl sulfonic acid MES (pH5) of 25mM washing magnetic bead.(carbodiimide, 50mg/ml) (N-hydroxy-succinamide 50mg/ml), mixes, room temperature reaction 30min with 500 μ l NHS to add 500 μ l EDC again.How anti-joining through the activatory magnetic bead with 200 μ l6.39mg/ml adds MES again and supplies 1ml, mixes, and cultivates 3h under the room temperature.Add 1ml confining liquid (PBS that contains 1% bovine serum albumin BSA), sealing unreacted site 30min.Add 1ml PBS washed twice.Add preservation liquid and (contain 0.02% sodiumazide NaN
3, the PBS of 0.1% bovine serum albumin BSA) and 1.5ml, being configured to final concentration is the immunomagnetic beads solution of 15mg/ml, 4 ℃ of refrigerators are preserved subsequent use.
Three, use
system (Britain Matrix company) and Vibrio parahemolyticus is carried out enrichment catch, step is following:
1. get immunomagnetic beads solution 100 μ l in the 2ml centrifuge tube of sterilization, place on the magnetic force frame, leave standstill 2min, treat that magnetic bead is adsorbed on a rear flank in band magnetic field fully, remove supernatant, add the PBS of same volume again, mix, on vertical mixed instrument, wash 5min.
2. get Tris-HCl (pH 6.4) 50ml and place disposable sample hose (sample tube); Add the 1ml test with bacterium liquid and 100 μ l magnetic beads; In wash-out pipe (elution tube), add the PBST (PBS+0.05%Tween-20) of 35ml, disposal package packing (consumable) is connected with aseptic pipeline.
3. open
instrument; When
to be shown; Can take off reactive tank (cartridge); Mounted disposal package packing is placed in the reactive tank; Press red button, behind the 15min, when treating the traffic lights Alternation Display; By once, accomplish IMS behind about 1min again.
4. take off complete assembly, take out the wash-out pipe, place on the magnetic force pipe support, when treating that magnetic bead is attracted to a side, remove supernatant, add 200 μ l PBS, be kept at-20 ℃.
Four. the detection of Vibrio parahemolyticus quantity of the catch
To the somatic cells that captures and the mixture of magnetic bead, get the resuspended liquid of 100 μ l respectively and directly be coated with the TCBS flat board, carry out enumeration after in 37 ℃ of constant incubators, cultivating 16-18h, calculate quantity of the catch.
Embodiment 2
The immunomagnetic beads optimization selection of enrichment Vibrio parahemolyticus:
The preparation process of carboxyl immunomagnetic beads is with reference to embodiment 1.The preparation process of tosyl group magnetic bead D-280-T, streptavidin magnetic bead D-280-S and D-100-S is consulted and used explanation (three kinds magnetic bead all available from American I nvitrogen company).Adopt
system's enrichment catch Vibrio parahemolyticus and to its colour developing culturing step that carries out with reference to embodiment 1.
Immunomagnetic beads is seen table 1 to the detected result of Vibrio parahemolyticus capture ability, and when the starter bacteria liquid measure was 6.64log CFU/ml, the amount of each magnetic capture Vibrio parahemolyticus was all in 4-5log CFU/ml level.Carboxyl magnetic capture ability is optimum, is 5.76log CFU/ml, therefore selects carboxyl immunomagnetic beads enrichment Vibrio parahemolyticus for use.
Table 1: the detection of immunomagnetic beads capture ability
The optimization selection of
system response condition:
The optimization of IMBs consumption: select IMBs consumption 0 μ l; 20 μ l, 50 μ l, 100 μ l; 150 μ l; 200 μ l,
system of employing carries out enrichment Vibrio parahemolyticus (specifically seeing embodiment 1), considers in conjunction with experimental result and economic optimum principle; The IMBs consumption of confirming 100 μ l at last is optimum, sees table 2.
Table 2:IMBs consumption is to catching the influence of Vibrio parahemolyticus
The optimization of reaction solution: the Tris-HCl (pH6.4 that selects different pH; PH7.4; PH, 8.4) and PBS as reaction solution,
system of employing carries out enrichment Vibrio parahemolyticus (specifically seeing embodiment 1); Confirm Tris-HCl (pH6.4) optimum at last, see table 3.
Table 3: reaction solution is caught the influence of Vibrio parahemolyticus to IMBs
The optimization of elutriant: select elutriant PBS; PBST1 (PBS+0.05%Tween-20); PBST2 (PBS+0.1%Tween-20);
system of employing carries out enrichment Vibrio parahemolyticus (specifically seeing embodiment 1); Confirm that at last PBST1 (PBS+0.05%Tween-20) is an optimal conditions, sees table 4.
Table 4: elutriant is caught the influence of Vibrio parahemolyticus ability to IMBs
Immunomagnetic beads and the reaction system of utilizing above-mentioned optimal conditions to set up the operation of suitable
system are: adopt carboxyl magnetic bead 100 μ l; Reaction solution is Tris-HCl (pH6.4), and elutriant is PBS+0.05%Tween-20.
Embodiment 3
The specificity test:
The method of application implementation example 1 singly increases listeria spp to 25 strain Vibrio parahemolyticus, 1 strain streptococcus aureus, 1 strain Huo Shi enterobacteria, 1 strain vibrio cholerae, 1 strain Salmonella enteritidis, 1 strain and adopts
system's high-throughput enrichment to catch; Using P CR method then detects the species specificity gene tlh gene of Vibrio parahemolyticus.The result finds that the specific amplification band all appears in 25 strain Vibrio parahemolyticus, and the specific amplification band does not all appear in the non-Vibrio parahemolyticus of 5 strains, shows this method high specificity.
Claims (5)
1. a high-throughput enrichment, the method for catching Vibrio parahemolyticus; It is characterized in that: the polyclonal antibody that adopts Vibrio parahemolyticus; Utilize the EDC/NHS activation method that the polyclonal antibody of said Vibrio parahemolyticus is coupled at pan coating and on the magnetic bead of carboxyl, obtain the immunomagnetic beads of preparation Vibrio parahemolyticus; The immunomagnetic beads that resuspended coupling resists more; It is joined in the reaction tubes; Add reaction solution and sample liquid again; In the wash-out pipe, add washings,
system of employing carries out immunity enrichment.
2. a kind of high-throughput enrichment according to claim 1, the method for catching Vibrio parahemolyticus, it is characterized in that: the preparation method of said immunomagnetic beads is:
Magnetic bead activation: wash magnetic bead 1-3 time with 2-(N-morpholino) ethyl sulfonic acid (pH 5), add the carbodiimide solution of 50mg/ml and the N-hydroxy-succinamide solution of 50mg/ml then, room temperature reaction 20-40 minute, obtain the activatory magnetic bead;
Magnetic bead and how anti-coupling: join in the activatory magnetic bead incubated at room temperature 2-4 hour with Vibrio parahaemolyticus is how anti-; Add the PBS solution that contains 1% (w/v) bovine serum albumin, capping 20-40 minute; Adding is to contain 0.02% (w/v) sodiumazide NaN
3, 0.1% (w/v) bovine serum albumin BSA PBS as preserving liquid, being made into final concentration is the immunomagnetic beads solution of 15mg/ml.
3. a kind of high-throughput enrichment according to claim 1, the method for catching Vibrio parahemolyticus is characterized in that: the reaction process that Vibrio parahemolyticus is caught in application
system's high-throughput enrichment is following:
A. get immunomagnetic beads solution repetitive scrubbing 1-3 time in the PBS lavation buffer solution, add the resuspended immunomagnetic beads of 100 μ l PBS again;
B., reaction system is installed: with the 50ml reaction solution and such as steps A the immunomagnetic beads solution after the processing place sample hose, add detected sample and pat liquid, in the wash-out pipe, add the elutriant of 35ml;
C. open
instrument; To react suit and be installed in the reaction support, carry out immunity enrichment.
4. a kind of high-throughput enrichment according to claim 3, the method for catching Vibrio parahemolyticus is characterized in that:
system that uses carries out the high-throughput enrichment and catches the reaction conditions of Vibrio parahemolyticus and be: the consumption of immunomagnetic beads is for being no less than 0.75mg; Reaction solution is Tris-HCl or PBS; Elutriant is PBS, PBS+0.05%Tween-20 or PBS+0.1%Tween-20.
5. a kind of high-throughput enrichment according to claim 4, the method for catching Vibrio parahemolyticus; It is characterized in that: use
optimizing reaction conditions that Vibrio parahemolyticus is caught in system's high-throughput enrichment, be specially: the consumption of immunomagnetic beads is 1.5mg; Reaction solution is the Tris-HCl of pH 6.4; Elutriant is PBS+0.05%Tween-20.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104651482A (en) * | 2013-11-19 | 2015-05-27 | 北京市理化分析测试中心 | Method for detecting living bacteria body of vibrio parahemolyticus in sample |
| CN104805010A (en) * | 2015-04-22 | 2015-07-29 | 镇江吉蕊生物科技有限公司 | Chip for rapid enrichment of pathogenic bacteria and preparation method of chip |
| CN105242037A (en) * | 2015-09-09 | 2016-01-13 | 上海市疾病预防控制中心 | Preparation method and application of immunomagnetic beads for enriching TDH pathogenic vibrio parahaemolyticus |
| CN107478822A (en) * | 2017-07-11 | 2017-12-15 | 广东省湛江市质量计量监督检测所 | A kind of immunomagnetic beads preparation method and application for being used to be enriched with vibrio parahemolyticus |
| CN107561267A (en) * | 2017-07-11 | 2018-01-09 | 广东省湛江市质量计量监督检测所 | For capturing the NHS immunomagnetic beads preparation method and applications of vibrio parahemolyticus |
| CN108333358A (en) * | 2017-01-19 | 2018-07-27 | 深圳市新产业生物医学工程股份有限公司 | A kind of screening technique of immunoaffinity purification eluent |
| CN112760356A (en) * | 2020-12-23 | 2021-05-07 | 浙江农林大学 | Vibrio parahaemolyticus detection method |
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2012
- 2012-03-12 CN CN2012100632144A patent/CN102586157A/en active Pending
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104651482A (en) * | 2013-11-19 | 2015-05-27 | 北京市理化分析测试中心 | Method for detecting living bacteria body of vibrio parahemolyticus in sample |
| CN104805010A (en) * | 2015-04-22 | 2015-07-29 | 镇江吉蕊生物科技有限公司 | Chip for rapid enrichment of pathogenic bacteria and preparation method of chip |
| CN104805010B (en) * | 2015-04-22 | 2017-01-04 | 江苏微全芯生物科技有限公司 | A kind of pathogenic bacterium fast enriching chip and preparation method thereof |
| CN105242037A (en) * | 2015-09-09 | 2016-01-13 | 上海市疾病预防控制中心 | Preparation method and application of immunomagnetic beads for enriching TDH pathogenic vibrio parahaemolyticus |
| CN108333358A (en) * | 2017-01-19 | 2018-07-27 | 深圳市新产业生物医学工程股份有限公司 | A kind of screening technique of immunoaffinity purification eluent |
| CN107478822A (en) * | 2017-07-11 | 2017-12-15 | 广东省湛江市质量计量监督检测所 | A kind of immunomagnetic beads preparation method and application for being used to be enriched with vibrio parahemolyticus |
| CN107561267A (en) * | 2017-07-11 | 2018-01-09 | 广东省湛江市质量计量监督检测所 | For capturing the NHS immunomagnetic beads preparation method and applications of vibrio parahemolyticus |
| CN112760356A (en) * | 2020-12-23 | 2021-05-07 | 浙江农林大学 | Vibrio parahaemolyticus detection method |
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Application publication date: 20120718 |