CN105242037A - Preparation method and application of immunomagnetic beads for enriching TDH pathogenic vibrio parahaemolyticus - Google Patents

Preparation method and application of immunomagnetic beads for enriching TDH pathogenic vibrio parahaemolyticus Download PDF

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CN105242037A
CN105242037A CN201510567026.9A CN201510567026A CN105242037A CN 105242037 A CN105242037 A CN 105242037A CN 201510567026 A CN201510567026 A CN 201510567026A CN 105242037 A CN105242037 A CN 105242037A
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tdh
immunomagnetic beads
vibrio parahemolyticus
magnetic bead
pathogenic
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张红芝
陈敏
张曦
朱颖莹
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SHANGHAI DISEASE PREVENTION AND CONTROL CENTRE
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    • G01—MEASURING; TESTING
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    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria

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Abstract

The invention relates to a preparation method and an application of pathogenic bacterial immunomagnetic beads and especially relates to a preparation method and an application of immunomagnetic beads for enriching TDH pathogenic vibrio parahaemolyticus. The invention mainly solves the technical field that a detection method in the prior art only allows qualitative analysis to the vibrio parahaemolyticus and cannot achieve separation of the pathogenic vibrio parahaemolyticus. In the technical scheme, a virulence factor TDH in the vibrio parahaemolyticus is expressed and purified and then is used for immunizing rabbits to obtain a monoclonal antibody. The monoclonal antibody then is coupled with magnetic beads to obtain the immunomagnetic beads combined with a marked secondary antibody. The immunomagnetic bead then is used for directly capturing the vibrio parahaemolyticus which produces thermostable direct hemolysin. The immunomagnetic beads are suitable in the fields of entry and exit inspection and quarantine, aquaculture industry, food sanitation and disease prevention and control, and the detection and separation works which are developed in relative laboratories and are based on the vibrio parahaemolyticus which produces thermostable direct hemolysin.

Description

Enrichment TDH pathogenic vibrio parahemolyticus immunomagnetic beads preparation method and application
Technical field
The present invention relates to a kind of Synthesis and applications of pathogen immunomagnetic beads, in particular to the preparation method of immunomagnetic beads of pathogenic vibrio parahemolyticus and the application of producing heat-resisting direct hemolysin (TDH) for enrichment, it take magnetic bead as carrier, polyclonal antibody prepared by immune rabbit is middle identity, process through coupling-washing prepares immunomagnetic beads, efficient capture can produce the pathogenic vibrio parahemolyticus of TDH under suitable conditions, concentration and separation and the detection of pathogenic vibrio parahemolyticus in environment and import and export food inspection can be widely used in.
Background technology
Vibrio parahemolyticus is the important pathogen causing a lot of national especially coastland food origin disease in the world, and it is extensively present in inshore seawater, marine bottom sediment and fish and shrimp, shellfish.The food poisoning symptom that it causes is mainly based on the acute gastroenteritis of stomachache, diarrhoea, Nausea and vomiting, heating etc.At present, the clinical case caused by vibrio parahemolyticus quantitatively exceedes salmonellal clinical case, become the foodborne bacterial pathogens that China is primary, particularly some coastal cities, account for the ratio of food posioning up to more than 60% by this microbial food poisoning.
The bacterial strain that virulence factor is only carried in research display just has pathogenic.But large quantity research shows, nearly all vibrio parahemolyticus be separated from seawater and aquatic products is atoxigenic bacterial strain, although this type of bacterial strain also has the report causing food poisoning, the overwhelming majority is non-pathogenic.And along with detection technique development and application confirm, the content of pathogenic vibrio parahemolyticus really in environment and food is lower, the mixed distribution of pathogenic bacterial strains and non-pathogenic bacterial strains most diverse is in littoral seawater, Haihe River intersection and marine product, and pathogenic bacterial strains only accounts for 1/100-1/1000.The vibrio parahaemolytious great majority utilizing traditional plate isolation method to be separated from environment and food are non-pathogenic bacterial strains, are difficult to be separated to pathogenic bacterial strains.But the vibrio parahemolyticus be separated in clinical patient is all pathogenic bacterial strains more than 90%.
Monitoring not for pathogenic vibrio parahemolyticus just can not effectively early warning and the generation controlling food poisoning.The Pacific Northwest banks in the U.S. in 1997 and all there occurs because the vibrio parahemolyticus caused by edible oyster breaks out case in the oyster bay of the U.S. in 1998, epidemiology survey shows that the vibrio parahemolyticus sum detected in the sample eaten is less than 200cfu/g, according to this result of FDA standard well below 1 × 10 4cfu/g, belongs to " normal qualified samples ".The research of the people such as Kara-Kudo also confirms that the amount of vibrio parahemolyticus total in marine product correctly can not reflect the situation of pathogenic vibrio parahemolyticus, direct relation is not had between the amount of pathogenic vibrio parahemolyticus and the amount of higher total vibrio parahemolyticus, because in the sample of the TDH positive detected, the amount of its total vibrio parahemolyticus is less than 1 × 10 4cfu/g, this prompting, total vibrio parahemolyticus is not a reliable index of the pathogenic vibrio parahemolyticus of prediction [5].Therefore, the detection method set up for pathogenic vibrio parahemolyticus has important meaning for prevention and corntrol food origin disease.
Molecule epidemic disease-ology research finds, in pathogenic vibrio parahemolyticus, Major Virulence Factors is heat-resisting direct hemolysin (TDH), pathogenic bacterial strains more than 90% carries TDH, little pathogenic bacterial strains is only had to carry Thermostable direct hemolysin-related hemolysin (TRH), therefore usual using the mark of TDH as the pathogenic vibrio parahemolyticus of qualification.At present, for the method mainly Molecular Detection that pathogenic vibrio parahemolyticus detects, relatively common are Standard PCR, real-time fluorescence PCR and genetic chip, these detection methods have high sensitivity, simple to operate, the advantage such as high specific and high flux, but these methods can only carry out qualitative analysis to it, can not accomplish to be separated pathogenic vibrio parahemolyticus, be unfavorable for the further investigation to it.
Namely on this basis, expression and purification virulence factor TDH, prepares polyclonal antibody, immunomagnetic beads, and the vibrio parahemolyticus of TDH is carried in enrichment in this research.
Summary of the invention
The present invention aims to provide a kind of enrichment TDH pathogenic vibrio parahemolyticus immunomagnetic beads preparation method and application.Mainly solve current detection method and can only carry out qualitative analysis to it, the technical matters being separated pathogenic vibrio parahemolyticus can not be accomplished.Thinking of the present invention is: utilize virulence factor TDH in the heat-resisting direct hemolysin vibrio parahemolyticus of product to prepare polyclonal antibody as antigen, the latter's binding immunoassay magnetic bead carries out enrichment to food object bacterium, then utilize vibrios colour developing flat board to be separated, thus reach the object detecting targetedly and produce heat-resisting direct hemolysin vibrio parahemolyticus in food.
Realizing technical scheme of the present invention is: a kind of enrichment TDH pathogenic vibrio parahemolyticus immunomagnetic beads preparation method, comprise the following steps: by virulence factor TDH in expression and purification vibrio parahemolyticus, by its immune White Rabbit, obtain polyclonal antibody, then polyclonal antibody is coupled on magnetic bead, obtain the immunomagnetic beads that incorporation of markings two is anti-, then produce heat-resisting direct hemolysin vibrio parahemolyticus in Direct Acquisition food.
Concrete steps are as follows:
1. the expression and purification of virulence factor TDH:
According to the whole genome sequence of the online vibrio parahemolyticus RIMD2210633 bacterial strain announced, software Oligo6.0 is utilized to design the expression primer of virulence factor TDH, and the restriction enzyme site added as required required for clone and protection base.Primer is: FP- cGGGATCCaTGAAGTACCGATATTTTGC; RP- cCCAAGCtTTTATTGTTGATGTTTAC.Through pcr amplification, be cloned into expression plasmid pET-30a, set up recombinant plasmid, proceeded to and express in bacterium BL21, have children outside the state plan centrifugal after abduction delivering, supernatant HisBindPurificationKit (Novagen company) purification of recombinant proteins.
2. the preparation of polyclonal antibody:
With the subcutaneous multi-point injection adult New Zealand rabbits of recombinant protein obtained, dosage is 1mg/ for the first time.14th day, carry out second time immunity, dosage is 0.5mg/.28th day, carry out third time immunity, dosage is 0.5mg/.Within 35th day, carry out the 4th immunity, dosage is 0.5mg/, and immunity once, is total to immunity 7 times week about more afterwards.After immunity is complete, carry out the purifying of rabbit blood.Rabbit blood system good for purifying is filled, frozen for subsequent use in-20 DEG C.
3. the preparation of immunomagnetic beads:
The preparation of immunomagnetic beads is carried out according to magnetic bead instructions.Get 100 μ l bead suspension (being about 1mg containing magnetic bead) in centrifuge tube, centrifuge tube is put on magnetic frame, become after clarification until supernatant liquid, retain the magnetic bead of bottom, discard supernatant, add 1ml magnetic bead coupling buffer, after resuspended magnetic bead, under magnetic force condition, magnetic bead is precipitated; Successively add 100 μ l magnetic bead coupling buffers and 2 μ l antibody (containing antibody about 7 μ g) mixing, hatch 30min, magnetic bead precipitates by magnetic force, abandons supernatant; Add 1ml washing lotion, magnetic bead precipitates by magnetic force; Repeat this step 3 time; Finally add 100 μ l washing lotions, gravity treatment magnetic bead, be put in 4 DEG C of refrigerators for subsequent use.
Advantage of the present invention is: virulence factor TDH main in 1 expression and purification vibrio parahemolyticus, it can be used as antigen, prepare polyclonal antibody, and ensure the high degree of specificity be separated, this is less than cellar culture separation method; 2 utilize immunomagnetic ca pture technology can quick collection, the object bacterium in concentrated environment and food, easy and simple to handle, fast.
Accompanying drawing explanation
Fig. 1 is that immunomagnetic ca pture produces antigen-antibody optimal combination experimental result in the experiment of heat-resisting direct hemolysin vibrio parahemolyticus.
Fig. 2 is the specificity experiments result that immunomagnetic ca pture produces heat-resisting direct hemolysin vibrio parahemolyticus.
Fig. 3 is without producing heat-resisting direct hemolysin vibrio parahemolyticus experimental result in immunomagnetic ca pture aquatic products sample.
Fig. 4 for produce heat-resisting direct hemolysin vibrio parahemolyticus experimental result in immunomagnetic ca pture aquatic products sample.
Embodiment
Enrichment TDH pathogenic vibrio parahemolyticus immunomagnetic beads preparation method, comprises the following steps:
1. the expression and purification of virulence factor TDH:
According to the whole genome sequence of the online vibrio parahemolyticus RIMD2210633 bacterial strain announced, software Oligo6.0 is utilized to design the expression primer of virulence factor TDH, and the restriction enzyme site added as required required for clone and protection base.Primer is: FP- cGGGATCCaTGAAGTACCGATATTTTGC; RP- cCCAAGCtTTTATTGTTGATGTTTAC.Through pcr amplification, be cloned into expression plasmid pET-30a, set up recombinant plasmid, proceeded to and expressed in bacterium BL21, have children outside the state plan centrifugal after abduction delivering, supernatant HisBindPurificationKit (fusion tag protein purification kit, Novagen company) purification of recombinant proteins.
2. the preparation of polyclonal antibody:
With the subcutaneous multi-point injection adult New Zealand rabbits of recombinant protein obtained, dosage is 1mg/ for the first time.14th day, carry out second time immunity, dosage is 0.5mg/.28th day, carry out third time immunity, dosage is 0.5mg/.Within 35th day, carry out the 4th immunity, dosage is 0.5mg/, and immunity once, is total to immunity 7 times week about more afterwards.After immunity is complete, carry out the purifying of rabbit blood.Rabbit blood system good for purifying is filled, frozen for subsequent use in-20 DEG C.
3. the preparation of immunomagnetic beads:
The preparation of immunomagnetic beads is carried out according to magnetic bead instructions.Get the 100 μ l bead suspension (DynabeadsM-280Sheepanti-RabbitIgG that Dynal company produces, 1mg is about containing magnetic bead) in centrifuge tube, centrifuge tube is put on magnetic frame, become after clarification until supernatant liquid, retain the magnetic bead of bottom, discard supernatant, add 1ml magnetic bead coupling buffer, after resuspended magnetic bead, under magnetic force condition, magnetic bead is precipitated; Successively add 100 μ l magnetic bead coupling buffers and 2 μ l antibody (containing antibody about 7 μ g) mixing, hatch 30min, magnetic bead precipitates by magnetic force, abandons supernatant; Add 1ml washing lotion, magnetic bead precipitates by magnetic force; Repeat this step 3 time; Finally add 100 μ l washing lotions, gravity treatment magnetic bead, be put in 4 DEG C of refrigerators for subsequent use.
4. the application of immunomagnetic beads:
Sterile working gets sample in 225ml basic peptone water, light and slow vibration 30min; Get sample supernatant in the centrifugal 2min of 3000rpm, supernatant in the centrifugal 2min of 5000rpm, abandons liquid again, and precipitation is suspended from 1ml washing lotion.Get sample suspension in the tubule that 100 μ l immunomagnetic beads suspensions are housed, the light and slow rotational oscillation 30min of room temperature; Tubule is placed in 1min on magnetic frame, carefully shifts out liquid in pipe; Add the mixing of 1ml washing lotion, be placed in 2min on magnetic frame, shift out washing lotion, repeated washing 3-5 time, add 100 μ l washing lotions and make the magnetic bead of absorption vibrio parahemolyticus product strain wherein resuspended, the dull and stereotyped TCBS of coating colour developing.Immunomagnetic ca pture produces heat-resisting direct hemolysin vibrio parahemolyticus and obtains susceptibility results in table 1.
Table 1 immunomagnetic ca pture produces heat-resisting direct hemolysin vibrio parahemolyticus and obtains susceptibility results
Different bacterial concentration The growth conditions CFU/50ul of bacterium
4.3×10 4 113/ is dull and stereotyped
4.3×10 3 43/ is dull and stereotyped
4.3×10 2 5/ is dull and stereotyped
4.3×10 1 1/ is dull and stereotyped
All the other test findings are shown in Fig. 1-4, and as shown in Figure 1, and the concentration of non-antibody is the bigger the better, and when 2 μ l antibody, i.e. 6.34 μ g antibody, be combined with 50 μ l immunomagnetic beadses, capture rate is now the highest.As shown in Figure 2, salmonella, Escherichia coli, Vibrio vulnificus, staphylococcus aureus and Listeria monocytogenes be totally 5 kinds of foodborne bacterial pathogenses, being diluted to finite concentration adds in the bacterium liquid of the ATCC33847 carrying TDH virulence factor, and immunomagnetic beads specificly can be enriched to ATCC33847.Last supernatant clump count on TCBS flat board of immunomagnetic beads washing, lower than 10 or not growth, illustrates immunomagnetic beads damping fluid by non-specific target wash clean.Immunomagnetic beads after enrichment all has green bacterium colony to occur on TCBS flat board, through being accredited as the heat-resisting direct hemolysin vibrio parahemolyticus of the product carrying TDH.As shown in Figure 3, without the testing result of immunomagnetic beads enrichment, vibrio parahemolyticus is not had to detect.As shown in Figure 4, after immunomagnetic beads enrichment, flat board detects 4 strain vibrio parahemolyticus, be accredited as through PCR and produce heat-resisting direct hemolysin vibrio parahemolyticus.
The present invention utilizes the object bacterium in anti-pathogenic vibrio parahemolyticus virulence factor TDH immunomagnetic beads Direct Acquisition sample, and recycling colour developing is dull and stereotyped to be separated its vibrio parahemolyticus.The magnetic capture that this method adopts combines colour developing plate technique, improve in food the recall rate and sensitivity of producing heat-resisting direct hemolysin vibrio parahemolyticus, solve in food a detection difficult problem of producing heat-resisting direct hemolysin vibrio parahemolyticus, reach accurately, fast, sensitive requirement.

Claims (5)

1. an enrichment TDH pathogenic vibrio parahemolyticus immunomagnetic beads preparation method, it is characterized in that comprising the following steps, by virulence factor TDH in expression and purification vibrio parahemolyticus, by its immune White Rabbit, obtain polyclonal antibody, then polyclonal antibody is coupled on magnetic bead, obtains the immunomagnetic beads that incorporation of markings two is anti-.
2. enrichment TDH according to claim 1 pathogenic vibrio parahemolyticus immunomagnetic beads preparation method, it is characterized in that described virulence factor TDH expression and purification: according to the whole genome sequence of the online vibrio parahemolyticus RIMD2210633 bacterial strain announced, software Oligo6.0 is utilized to design the expression primer of virulence factor TDH, and the restriction enzyme site added as required required for clone and protection base; Primer is: FP- cGGGATCCaTGAAGTACCGATATTTTGC; RP- cCCAAGCtTTTATTGTTGATGTTTAC; Through pcr amplification, be cloned into expression plasmid pET-30a, set up recombinant plasmid, proceeded to and express in bacterium BL21, have children outside the state plan centrifugal after abduction delivering, supernatant fusion tag protein purification kits recombinant protein.
3. enrichment TDH according to claim 1 pathogenic vibrio parahemolyticus immunomagnetic beads preparation method, is characterized in that the acquisition of described polyclonal antibody: with the subcutaneous multi-point injection adult New Zealand rabbits of recombinant protein obtained, and dosage is 1mg/ for the first time; 14th day, carry out second time immunity, dosage is 0.5mg/; 28th day, carry out third time immunity, dosage is 0.5mg/; Within 35th day, carry out the 4th immunity, dosage is 0.5mg/, and immunity once, is total to immunity 7 times week about more afterwards; After immunity is complete, carry out the purifying of rabbit blood; Rabbit blood system good for purifying is filled, frozen for subsequent use in-20 DEG C.
4. enrichment TDH according to claim 1 pathogenic vibrio parahemolyticus immunomagnetic beads preparation method, it is characterized in that the acquisition of described immunomagnetic beads: get 100 μ l bead suspension in centrifuge tube, centrifuge tube is put on magnetic frame, become after clarification until supernatant liquid, retain the magnetic bead of bottom, discard supernatant, add 1ml magnetic bead coupling buffer, after resuspended magnetic bead, under magnetic force condition, magnetic bead is precipitated; Successively add 100 μ l magnetic bead coupling buffers and the mixing of 2 μ l antibody, hatch 30min, magnetic bead precipitates by magnetic force, abandons supernatant; Add 1ml washing lotion, magnetic bead precipitates by magnetic force; Repeat this step 3 time; Finally add 100 μ l washing lotions, gravity treatment magnetic bead, be put in 4 DEG C of refrigerators for subsequent use.
5. an application process for the pathogenic vibrio parahemolyticus immunomagnetic beads of enrichment TDH described in claim 1, is characterized in that comprising the following steps sterile working gets sample in 225ml basic peptone water, light and slow vibration 30min; Get sample supernatant in the centrifugal 2min of 3000rpm, supernatant in the centrifugal 2min of 5000rpm, abandons liquid again, and precipitation is suspended from 1ml washing lotion; Get sample suspension in the tubule that 100 μ l immunomagnetic beads suspensions are housed, the light and slow rotational oscillation 30min of room temperature; Tubule is placed in 1min on magnetic frame, carefully shifts out liquid in pipe; Add the mixing of 1ml washing lotion, be placed in 2min on magnetic frame, shift out washing lotion, repeated washing 3-5 time, add 100 μ l washing lotions and make the magnetic bead of absorption vibrio parahemolyticus product strain wherein resuspended, the dull and stereotyped TCBS of coating colour developing.
CN201510567026.9A 2015-09-09 2015-09-09 Preparation method and application of immunomagnetic beads for enriching TDH pathogenic vibrio parahaemolyticus Pending CN105242037A (en)

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CN105755118A (en) * 2016-03-10 2016-07-13 杭州市疾病预防控制中心 Method for quickly detecting vibrio parahaemolyticus by aid of immunomagnetic beads and loop-mediated isothermal amplification processes
CN105859883A (en) * 2016-04-15 2016-08-17 汕头大学 Immunomagnetic bead containing OmpU antibody, preparation of immunomagnetic bead and method for capturing pathogenic vibrios and detecting pathogenic vibrios through multiplex PCR by utilizing immunomagentic bead
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

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Cited By (4)

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Publication number Priority date Publication date Assignee Title
CN105755118A (en) * 2016-03-10 2016-07-13 杭州市疾病预防控制中心 Method for quickly detecting vibrio parahaemolyticus by aid of immunomagnetic beads and loop-mediated isothermal amplification processes
CN105755118B (en) * 2016-03-10 2020-01-21 杭州市疾病预防控制中心 Method for rapidly detecting vibrio parahaemolyticus by immunomagnetic bead loop-mediated isothermal amplification method
CN105859883A (en) * 2016-04-15 2016-08-17 汕头大学 Immunomagnetic bead containing OmpU antibody, preparation of immunomagnetic bead and method for capturing pathogenic vibrios and detecting pathogenic vibrios through multiplex PCR by utilizing immunomagentic bead
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

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