CN103160603B - LAMP (loop-mediated isothermal amplification) detection kit of vibrio parahaemolyticus and detection method thereof - Google Patents
LAMP (loop-mediated isothermal amplification) detection kit of vibrio parahaemolyticus and detection method thereof Download PDFInfo
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Abstract
The invention discloses an LAMP (loop-mediated isothermal amplification) detection kit of vibrio parahaemolyticus and a detection method thereof and specifically relates to a group of primers for detecting the vibrio parahaemolyticus, which have oligonucleotide sequences shown in SEQ ID No. 1 to SEQ ID No. 6 in a sequence table, as well as the kit containing the primers and the detection method thereof. The kit disclosed by the invention is high in sensitivity, strong in specificity, low in cost and simple and convenient to operate.
Description
Technical field
The present invention relates to LAMP detection kit and the detection method thereof of a kind of Vibrio parahemolyticus, belong to biological technical field.
Background technology
Vibrio parahemolyticus (Vibrio parahaemolyticus, be called for short Vp) be a kind of Gram-negative halophilic bacterium, be distributed widely in the sea-foods such as greater coasting area, salt lake and fish, shellfish, it is the important pathogen that coastland causes food poisoning, can cause the typical gastro-enteritis reactions such as patient occurs diarrhoea, enterospasm, feels sick, vomiting, fever, severe patient can cause septicemia.In recent years, become one of food source property public health problem serious in world wide by the food poisoning that infects Vibrio parahemolyticus initiation.In Japan, account for 20%～30% of whole nation food poisoning number of cases by this microbial food poisoning; Country of China food origin disease monitoring net data presentation maritime provinces Vibrio parahemolyticus causes that the generation scale of food poisoning and crowd's exposure scale are obvious ascendant trend, have been in the poisoning first place of microorganism food.For guaranteeing food safety, protection people's health, the developed countries such as European Union, the U.S. have made clear and definite regulation to the Vibrio parahemolyticus in fishery products.As European Union regulation Vibrio parahemolyticus must not detect, and U.S.'s regulation is less than l × 10
4cFU/g.China classifies this bacterium as main inspection or monitoring target.At present, this bacterium is most one of pathogenic bacterium that fishery products must examine of importing and exporting.
The method that at present detects pathogenic microorganism in food is mainly taking traditional method as main, i.e. isolation and identification method, and the method required time is long, generally needs 5～7 days, sometimes reaches 10～15 days, is difficult to meet the needs of Rapid identification; The round pcr growing up in recent years, is a kind of quick, sensitive, technology that specificity is good, but this technology still depends on the front increasing bacterium step of traditional method at present, often contains PCR inhibitor, thereby affect the amplification of PCR in enrichment liquid; ELISA method detects fast, has quick, sensitive feature as screening method, be to be subject to the extensively screening method of welcome, but the test kit using is all from abroad, expensive, and need to be equipped with its special instrument.Ring mediated isothermal amplification (LAMP) is a kind of new amplification technique based on detecting hereditary material DNA that round pcr grows up that continues.This technology depends on primer and a kind of archaeal dna polymerase with strand displacement characteristic that can identify 6 special regions on target sequence, can be efficiently under isothermal condition, fast, high amplified target sequence specifically; Be characterized in without special, expensive detecting instrument, only need simple thermostatic equipment or heat block, can complete pcr amplification, can meet the needs of Site Detection.
Nanometer magnetic bead is to use nanotechnology, on the basis of traditional biological and Modern Molecular Biotechnology, carries out the research of the aspect such as bio-science, medical science.In molecule, cell and individual level for food-borne pathogens detects and the aspect such as the diagnosis of disease provides novel material, new technology and method.Nanometer magnetic bead has significant application value in the compartment analysis of biological sample: (1) efficiently concentrating: nanometer magnetic bead specific surface area is large, and significantly increasing reaction interface can efficiently concentrating biological sample; (2) inhibitor in very effective removal nucleic acid amplification reaction: with present detection method, pathogenic microorganism not of the same race adopts different enrichment liquids, additive in enrichment liquid often has stronger restraining effect to nucleic acid amplification, and the method that adopts magnetic bead to separate can very effective removal inhibitor; (3) operability: nanometer magnetic bead has superparamagnetic effect, externally-applied magnetic field can move and separate it fast; (4) scalar nature: biomolecule labeling method is simple, reliable.Antibody is the important biological active materials of a class, be widely used in the fields such as inspection and quarantine, medical diagnosis on disease, drug screening, national defence, space flight, judicial expertise, food sanitation, environmental monitoring and military detection, become the valuable source of Development of Novel immunoassay technology and the fast development of promotion detection quarantine industry.The nanometer magnetic bead of the present invention's application and the coupling of anti-Vibrio parahemolyticus monoclonal antibody, be immunomagnetic beads (Immunomagnetic Beads, IMB), it is good that it has selectivity, high specificity, can play the effect of the concentrated bacterium of enrichment, effectively avoid or reduce undetectedly, and can remove the composition that suppresses nucleic acid amplification in food samples.Immunomagnetic beads is separated to (Immunomagnetic separation, IMS) and combine with LAMP technology, set up IMS-LAMP detection method, can greatly improve detection efficiency.And developed thus Vibrio parahemolyticus IMS-LAMP detection kit, have broad application prospects.
Summary of the invention
First technical problem that the present invention will solve is to provide strong, the highly sensitive LAMP primer for detection of Vibrio parahemolyticus of a group-specific.
Second technical problem that the present invention will solve is to provide a kind of simple to operate, result LAMP detection kit of Vibrio parahemolyticus accurately.
The 3rd technical problem that the present invention will solve is to provide the IMS-LAMP detection method of a kind of Vibrio parahemolyticus.
For achieving the above object, the present invention is by the following technical solutions:
The invention provides one group and detect the primer of Vibrio parahemolyticus, this group primer is the gene order design corresponding according to the NCBI number of including M36437, is made up of to the oligonucleotide of base sequence shown in sequence table SEQ ID No.6 sequence table SEQ ID No.1; Wherein SEQ ID No.1 is outside upstream primer, SEQ ID No.2 is outside downstream primer, and SEQ ID No.3 is inner side upstream primer, and SEQ ID No.4 is inner side downstream primer, SEQ ID No.5 is ring-type upstream primer, and SEQ ID No.6 is ring-type downstream primer.Refer to table 1.
Table 1 primer sequence
Primer provided by the present invention has the following advantages: (1) efficient and sensible.The introducing Loop primer that studies show that in recent years will contribute to improve detection sensitivity, Reaction time shorten.(2) high specificity.
The LAMP detection kit that the invention provides a kind of Vibrio parahemolyticus, it comprises following material:
(1) coupling has the immunomagnetic beads of anti-Vibrio parahemolyticus monoclonal antibody;
Wherein, anti-Vibrio parahemolyticus monoclonal antibody can be disclosed anti-Vibrio parahemolyticus monoclonal antibody in prior art, preferably this monoclonal antibody is to be the Vibrio parahemolyticus flagellin monoclonal antibody of the hybridoma cell strain secretion generation of CGMCC No.6061 by deposit number, and specifying information and preparation method are shown in the content in Chinese invention patent application CN102659942A.By using coupling to have the immunomagnetic beads of anti-Vibrio parahemolyticus monoclonal antibody, the effectively Vibrio parahemolyticus in enrichment sample to be checked, improves the specificity and the sensitivity that detect.
(2) LAMP reaction solution, it comprises the outside upstream primer (F3) shown in sequence table SEQ ID No.1, outside downstream primer (B3) shown in sequence table SEQ ID No.2, inner side upstream primer (FIP) shown in sequence table SEQ ID No.3, inner side downstream primer (BIP) shown in sequence table SEQ ID No.4, ring-type upstream primer (LF) shown in sequence table SEQ ID No.5, the ring-type downstream primer (LB) shown in sequence table SEQ ID No.6; Described primer all entrusts Dalian precious biotechnology company limited synthetic;
(3) Bst archaeal dna polymerase: 8U/ μ L, purchased from NEB company;
(4) negative control: DEPC water;
(5) positive control: the nucleic acid being extracted by Vibrio parahemolyticus reference culture ATCC17802, as positive control, adopts bacterial genomes DNA extraction test kit and extracts 1mL sample gene group DNA by its operation instructions, and nucleic acid concentration is about 80ug/ml;
(6) nitrite ion: SYBR Green I dyestuff, purchased from Invitrogen company.
Further, above-mentioned LAMP reaction solution also comprises ThermoPol damping fluid, dNTPs and MgSO
4; Wherein ThermoPol damping fluid, purchased from NEB company, 1 × ThermoPol damping fluid is containing 0.1%TritonX-100,10mM (NH4)
2sO
4, 10mM KCl, 20mM Tris-HCl (pH8.8); DNTPs: purchased from sky root.
The present invention also provides the detection method of a kind of Vibrio parahemolyticus, and the method comprises the following steps:
(1) immunomagnetic beads enrichment thalline: get 1.5mL immunomagnetic beads in centrifuge tube, be placed in after magnetic separator frame absorption 1min, abandon supernatant, immunomagnetic beads is resuspended with 30 μ L PBS; Get 1mL sample to be checked or bacteria suspension and add in immunomagnetic beads suspension, put upside down to mix and make magnetic bead in suspended state; After 30min, magnetic separator frame absorption magnetic bead 1min, with enrichment thalline, abandons supernatant, immunomagnetic beads PBS washed twice, and each 30s, finally uses the resuspended magnetic bead of 50 μ L DEPC water, obtains the thalline sample after enrichment;
Wherein, 10 × PBS buffered soln is: NaCl80g, NaHPO
4× 12H
2o29g, KCl2g, KH
2pO
42g, is first dissolved in 900ml deionized water, after fully dissolving, adds deionized water to 1000ml again, and room temperature preservation, carries out 1:10 dilution when use.
(2) DNA of the thalline sample that extraction step (1) obtains, it is template DNA, can utilize method well known in the prior art or test kit to extract, for example bacterial genomes DNA extraction test kit or equivalent agent box, or method for boiling extracts sample gene group DNA;
(3) carry out LAMP reaction, this LAMP reaction system sees the following form:
Table 2LAMP reaction system
(4) reaction conditions: 65 DEG C of isothermal reaction 60min, 85 DEG C of heating 2min make enzyme deactivation, and reaction finishes;
(5) result is judged:
Colour-change: adding 1 μ L nitrite ion to reaction end-body system is SYBR Green I fluorescence dye, and positive reaction is fluorescent green, and negative reaction keeps the fluorescent orange of SYBR Green I dyestuff.
Electrophoresis detection: the amplified production of LAMP method is the stem-ring texture DNA of various different lengthss, therefore positive reaction is detected and is trapezoid-shaped strips by 1.5% agarose electrophoresis, and negative reaction does not have trapezoidal amplified band to occur.
Advantage of the present invention is:
The immunomagnetic beads of the present invention application and the coupling of anti-Vibrio parahemolyticus monoclonal antibody, has selectivity good, and high specificity, can play the effect of the concentrated bacterium of enrichment, effectively avoids or reduces undetectedly, and can remove the composition that suppresses nucleic acid amplification in food samples.The present invention designs the identification in 6 distinguished sequence districts of 6 primer pair target sequences, has ensured the high degree of specificity of LAMP amplification.Be that LAMP can, from only differing the gene sample of 1 Nucleotide, find out corresponding target sequence and increase.And the present invention increases under isothermal condition, can be because temperature change causes leeway, and be subject to the impact of non-target sequence little.Simultaneously consuming time short, in 1h, target sequence can be expanded to 10
9doubly.
Below in conjunction with specification drawings and specific embodiments, the invention will be further described, all any this areas of doing according to the disclosure of invention be equal to replacement, all belong to protection scope of the present invention.
Brief description of the drawings
Fig. 1 IMS-LAMP detection specificity figure; (wherein M: molecular weight standard, DL2000; 1 Vibrio parahemolyticus nucleic acid amplification result; 2 vibrio alginolyticus nucleic acid amplification results; 3 Wound vibrio amplifications; 4 vibrio fluvialis nucleic acid amplification results; 5 take Nice vibrios nucleic acid amplification result; 6 vibrio proteolyticus nucleic acid amplification results; 7 single listeria spp nucleic acid amplification results that increase; 8 enterobacter cloacae nucleic acid amplification results; 9 streptococcus aureus nucleic acid amplification results; 10 Salmonella paratyphi A nucleic acid amplification results; 11 Escherichia coli nucleic acid amplifications; 12 Salmonella enteritidis nucleic acid amplification results; 13 shigella flexneri nucleic acid amplification results; 14 Salmonella typhimurium nucleic acid amplification results; 15 Enterobacter sakazakii nucleic acid amplification results; 16 enterococcus faecalis nucleic acid amplification results; The negative contrast of NC.）
Fig. 2 IMS-LAMP detection sensitivity figure; (wherein M: molecular weight standard, DL2000; PC: Vibrio parahemolyticus positive control;-1～-7: amplification after Vibrio parahemolyticus positive control doubling dilution; The negative contrast of NC.）
Fig. 3 IMS-LAMP detects matrix and adds lab diagram; (wherein M: molecular weight standard, DL2000; PC: Vibrio parahemolyticus positive control; 2h～12h: detected result after corresponding time increasing bacterium; The negative contrast of NC.）
Embodiment
Below in conjunction with specific embodiment, enter-walk to set forth the present invention.Be not used in and limit the scope of the invention but these embodiment only limit to the present invention is described.
Experimental technique in following examples is ordinary method if no special instructions.
In following examples, material therefor, reagent etc. all can obtain if no special instructions from commercial channels.
Embodiment 1: coupling has the preparation of the immunomagnetic beads of anti-Vibrio parahemolyticus monoclonal antibody
The acquisition of anti-Vibrio parahemolyticus monoclonal antibody
The present invention can use in prior art disclosed anti-Vibrio parahemolyticus monoclonal antibody, in order to reach better detection effect, preferably, the Vibrio parahemolyticus flagellin monoclonal antibody that the hybridoma cell strain secretion that it is CGMCC No.6061 that the present invention uses by deposit number produces, the specifying information of this monoclonal antibody and preparation method are shown in the content in Chinese invention patent application CN102659942A.
Coupling has the preparation of the immunomagnetic beads of this anti-Vibrio parahemolyticus monoclonal antibody
1. the preparation of nanometer magnetic bead
The preparation method of immunomagnetic beads of the present invention comprises the preparation of nanometer Superparamagnetic Fe_3O_4 and the finishing of specific antibody thereof, specifically provides a kind of preferred preparation method at this:
2. the preparation of the stable Superparamagnetic Fe_3O_4 of polyacrylic acid
(1) preparation of source of iron: accurately take the polyacrylic acid (Mw=～1800) of 0.8mmol FeCl3 and 8mmol in 34ml Diethylene Glycol solvent, under the condition stirring at Quick mechanical, mixed solution is heated to 220 DEG C in argon gas atmosphere, keep heating 30min.
(2) preparation of sodium hydroxide solution: accurately take 2.0gNaOH in 20mL Diethylene Glycol solvent, under argon gas atmosphere, mixed solution is heated to 120 DEG C, keep 1h, then temperature is reduced to 70 DEG C, stand-by.
(3) get 1.75mL sodium hydroxide solution, be injected into fast in the mixed solution of source of iron, then temperature of reaction is adjusted to 210 DEG C, under mechanical stirring, keeping 1h fast, then cool to room temperature, sample is cleaned.
(4) sample clean: taking ethanol as precipitation agent, (volume ratio 2:1) carries out eccentric cleaning (10000r/min) to gained sample, repeated washing three times, finally by sample dispersion in deionized water.
3. the modification of specific antibody
The modification of antibody comprises the activation on magnetic particle surface, and the coupling of specific antibody is modified and the BSA in non-specific site seals.
(1) surface active of magnetic particle: get the prepared magnetic nano-particle of 2mg, the method separating by magnetic in the phosphate buffer solution of 1mL PH=6.0 is cleaned twice particle.
(2) accurately take 1.38mg Sufo-NHS and 3mg EDC in 1mL2mg/mL magnetic nano-particle dispersion liquid, in ice bath, under concussion condition, keep 30min to carry out surface active.Then mixed solution is carried out to magnetic and separate twice, wash excessive Sufo-NHS and EDC activator off.
(3) magnetic nano-particle after activation is distributed in the phosphate buffer solution of pH=7.9, in dispersion liquid, the concentration of magnetic nano-particle is 2mg/mL.Get 100 μ g specific antibodies in the magnetic nano-particle solution of 1mL activation, under room temperature, in shaking table (200r/min), keep 4h, finally separate and clean by magnetic, remove the excessive specific antibody that is not coupled to magnetic particle surface.
(4) particle after cleaning in 2mg step 3 is distributed in the phosphate buffer solution that 2mL BSA mass concentration is 1% (pH=7.4), under room temperature, in shaking table (200r/min), keep 10h to carry out the sealing in non-specific site, then separate gained particle is separated by magnetic, be finally distributed to (pH=7.4) in the phosphate buffer solution that 2mL BSA mass concentration is 0.1% and preserve.
(5) particle diameter of gained immunomagnetic beads is in 90nm left and right, and saturation magnetization can reach 64emu/g, and superparamagnetic performance is good, and magnetic responsiveness is strong, and dispersion stabilization is good, and biological immune activity is high.
Embodiment 2: the composition of the LAMP detection kit of Vibrio parahemolyticus
(1) coupling has the immunomagnetic beads of anti-Vibrio parahemolyticus monoclonal antibody; The for example immunomagnetic beads of preparation in embodiment 1;
(2) LAMP reaction solution, it comprises the outside upstream primer (F3) shown in sequence table SEQ ID No.1, outside downstream primer (B3) shown in sequence table SEQ ID No.2, inner side upstream primer (FIP) shown in sequence table SEQ ID No.3, inner side downstream primer (BIP) shown in sequence table SEQ ID No.4, ring-type upstream primer (LF) shown in sequence table SEQ ID No.5, the ring-type downstream primer (LB) shown in sequence table SEQ ID No.6; Described primer all entrusts Dalian precious biotechnology company limited synthetic;
Further, described LAMP reaction solution also comprises ThermoPol damping fluid, dNTPs and MgSO
4; Wherein ThermoPol damping fluid, purchased from NEB company, 1 × ThermoPol damping fluid is containing 0.1%TritonX-100,10mM (NH4)
2sO
4, 10mM KCl, 20mM Tris-HCl (pH8.8); DNTPs: purchased from sky root;
(3) Bst archaeal dna polymerase: 8U/ μ L, purchased from NEB company;
(4) negative control: DEPC water;
(5) positive control: the nucleic acid being extracted by Vibrio parahemolyticus reference culture ATCC17802, as positive control, adopts bacterial genomes DNA extraction test kit and extracts 1mL sample gene group DNA by its operation instructions, and nucleic acid concentration is about 80ug/ml;
(6) nitrite ion: SYBR Green I dyestuff, purchased from Invitrogen company.
Embodiment 3: detect specificity and the susceptibility test of Vibrio parahemolyticus test kit
1. detection specificity analysis
In his-and-hers watches 3, listed bacterial strain carries out respectively IMS-LAMP detection, and result shows that the amplified production of Vibrio parahemolyticus reference culture, through the aobvious fluorescent green of SYBR Green I dyeing, detects and occurs typical scalariform amplified band through agarose gel electrophoresis; And all the other bacterial strains are all without specific amplification (see figure 1), occur without any false positive and false negative result, illustrate that the method detects Vibrio parahemolyticus and has good specificity.
Table 3 experiment bacterial strain list
| Sequence number | Strain name | Numbering |
| 1 | Vibrio parahemolyticus | ATCC17802 |
| 2 | Vibrio alginolyticus | ATCC1833 |
| 3 | Vibrio vulnificus | ATCC1758 |
| 4 | Vibrio fluvialis | ATCC1.1611 |
| 5 | Take Nice vibrios | ATCC1.1612 |
| 6 | Vibrio proteolyticus | ATCC1.1826 |
| 7 | Single listeria spp that increases | ATCC15313 |
| 8 | Enterobacter cloacae | CGMCC1.57 |
| 9 | Streptococcus aureus | ATCC25923 |
| 10 | Salmonella paratyphi A | CMCC50001 |
| 11 | Intestinal bacteria | ATCC25912 |
| 12 | Salmonella enteritidis | CMCC50041 |
| 13 | Shigella flexneri | CMCC51571 |
| 14 | Salmonella typhimurium | CMCC50115 |
| 15 | Enterobacter sakazakii | ATCC29544 |
| 16 | Enterococcus faecalis | CGMCC1.2135 |
2. detection sensitivity analysis
The bacteria suspension of Vibrio parahemolyticus reference culture ATCC17802 is carried out to 10 times of gradient dilutions, get respectively the each gradient dilution liquid of 1mL and carry out IMS-LAMP detection.Found that LAMP can detect-5 extent of dilution (Fig. 2), corresponding Vibrio parahemolyticus cell concentration is 6.8 × 10
2cFU/mL, i.e. the sensitivity of this detection method is 6.8 × 10
2cFU/mL.
IMS-LAMP detection method is described above.
Embodiment 4: the food substrate that detects Vibrio parahemolyticus test kit adds experiment
(1) Vibrio parahemolyticus reference culture ATCC17802 gradient dilution plate count for bacteria suspension, the bacteria suspension of getting concentration and be 0～10CFU/mL does lower concentration and adds experiment.
(2) learn from else's experience traditional method (GB4789.7) checking without the scallop 25g of Vibrio parahemolyticus, add (sterilizing) in the wide-necked bottle that 225mL basic peptone water (APW) is housed, add 1mL to add the bacteria suspension that concentration is 0～10CFU/mL.After fully mixing, be placed in 36 DEG C of cultivations.
(3) get 1mL nutrient solution respectively at 2h, 4h, 6h, 8h, 10h, 12h and carry out IMS-LAMP detection, IMS-LAMP detection method is described above.
Through counting, be 1CFU/mL for the bacteria suspension concentration of adding experiment, show that it is 1CFU/25g sample that food substrate adds the interpolation concentration of testing.After increasing bacterium 8h, sample, available IMS-LAMP detection method detects Vibrio parahemolyticus (Fig. 3).
Result shows that detection Vibrio parahemolyticus IMS-LAMP test kit of the present invention is in the detection of food samples, and sensitivity can reach 1CFU/25g sample.
Obviously, the above embodiment of the present invention is only for example of the present invention is clearly described, and is not the restriction to embodiments of the present invention.For those of ordinary skill in the field, can also make other changes in different forms on the basis of the above description.Here cannot give exhaustive to all embodiments.Everyly belong to apparent variation or the still row in protection scope of the present invention of variation that technical scheme of the present invention extends out.
Claims (3)
1. a LAMP detection kit for Vibrio parahemolyticus, is characterized in that, this test kit comprises following material:
(1) coupling has the superparamagnetism Fe of anti-Vibrio parahemolyticus monoclonal antibody
3o
4nanoparticle;
(2) LAMP reaction solution, it comprises: the outside upstream primer shown in sequence table SEQ ID No.1, outside downstream primer shown in sequence table SEQ ID No.2, inner side upstream primer shown in sequence table SEQ ID No.3, inner side downstream primer shown in sequence table SEQ ID No.4, ring-type upstream primer shown in sequence table SEQ ID No.5, the ring-type downstream primer shown in sequence table SEQ ID No.6;
(3) Bst archaeal dna polymerase;
(4) negative control;
(5) positive control;
(6) nitrite ion: SYBR Green I dyestuff;
Wherein, described coupling has the superparamagnetism Fe of anti-Vibrio parahemolyticus monoclonal antibody
3o
4nanoparticle is prepared by following method:
(a) preparation of source of iron: take 0.8mmol FeCl
3in 34ml Diethylene Glycol solvent, under churned mechanically condition, in argon gas atmosphere, mixed solution is heated to 220 DEG C with the polyacrylic acid of 8mmol, keeps heating 30min;
(b) preparation of sodium hydroxide solution: take 2.0gNaOH in 20mL Diethylene Glycol solvent, under argon gas atmosphere, mixed solution is heated to 120 DEG C, keep 1h, then temperature is reduced to 70 DEG C, stand-by;
(c) get 1.75mL sodium hydroxide solution, be injected into fast in the mixed solution of source of iron, then temperature of reaction is adjusted to 210 DEG C, under mechanical stirring, keep 1h, then cool to room temperature, obtain magnetic nano-particle;
(d) taking ethanol as precipitation agent, gained magnetic nano-particle is carried out to eccentric cleaning, finally magnetic nano-particle is dispersed in deionized water, for subsequent use;
(e) get magnetic nano-particle prepared by 2mg step (d), the method separating by magnetic in the phosphate buffer solution of 1mL pH=6.0 is cleaned particle;
(f) take 1.38mg Sufo-NHS and 3mg EDC in the solution of 1mL2mg/mL step (e), in ice bath, under concussion condition, keep 30min to carry out surface active, then mixed solution is carried out to magnetic and separate twice, wash excessive Sufo-NHS and EDC activator off;
(g) magnetic nano-particle after activation is distributed in the phosphate buffer solution of pH=7.9, in dispersion liquid, the concentration of magnetic nano-particle is 2mg/mL, get 100 μ g and secrete the Vibrio parahemolyticus flagellin monoclonal antibody producing in the magnetic nano-particle solution of 1mL activation by the hybridoma cell strain of CGMCC No.6061, under room temperature, in shaking table, keep 4h, shaking speed is 200r/min, finally separate and clean by magnetic, remove the excessive antibody that is not coupled to magnetic particle surface;
(h) particle after cleaning in 2mg step (g) being distributed to 2mL BSA mass concentration is 1%, in the phosphate buffer solution of pH=7.4, under room temperature, in shaking table, keep 10h to carry out the sealing in non-specific site, shaking speed is 200r/min, then separate gained particle is separated by magnetic, finally be distributed in the phosphate buffer solution that 2mL BSA mass concentration is 0.1%, pH=7.4 and preserve.
2. test kit according to claim 1, is characterized in that, described LAMP reaction solution also comprises ThermoPol damping fluid, dNTPs and MgSO
4.
3. test kit according to claim 2, is characterized in that, in described LAMP reaction solution, the use final concentration of various compositions is 1 × ThermoPol damping fluid, 1.6mM dNTPs, 2.0mM MgSO
4, downstream primer, 1.0 μ M ring-type upstream primers, 1.0 μ M ring-type downstream primers inside upstream primer, 2.0 μ M inside downstream primer, 2.0 μ M outside upstream primer, 0.2 μ M outside 0.2 μ M.
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| CN109112224A (en) * | 2018-08-24 | 2019-01-01 | 暨南大学 | Primer and its kit and method based on digital LAMP technology detection vibrio parahaemolytious |
| CN114350827A (en) * | 2022-01-21 | 2022-04-15 | 国科宁波生命与健康产业研究院 | CDA primer group and kit for detecting vibrio parahaemolyticus and application of CDA primer group and kit |
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| P. Prompamorn等.The development of loop-mediated isothermal amplification combined with lateral flow dipstick for detection of Vibrio parahaemolyticus.《Letters in Applied Microbiology》.2011,第52卷 * |
| The development of loop-mediated isothermal amplification combined with lateral flow dipstick for detection of Vibrio parahaemolyticus;P. Prompamorn等;《Letters in Applied Microbiology》;20111231;第52卷;第346页表2 * |
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