CN103866033B - Staphylococcus aureus immune PCR (polymerase chain reaction) detection kit - Google Patents
Staphylococcus aureus immune PCR (polymerase chain reaction) detection kit Download PDFInfo
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- CN103866033B CN103866033B CN201410130783.5A CN201410130783A CN103866033B CN 103866033 B CN103866033 B CN 103866033B CN 201410130783 A CN201410130783 A CN 201410130783A CN 103866033 B CN103866033 B CN 103866033B
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- 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
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- 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
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
- C12Q1/14—Streptococcus; Staphylococcus
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Abstract
The invention discloses a PCR (polymerase chain reaction) tube coated by a specific antibody and an immune PCR (polymerase chain reaction) detection kit, which are used for detecting staphylococcus aureu. A monoclonal antibody capable of specifically enriching staphylococcus aureu in a sample is pre-coated on the PCR tube, the detection kit disclosed by the invention is capable of fast, accurately and sensitively detecting staphylococcus aureu from samples such as food, the sensitivity can achieve 10<3>-10<4>cfu/ml, and is improved by 10-100 times in comparison with that of the conventional PCR. The immunology and a molecular biology method are organically combined to enrich and detect the staphylococcus aureu in the sample in the PCR tube, the operation is simple, the cost is low, the detection is fast and the result is accurate.
Description
Technical field
The invention belongs to biotechnology and field of immunology, be specifically related to a kind of immuno-PCR detection kit detecting streptococcus aureus.
Background technology
Streptococcus aureus (Staphylococcus aureus) is modal food-borne pathogens, is extensively present in physical environment.Streptococcus aureus mainly pollutes the animal products such as milk, meat, egg, fish and goods thereof, comprise beverage, cold drink, cake that milk makes, cold cuts, meat product can, leftovers, fried egg, glutinous rice cakes, bean jelly, surplus rice and rice wine etc., popular name " addicted to meat bacterium ".Streptococcus aureus mainly can cause human body illness after infecting human body, first affecting conditions as caused pyogenic infection, as wound suppuration, phlegmon, pneumonia, meningitis, pericarditis, septicemia etc.; It two is the toxin diseases produced by streptococcus aureus, cause dizziness after people is edible, feel sick, diarrhoea, the food poisoning symptoms such as acute gastroenteritis such as vomiting, and the shock-syndrome caused by toxin shows as high heat, ypotension, diarrhoea, fash, shock.The detection of current streptococcus aureus depends on the biochemical identification of GB defined, and its shortcoming is that complex operation, sense cycle are longer, cannot adapt to a large amount of sample examinations.
Immunology detection is quick, accurate, the most stable rapid detection means occurred in recent years; but the whole dependence on import of testing product; China there is no at present and has complete independent intellectual property right; and the rapid detection product detecting practice can be applied to; this patent is detect target with streptococcus aureus, and technology required for protection relates to streptococcus aureus rapid detection product.
Summary of the invention
The object of this invention is to provide a kind of PCR reaction tubes for detecting streptococcus aureus.
Another object of the present invention is to provide a kind of immuno-PCR detection kit for detecting streptococcus aureus.
A first aspect of the present invention there is provided a kind of PCR reaction tubes for detecting streptococcus aureus, and described PCR reaction tubes comprises:
Immuno-PCR pipe; With
Be coated in the anti-Staphylococcus aureus monoclonal antibody of described immuno-PCR pipe inboard wall of tube body, described anti-Staphylococcus aureus monoclonal antibody is by mouse hybridoma cell system 5D2G2D9C1, CGMCC No.8763 or 5D6D9G3B4, CGMCC No.8764 produces.
A second aspect of the present invention there is provided a kind of detection kit, and described test kit contains described PCR reaction tubes.
In a preference, described test kit also containing container a, is equipped with described PCR reaction tubes in described container a.
In another preference, described test kit is also containing damping fluid, dNTP, Taq DNA polymerase, ddH
2o, primer, positive control and negative control.
In another preference, described primer comprises forward primer and reverse primer.
In another preference, described positive control is the streptococcus aureus bacterium liquid of deactivation, and described negative control is the brain heart leach liquor meat soup of sterilizing.
The details of all respects of the present invention is able to detailed description by chapters and sections subsequently.By hereafter and the description of claim, feature of the present invention, object and advantage will be more obvious.
Accompanying drawing explanation
Fig. 1 is use flow process and the schematic diagram of test kit of the present invention.
Fig. 2 is streptococcus aureus Direct PCR gradient dilution detectability result of study.
Wherein, M:1000bp DNA ladder; N: negative control; The bacterial concentration that 1-8 swimming lane is corresponding is: 1.24 × 10
8~ 1.24 × 10cfu/mL.
Fig. 3 is that staphylococcus aureus immunity PCR detection kit detects streptococcus aureus gradient dilution detectability result of study.
Wherein, M:1000bp DNA ladder; N: negative control; The bacterial concentration that 1-8 swimming lane is corresponding is: 1.24 × 10
8~ 1.24 × 10cfu/mL.
Fig. 4 is the specific result of detection kit.
Wherein, M:1000bp DNA ladder; N: negative control; 1-11 swimming lane is respectively: streptococcus aureus ATCC82346, streptococcus aureus ATCC27660, Shu Shi salmonella typhi ATCC22956, Salmonella enteritidis ATCC13076, Listeria monocytogenes ATCC43251, yersinia entero-colitica ATCC23715, shigella flexneri ATCC12022, bacillus ceylonensis A ATCC25931, shigella dysenteriae ATCC51329, beta hemolytic streptococcus ATCC10373, Enterobacter sakazakii ATCC29004.
Fig. 5 simulates milk sample immuno-PCR test kit detectability result of study of carrying disease germs.
A(1) milk sample Direct PCR detectability result of study of carrying disease germs is simulated;
A(2) milk sample immunocapture PCR detection kit detectability result of study of carrying disease germs is simulated;
Wherein, M:1000bp DNA ladder; N: negative control; No. 1 swimming lane is milk sample (not adding bacterium liquid); The bacterial concentration that 2-8 swimming lane is corresponding is: 1.24 × 10
2~ 1.24 × 10
8cfu/mL.
Fig. 6 is analog band bacteria yoghurt sample immuno-PCR test kit detectability result of study.
B(1) analog band bacteria yoghurt sample Direct PCR detectability result of study;
B(2) analog band bacteria yoghurt sample immunocapture PCR detection kit detectability result of study;
Wherein, M:1000bp DNA ladder; N: negative control; No. 1 swimming lane is yoghurt example (not adding bacterium liquid); The bacterial concentration that 2-8 swimming lane is corresponding is: 1.24 × 10
2~ 1.24 × 10
8cfu/mL.
Fig. 7 simulates sausage sample immuno-PCR test kit detectability result of study of carrying disease germs;
C(1) sausage sample Direct PCR detectability result of study of carrying disease germs is simulated;
C(2) sausage sample immunocapture PCR detection kit detectability result of study of carrying disease germs is simulated;
Wherein, M:1000bp DNA ladder; N: negative control; No. 1 swimming lane is sausage sample (not adding bacterium liquid); The bacterial concentration that 2-8 swimming lane is corresponding is: 1.24 × 10
2~ 1.24 × 10
8cfu/mL.
Fig. 8 simulates cake sample immuno-PCR test kit detectability result of study of carrying disease germs.
D(1) cake sample Direct PCR detectability result of study of carrying disease germs is simulated;
D(2) cake sample immunocapture PCR detection kit detectability result of study of carrying disease germs is simulated;
Wherein, M:1000bp DNA ladder; N: negative control; No. 1 swimming lane is cake sample (not adding bacterium liquid); The bacterial concentration that 2-8 swimming lane is corresponding is: 1.24 × 10
2~ 1.24 × 10
8cfu/mL.
Fig. 9 simulates samples of juice immuno-PCR test kit detectability result of study of carrying disease germs.
E(1) samples of juice Direct PCR detectability result of study of carrying disease germs is simulated;
E(2) samples of juice immunocapture PCR detection kit detectability result of study of carrying disease germs is simulated;
Wherein, M:1000bp DNA ladder; N: negative control; No. 1 swimming lane is samples of juice (not adding bacterium liquid); The bacterial concentration that 2-8 swimming lane is corresponding is: 1.24 × 10
2~ 1.24 × 10
8cfu/mL.
Figure 10 simulates egg sample immuno-PCR test kit detectability result of study of carrying disease germs.
F(1) egg sample Direct PCR detectability result of study of carrying disease germs is simulated;
F(2) egg sample immunocapture PCR detection kit detectability result of study of carrying disease germs is simulated;
Wherein, M:1000bp DNA ladder; N: negative control; No. 1 swimming lane is egg sample (not adding bacterium liquid); The bacterial concentration that 2-8 swimming lane is corresponding is: 1.24 × 10
2~ 1.24 × 10
8cfu/mL.
Embodiment
The research of the present inventor shows, using streptococcus aureus as immunogen, immunity Balb/c mouse, separation and purification obtains anti-Staphylococcus aureus monoclonal antibody 5D2G2D9C1 and 5D6D9G3B4, its antibody titer can reach 1:100000, its can specificity, be combined with streptococcus aureus efficiently, detection sensitivity reaches 10
5cfu/ml.With singly increase Liszt, hog cholera sramana, mouse typhus sramana, enteritis sramana, Fu Shi will is congratulated, Song Nei Shi will is congratulated, Boydii will is congratulated, enterohemorrhagic Escherichia coli O 157: H7, Enterobacter sakazakii, small intestine Yersinia, streptococcus pneumoniae, bacillus cereus etc. amount to 74 kinds of equal no cross reactions of pathogenetic bacteria.
On this basis, the present inventor and then pathogenic bacterium immunity enrichment technology and gene level detection technique are effectively combined, namely first by anti-Staphylococcus aureus monoclonal antibody of the present invention by the pathogenic bacteria specific immunity enrichment in testing sample, and then carry out pcr amplification, thus provide a kind of can fast, the detection kit of efficient detection food source property streptococcus aureus.
Antibody
Anti-Staphylococcus aureus monoclonal antibody of the present invention can utilize mouse hybridoma cell system 5D2G2D9C1(CGMCC No.8763) or 5D6D9G3B4(CGMCC No.8764) secretion generation.The present invention includes the monoclonal antibody of the corresponding aminoacid sequence with anti-Staphylococcus aureus monoclonal antibody 5D2G2D9C1 or 5D6D9G3B4, and there is other protein of these chains or protein conjugate and fusion expressed product.Particularly, the present invention includes and have containing hypervariable region (complementary determining region, any protein of light chain CDR) and heavy chain or protein conjugate and fusion expressed product (i.e. immune conjugate and fusion expressed product), as long as this hypervariable region is identical with the hypervariable region of heavy chain with light chain of the present invention or at least 90% homology, preferably at least 95% homology.As is known to the person skilled in the art, immune conjugate and fusion expressed product comprise: that medicine, toxin, cytokine (cytokine), radionuclide, enzyme and other diagnosis or treatment molecule are combined with anti-Staphylococcus aureus monoclonal antibody or its fragment and the conjugate that formed.The present invention also comprises the cell surface marker thing or antigen that are combined with anti-Staphylococcus aureus monoclonal antibody or its fragment.
For anti-Staphylococcus aureus monoclonal antibody heavy of the present invention and sequence of light chain, can measure by ordinary method.The hypervariable region of anti-Staphylococcus aureus monoclonal antibody V chain or complementary determining region (complementarity determining region, CDR) interesting especially because relate to conjugated antigen at least partly in them.Therefore, the present invention includes those and there is the band light chain immunoglobulin of CDR and the molecule of weight chain variable chain, as long as its CDR and anti-Staphylococcus aureus monoclonal antibody CDR has the homology of more than 90% (preferably more than 95%).The present invention not only comprises complete monoclonal antibody, also comprises and has immunocompetent antibody fragment, as Fab or (Fab ')
2fragment; Heavy chain of antibody; Light chain of antibody.
Present invention also offers the DNA molecular of above-mentioned immunoglobulin (Ig) or its fragment.The sequence of these DNA moleculars can use routine techniques, utilizes hybridoma cell line 5D2G2D9C1(CGMCC No.8763) or 5D6D9G3B4(CGMCC No.8764) obtain.In addition, also the encoding sequence of light chain and heavy chain can be merged, form single-chain antibody.
Once obtain relevant sequence, just relevant sequence can be obtained in large quantity with recombination method.This is normally cloned into carrier, then proceeds to cell, is then separated from the host cell after propagation by ordinary method and obtains relevant sequence.
In addition, also relevant sequence can be synthesized, when especially fragment length is shorter by the method for synthetic.Usually, by first synthesizing multiple small segment, and then carry out connect can obtain the very long fragment of sequence.
At present, the DNA sequence dna of code book invention albumen (or its fragment, or derivatives thereof) can be obtained completely by chemosynthesis.Then this DNA sequence dna can be introduced in the various existing DNA molecular (or as carrier) that in this area, oneself knows and cell.In addition, also by chemosynthesis, sudden change is introduced in protein sequence of the present invention.
The invention still further relates to the carrier comprising above-mentioned suitable DNA sequence dna and suitable promotor or control sequence.These carriers may be used for transforming suitable host cell, with can marking protein.
Host cell can be prokaryotic cell prokaryocyte, as bacterial cell; Or the eukaryotic cell such as low, as yeast cell; Or higher eucaryotic cells, as mammalian cell.
Can carry out with routine techniques well known to those skilled in the art with recombinant DNA transformed host cell.When host be prokaryotic organism as streptococcus aureus time, the competent cell that can absorb DNA can be gathered in the crops at exponential growth after date, uses CaC1
2method process, step used is well-known in this area.Another kind method uses MgC1
2.If needed, transform and also can be undertaken by the method for electroporation.When host is eukaryote, following DNA transfection method can be used: calcium phosphate precipitation, conventional mechanical methods as microinjection, electroporation, liposome packaging etc.
The transformant obtained can be cultivated by ordinary method, expresses the polypeptide of coded by said gene of the present invention.According to host cell used, substratum used in cultivation can be selected from various conventional medium.Cultivate under the condition being suitable for host cell growth.When after host cell growth to suitable cell density, the promotor selected with the induction of suitable method (as temperature transition or chemical induction), cultivates for some time again by cell.
Recombinant polypeptide in the above methods can be expressed or be secreted into extracellular in cell or on cytolemma.If needed, can utilize its physics, the albumen of being recombinated by various separation method abstraction and purification with other characteristic of chemistry.These methods are well-known to those skilled in the art.The example of these methods includes, but are not limited to: conventional renaturation process, combination by protein precipitant process (salting-out method), centrifugal, the broken bacterium of infiltration, supersound process, ultracentrifugation, sieve chromatography (gel-filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography (LC) technology and these methods.
Anti-Staphylococcus aureus monoclonal antibody 5D2G2D9C1 and 5D6D9G3B4 of the present invention, its height of tiring (can 1:100000 be reached), streptococcus aureus can be detected specifically, efficiently, with singly increase Liszt, hog cholera sramana, mouse typhus sramana, enteritis sramana, Fu Shi will is congratulated, Song Nei Shi will is congratulated, Boydii will is congratulated, enterohemorrhagic Escherichia coli O 157: H7, Enterobacter sakazakii, small intestine Yersinia, streptococcus pneumoniae, bacillus cereus etc. amount to 74 kinds of equal no cross reactions of pathogenetic bacteria, this is maximum innovative point of the present invention.
Detection kit
The present inventor is according to the principle of immuno-PCR, prepare a kind of test kit that can be used for detecting Gold Samples staphylococcus aureus, namely first with anti-Staphylococcus aureus monoclonal antibody 5D2G2D9C1 or 5D6D9G3B4 of the present invention by the immunity enrichment specifically of the pathogenic bacteria in testing sample, and then carry out pcr amplification, to improve sensitivity and the specificity of detection.
Specifically, first the present invention there is provided a kind of PCR reaction tubes for detecting streptococcus aureus, it comprises immuno-PCR pipe and is coated in anti-Staphylococcus aureus monoclonal antibody 5D2G2D9C1 or the 5D6D9G3B4 of described immuno-PCR pipe inboard wall of tube body, described anti-Staphylococcus aureus monoclonal antibody 5D2G2D9C1 and 5D6D9G3B4 is respectively by mouse hybridoma cell system 5D2G2D9C1, CGMCC No.8763 or 5D6D9G3B4, CGMCC No.8764 secretes generation.
On this basis, the present invention and then provide a kind of staphylococcus aureus immunity PCR detection kit, it contains the above-mentioned PCR reaction tubes for detecting streptococcus aureus;
As optimal way of the present invention, container a is also mounted with in described test kit, described container a can be valve bag or other forms of container, and the PCR reaction tubes being coated with anti-Staphylococcus aureus monoclonal antibody 5D2G2D9C1 or 5D6D9G3B4 of the present invention is wherein housed.
In addition, in order to make test kit of the present invention more convenient when detecting, preferably some other auxiliary reagent is also comprised in described test kit of the present invention, described auxiliary reagent is conventional some reagent used in immuno-PCR detection kit, and the characteristic of these reagent and their compound method are all well-known to those skilled in the art.Described reagent is (but being not limited to) such as: damping fluid (10 × PCR Buffer), dNTP, Taq DNA polymerase, ddH
2o(sterilizing distilled water), the Standard PCR reaction reagent such as primer.10 × PCR Buffer, dNTP, Taq DNA polymerase, ddH
2o is same with the Reagent evaluation used in conventional immuno-PCR detection kit.Meanwhile, in order to eliminate false positive and false negative, also can Quality Control (contrast) being set in PCR testing process, namely in test kit of the present invention, also including negative control and positive control etc.Preferably, described positive control solution is the streptococcus aureus bacterium liquid of deactivation, and negative control solution is brain heart leach liquor meat soup (BHI) of sterilizing.As well known to the skilled person, mentioned reagent and solution can be loaded in EP pipe or reagent bottle respectively.
In addition, in described test kit, also working instructions can be comprised, for illustration of the using method of the reagent wherein loaded.
Beneficial effect of the present invention: staphylococcus aureus immunity PCR detection kit of the present invention integrates that pathogenic bacteria is concentrated, specific antibody identification and pcr amplification, have that detected result accuracy is high, high specificity, highly sensitive and detect the advantages such as quick, easy to use, compensate for the deficiency that the time-consuming effort of existing food-borne pathogens detection technique, sensitivity are low.
Staphylococcus aureus immunity PCR detection kit of the present invention is applicable to the numerous areas such as bacterial strain Rapid identification, food borne pathogens in food detection, is particularly useful for the rapid detection of micro-pathogenic agent in sample.Quality supervision department, import and export sanitary authority etc. can be supplied for detecting the streptococcus aureus in food sample.
Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usual conveniently condition is as people such as Sambrook, molecular cloning: lab guide (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or according to the condition that manufacturer advises.Unless otherwise indicated, otherwise per-cent and number calculate by weight.
Unless otherwise defined, all specialties used in literary composition and scientific words and one skilled in the art the same meaning be familiar with.In addition, any method similar or impartial to described content and material all can be applicable in the present invention.The use that better implementation method described in literary composition and material only present a demonstration.
The preparation of embodiment 1. anti-Staphylococcus aureus monoclonal antibody 5D2G2D9C1 and 5D6D9G3B4
One, the preparation of immunogen and positive criteria product
Streptococcus aureus (ATCC No.27660) is inoculated in brain heart leach liquor meat soup (BHI), 37 DEG C, 150r/min shaking culture 17h, and counting, adds 0.3% formaldehyde solution room temperature deactivation 1 day.Streptococcus aureus (ATCCNo.27660) concentration to 5 × 10 are adjusted with physiological saline
9cfu/ml is as immunogen; Adjusting concentration with physiological saline is 10
8cfu/ml streptococcus aureus bacterium liquid is as positive control standard substance, and improvement brain heart leach liquor meat soup (BHI) is negative control standard substance.
Two, the preparation of monoclonal antibody
1) laboratory animal: select 38 week ages, about body weight 20g, female Balb/c mouse are laboratory animal.
2) immunization method: every mouse peritoneal injection 0.2ml immunogen, at interval of 2 weeks with same dosage booster shots once.
3) take a blood sample: from tail vein blood sampling after 3 booster immunizations, adopt indirect non-competing euzymelinked immunosorbent assay (ELISA) to measure antiserum titre.Wait to tire and no longer rise, abdominal injection same amount immunogen, conventionally carried out cytogamy after 3 days.
4) cytogamy: get that immune mouse spleen cell and SP2/0 myeloma cell are conventional under 50%PEG effect merges, is inoculated in 96 well culture plates respectively, is placed in 37 DEG C, 5%CO
2incubator is cultivated.
5) filtering hybridoma: adopt indirect non-competing euzymelinked immunosorbent assay (ELISA), the hybridoma in screening strong positive hole, transfers them to 24 well culture plates.
6) clone cultivates and antibody preparation: carry out colonized culture with limiting dilution assay.When Growth of Cells is to when to be paved with at the bottom of hole 1/10, then detect with same method, strong positive hole is cloned, 3-4 time so repeatedly, until positive rate reaches 100% again.By hybridoma enlarged culturing, be injected in through the pretreated Balb/c mouse peritoneal of paraffin oil, every 2 × 10
6individual hybridoma, 7 ~ 10 days mouse web portion protuberances, living body puncture extracts ascites.With caprylic acid-ammonium antibody purification from mouse ascites.
Staphylococcus aureus streptococcus aureus---GB: GB4789.10-2010
Brain heart leach liquor meat soup (BHI)
Composition: Trypsin matter peptone 10.0g, sodium-chlor 5.0g, Sodium phosphate dibasic (Na2HPO412H2O) 2.5g, glucose 2.0g, OX-heart leach liquor 500mL.
Method for making: heating for dissolving, regulates pH7.4 ± 0.2, puts 121 DEG C, 15min sterilizing.
Antibody titer measuring method:
(1) saturated culturing bacterium antigen is added 100 μ l together with substratum in the hole of correspondence, 4 DEG C spend the night (about wrapper sheet 36h).
(2) to turn liquid pat dry residual liquid, clean 3 times with 250 μ l washingss.
(3) 100 μ l1%BSA are added in each hole, 37 DEG C of closed 1h.
(4) to turn liquid pat dry residual liquid, clean 3 times with 250 μ l washingss.
(5) add 100 μ l serum in each hole, hatch 1h for 37 DEG C.
(6) to turn liquid pat dry residual liquid, add 250 μ lPBST washingss in each hole and wash 3 times.
(7) two anti-(sigma) that in each hole, the HRP of 50 μ l marks, incubated at room 1h.
(8) soak 5min with washings, turned letter liquid also pats dry residual liquid, adds 250 μ lPBST washingss and wash 3 times in each hole.
(9) add 100 μ l substrates in each hole, colour developing 30min, add stop buffer 100 μ l also immediately at OD
450reading.
Table 1. antibody titer measurement result
The CHARACTERISTICS IDENTIFICATION of embodiment 2. monoclonal antibody 5D2G2D9C1 and 5D6D9G3B4
One, monoclonal antibody subgroup identification
1, antigen coated: with 0.01M PBS bag by goat against murine two anti-igg+A+M, every hole 50 μ l, 4 DEG C of bags are spent the night, and discard liquid in hole next day, wash plate 3 times.
2, close: every hole adds 1%BSA200 μ l, close for 4 DEG C and spend the night.Next day pats dry plank and does not wash plate.
3, monoclonal antibody hybridoma cell supernatant is added, each sample 8 micropores, every hole 50 μ l.37 DEG C, hatch 1h.
4, after washing plate 4 times, the rabbit anti-mouse igg 1, IgG2a, IgG2b, IgG3, IgA, IgM, κ, λ, 37 DEG C adding specific combination respectively hatches 1h.
5, after washing plate 4 times, every hole adds horseradish peroxidase-labeled anti-rabbit two anti-igg (H+L) of having diluted, and 37 DEG C, hatches 30min.
6, after washing plate 4 times, 100 μ l substrate nitrite ions are added, 37 DEG C, lucifuge colour developing 10min.Result is read under 450nm wavelength.
Table 2. liang strain monoclonal antibody subgroup identification result
Two, the cross reaction test of monoclonal antibody 5D2G2D9C1 and 5D6D9G3B4
1, antigen coated: by 78 kind 10
8the pathogenic bacterium of cfu/ml bacterial concentration join in enzyme plate, each pathogenic bacterium bag 3 holes, and every hole 100 μ l, wraps and spent the night by 4 DEG C.
2, close: after washing plate 3 times, every hole adds 3%BSA200 μ l, hatches 2h for 37 DEG C.
3, plate is washed 3 times.Every hole adds the monoclonal antibody 100 μ l diluted, and hatches 1h for 37 degrees Celsius.
4, plate is washed 3 times.Adding the goat against murine two of having diluted resists in all micropores, and every hole 100 μ l, hatches 1h for 37 degrees Celsius.
5, plate is washed 4 times.Add substrate nitrite ion, 37 DEG C, lucifuge colour developing 15min.Result is read under 450nm wavelength.
Table 3. liang strain monoclonal antibody cross reaction detected result
The preparation of embodiment 3PCR reaction tubes
The concrete method for coating of the PCR reaction tubes for detecting streptococcus aureus of the present invention is as follows:
1, be buffered liquid with bag and dilute anti-Staphylococcus aureus monoclonal antibody 5D2G2D9C1 or 5D6D9G3B4 to 1:300 of the present invention doubly, antibody concentration is 10 μ g/mL, by the monoclonal antibody of having diluted often pipe 50uL add in immuno-PCR pipe, 4 DEG C of bag quilts that spend the night;
2, bag be buffered liquid (Carbonate Coating buffer(1 ×)) formula be: anhydrous sodium carbonate Na
2cO
31.59g, sodium bicarbonate NaHCO
32.93g, sodiumazide NaN
30.2g, is dissolved in 1000ml distilled water, adjusts pH to 9.6,4 DEG C of preservations.
The preparation of embodiment 4 staphylococcus aureus immunity PCR detection kit
Prepare staphylococcus aureus immunity PCR detection kit of the present invention, it contains:
PCR reaction tubes prepared by embodiment 3, loads in valve bag; EP pipe load 10 × PCR Buffer mono-manages, dNTP mono-manages, Taq DNA polymerase one is managed, ddH
2o mono-manages, forward primer one is managed, reverse primer one pipe; Positive control one bottle, negative control one bottle that reagent bottle loads.
10 × PCR Buffer loading amount is 300uL; The concentration of dNTP is 2.5mmol/L, and loading amount is 150uL; Taq DNA polymerase concentration is 5U/L, and loading amount is 60uL; Primer is for streptococcus aureus nuc gene design, and forward primer is 5 '-gcg attgat ggt gat acg gtt-3 ' (SEQ ID NO1), and primer concentration is 10umol/L, and amplification target fragment is 279bp; Loading amount is 150uL; Reverse primer is 5 '-agc caa gcc ttg acgaac taa agc-3 ' (SEQ ID NO2), and concentration is 10umol/L, and loading amount is 150uL.
Positive control is the streptococcus aureus bacterium liquid of deactivation, and negative control is brain heart leach liquor meat soup (BHI) of sterilizing.
The use flow process of detection kit of the present invention and schematic diagram are see Fig. 1.
Embodiment 5 gradient dilution streptococcus aureus pure bacterium liquid Direct PCR research experiment (Fig. 2)
Streptococcus aureus reference culture then by stroke-physiological saline solution successively 10 times of dilutions of successively decreasing, is labeled as 10 after cultivating 18h successively
-1, 10
-2, 10
-3, 10
-4, 10
-5, 10
-6, 10
-7, with 10
-5, 10
-6, 10
-7the bacterium liquid of gradient carries out plate count.The original bacterial concentration of enumeration is 1.24 × 10
9cfu/mL, dilutes original bacteria liquid successively, and bacterial concentration gradient is: 1.24 × 10
8~ 1.24 × 10cfu/mL.The each 5uL of bacterium liquid getting each gradient adds in detection PCR pipe of the present invention, and then add the 10 × PCR Buffer5uL in test kit of the present invention, dNTP2uL, forward primer 2uL, reverse primer 2uL, Taq DNA polymerase 1uL, ddH2O supply volume to 50uL.The amplification condition of PCR is 95 DEG C of 5min; 35 circulation (95 DEG C of 15s; 55 DEG C of 30s; 72 DEG C of 1min), 72 DEG C of 5min, 4 DEG C of preservations.The PCR primer of the getting 10uL agarose gel electrophoresis of 1.5% detects target stripe and gel imaging analysis.
As shown in Figure 2, in figure, the visible specific targets band of No. 1-4, swimming lane occurs result of implementation, and clip size is 279bp, and can see that brightness weakens successively.5-8 swimming lane and negative control have no specific band.Experimental result shows, and the sensitivity that streptococcus aureus pure bacterium liquid Direct PCR detects is 1.24 × 10
5cfu/mL.
This test kit of the pure bacterium liquid of embodiment 6 gradient dilution streptococcus aureus detects IC-PCR research experiment (Fig. 3)
Get the streptococcus aureus bacterium liquid (1.24 × 10 of gradient dilution
8~ 1.24 × 10cfu/mL) each 200uL adds in the detection PCR pipe in this test kit, be positioned over 37 DEG C and hatch 2h, then carefully suck bacterium liquid with pipettor, wash three times with PBST, aseptic filter paper is detained dry raffinate, then in PCR pipe, add the 10 × PCR Buffer5uL in test kit of the present invention, dNTP2uL, forward primer 2uL, reverse primer 2uL, Taq DNA polymerase 1uL, ddH2O supply volume to 50uL.The amplification condition of PCR is 95 DEG C of 5min; 35 circulation (95 DEG C of 15s; 55 DEG C of 30s; 72 DEG C of 1min), 72 DEG C of 5min, 4 DEG C of preservations.The PCR primer of the getting 10uL agarose gel electrophoresis of 1.5% detects target stripe and gel imaging analysis.
As shown in Figure 3, in figure, the visible specific targets band of No. 1-7, swimming lane occurs result of implementation, and clip size is 279bp, and can see that brightness weakens successively.8 swimming lanes and negative control have no specific band.Experimental result shows, and the sensitivity that staphylococcus aureus immunity PCR detection kit detects streptococcus aureus pure bacterium liquid detectability is 1.24 × 10
2cfu/mL.
Embodiment 7 test kits are to streptococcus aureus and non-streptococcus aureus cross reaction experiment (Fig. 4)
Strains tested has: streptococcus aureus ATCC82346, streptococcus aureus ATCC27660, Shu Shi salmonella typhi ATCC22956, Salmonella enteritidis ATCC13076, Listeria monocytogenes ATCC43251, yersinia entero-colitica ATCC23715, shigella flexneri ATCC12022, bacillus ceylonensis A ATCC25931, shigella dysenteriae ATCC51329, beta hemolytic streptococcus ATCC10373, Enterobacter sakazakii ATCC29004.Respectively getting strains tested 200ul adds in the detection PCR pipe of this test kit, be positioned over 37 DEG C and hatch 2h, then carefully suck bacterium liquid with pipettor, wash three times with PBST, aseptic filter paper is detained dry raffinate, then in PCR pipe, add the 10 × PCR Buffer5uL in test kit of the present invention, dNTP2uL, forward primer 2uL, reverse primer 2uL, Taq DNA polymerase 1uL, ddH2O supply volume to 50uL.The amplification condition of PCR is 95 DEG C of 5min; 35 circulation (95 DEG C of 15s; 55 DEG C of 30s; 72 DEG C of 1min), 72 DEG C of 5min, 4 DEG C of preservations.The PCR primer of the getting 10uL agarose gel electrophoresis of 1.5% detects target stripe and gel imaging analysis.
Result of implementation as shown in Figure 4, only has the swimming lane of streptococcus aureus to occur object band (279bp) in figure, other 9 strain control strains all do not have object band to occur, illustrate that this test kit has good specificity.
Embodiment 8 gradient dilution simulates streptococcus aureus Direct PCR research experiment (Fig. 5 to Figure 10) that carries disease germs
According to GB GB4789.10-2010, take food sample 25g(mL) join in 225mL physiological saline, homogeneous 2 ~ 3min on slap type homogenizer, as food homogenate, then to the pure bacterium liquid of streptococcus aureus of homogenate artificial contamination different concns gradient, in the food homogenate of artificial contamination, streptococcus aureus bacterial concentration is followed successively by: 1.24 × 10
8~ 1.24 × 10
2cfu/mL.The each 5uL of food homogenate getting the artificial contamination of different gradient adds in the detection PCR pipe of this test kit, then adds the 10 × PCR Buffer5uL in test kit of the present invention, dNTP2uL, forward primer 2uL, reverse primer 2uL, Taq DNA polymerase 1uL, ddH2O supply volume to 50uL.The amplification condition of PCR is 95 DEG C of 5min; 35 circulation (95 DEG C of 15s; 55 DEG C of 30s; 72 DEG C of 1min), 72 DEG C of 5min, 4 DEG C of preservations.The PCR primer of the getting 10uL agarose gel electrophoresis of 1.5% detects target stripe and gel imaging analysis.
Result of implementation is as the A(1 in Fig. 5 to Figure 10), B(1), C(1), D(1), E(1), F(1) shown in, can find out that from result the carry disease germs sensitivity of streptococcus aureus Direct PCR of food samples simulation can reach 1.24 × 10
5~ 1.24 × 10
6cfu/mL.
Embodiment 9 staphylococcus aureus immunity catches PCR kit sensitivity experiment (Fig. 5 to Figure 10)
According to GB GB4789.10-2010, take food sample 25g(mL) join in 225mL physiological saline, homogeneous 2 ~ 3min on slap type homogenizer, as food homogenate, then to the pure bacterium liquid of streptococcus aureus of homogenate artificial contamination different concns gradient, in the food homogenate of artificial contamination, streptococcus aureus bacterial concentration is followed successively by: 1.24 × 10
8~ 1.24 × 10
2cfu/mL.The each 200uL of food homogenate getting the artificial contamination of different gradient adds in the detection PCR pipe of this test kit, be positioned over 37 DEG C and hatch 2h, then carefully suck bacterium liquid with pipettor, wash three times with PBST, aseptic filter paper is detained dry raffinate, then in PCR pipe, add the 10 × PCR Buffer5uL in test kit of the present invention, dNTP2uL, forward primer 2uL, reverse primer 2uL, Taq DNA polymerase 1uL, ddH2O supply volume to 50uL.The amplification condition of PCR is 95 DEG C of 5min; 35 circulation (95 DEG C of 15s; 55 DEG C of 30s; 72 DEG C of 1min), 72 DEG C of 5min, 4 DEG C of preservations.The PCR primer of the getting 10uL agarose gel electrophoresis of 1.5% detects target stripe and gel imaging analysis.The results detailed in Fig. 5 to Figure 10.
Result of implementation is as the A(2 in Fig. 5 to Figure 10), B(2), C(2), D(2), E(2), F(2) shown in, as can be seen from result: staphylococcus aureus immunity PCR detection kit research food samples simulates the sensitivity of carrying disease germs can reach 1.24 × 10
3~ 1.24 × 10
4cfu/mL.
The preservation of biomaterial
Produce and be preserved in (No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) through the hybridoma cell strain 5D2G2D9C1 of the streptococcus aureus monoclonal antibody of above-mentioned qualification on January 9th, 2014, Institute of Microorganism, Academia Sinica, postcode: 100101), Classification And Nomenclature is the hybridoma cell strain producing anti-Staphylococcus aureus monoclonal antibody, and preserving number is CGMCC No.8763.
Produce and be preserved in (No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) through the hybridoma cell strain 5D6D9G3B4 of the streptococcus aureus monoclonal antibody of above-mentioned qualification on January 9th, 2014, Institute of Microorganism, Academia Sinica, postcode: 100101), Classification And Nomenclature is anti-Staphylococcus aureus hybridoma, and preserving number is CGMCC No.8764.
The all documents mentioned in the present invention are quoted as a reference all in this application, are just quoted separately as a reference as each section of document.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (6)
1. for detecting a PCR reaction tubes for streptococcus aureus, it is characterized in that, described PCR reaction tubes comprises:
Immuno-PCR pipe; With
Be coated in the anti-Staphylococcus aureus monoclonal antibody of described immuno-PCR pipe inboard wall of tube body, described anti-Staphylococcus aureus monoclonal antibody is by mouse hybridoma cell system 5D2G2D9C1, CGMCC No.8763 or 5D6D9G3B4, CGMCC No.8764 produces.
2. a detection kit, is characterized in that, described test kit is containing, for example PCR reaction tubes according to claim 1.
3. test kit as claimed in claim 2, is characterized in that, described test kit also containing container a, is equipped with PCR reaction tubes as claimed in claim 1 in described container a.
4. test kit as claimed in claim 2, is characterized in that, described test kit is also containing damping fluid, dNTP, Taq DNA polymerase, ddH
2o, primer, positive control and negative control.
5. test kit as claimed in claim 4, it is characterized in that, described primer comprises forward primer and reverse primer.
6. test kit as claimed in claim 4, it is characterized in that, described positive control is the streptococcus aureus bacterium liquid of deactivation, and described negative control is the brain heart leach liquor meat soup of sterilizing.
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CA2375276A1 (en) * | 2001-03-09 | 2002-09-09 | Her Majesty The Queen In Right Of Canada As Represented By The Minister Of Agriculture And Agri-Food Canada | Methods to isolate gene coding and flanking dna |
CN102304585A (en) * | 2011-09-22 | 2012-01-04 | 刘箐 | Immunocapture PCR (polymerase chain reaction) detection kit of staphylococcus aureus and using method of kit |
CN203144418U (en) * | 2012-12-11 | 2013-08-21 | 上海慧耘生物科技有限公司 | Immuno-PCR gene detection kit for escherichia coli O157:H7 |
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CA2375276A1 (en) * | 2001-03-09 | 2002-09-09 | Her Majesty The Queen In Right Of Canada As Represented By The Minister Of Agriculture And Agri-Food Canada | Methods to isolate gene coding and flanking dna |
CN102304585A (en) * | 2011-09-22 | 2012-01-04 | 刘箐 | Immunocapture PCR (polymerase chain reaction) detection kit of staphylococcus aureus and using method of kit |
CN203144418U (en) * | 2012-12-11 | 2013-08-21 | 上海慧耘生物科技有限公司 | Immuno-PCR gene detection kit for escherichia coli O157:H7 |
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