CN101580873A - Test kit and testing method for staphylococcus aureus toxin gene in food - Google Patents
Test kit and testing method for staphylococcus aureus toxin gene in food Download PDFInfo
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Abstract
The invention provides a test kit a testing method for staphylococcus aureus toxin gene in food. The kit comprises test solution A and test solution B, wherein the test solutions A and B respectively contain 10mM of Tris.Cl, 50mM of KCl, 25mM of MgCl2, 2.5mM of dNTP respectively, 5U/mu L Taq DNA polymerase, and 10 mu M of toxin gene primers of ETA, SEB, SEC, SEE, SEI and SEA respectively, or 10 mu M of toxin gene primers of SEJ, ETB, TSST, SED, SHE and SEG. The test kit and the testing method can high sensitively test 12 staphylococcus aureus toxin genes in food, have short testing time and simple operation, can save mass labor and financial power, and can meet the requirement of quick testing.
Description
Technical field
The present invention relates to be used for the detection kit and the detection method of food staphylococcus aureus toxin gene, relate in particular to quick detection kit and the detection method of PCR-based-DHPLC.
Background technology
Streptococcus aureus is under the condition of different foods, differing temps and pH value, and the enterotoxin of generation is different.Staphylococcus aureus enterotoxin (SE) has 12 serotypes, is respectively ET ETA (exfoliative toxin A), SEB, SEC, SEE, SEI, SEA, SEJ, ETB, poisoning shock toxin T SST (toxic shock syndrome toxin), SED, SEH, SEG.
Streptococcus aureus is lower than in the pH value under 5 the condition and seldom produces enterotoxin, and when the pH value was higher than 9.8, what type staphyloentero-toxin all can not produce.Staphylococcus produces toxin through 4h～8h under 20 ℃～37 ℃ on milk and flour congee, 18d produces toxin or do not produce toxin under 5 ℃～6 ℃ low temperature.In containing the more food of moisture, protein and starch, staphylococcus more easily breeds and produces toxin, as germ-carrying raw meat filling, under 37 ℃, produce toxin through 18h～19h, if in meat stuffing, add a small amount of steamed bun chip, 8h just can produce toxin in following process of same temperature, and visible starch has promoter action to forming toxin.
Cause the food that Staphylococcus aureus enterotoxin is poisoned: be mainly animal foods such as meat, milk, fish, eggs and goods thereof.Cake, cold and dressed with sauce powder, surplus boiled rice and the rice wine etc. cut also once caused poisoning; The cold cuts class as at ripe after stain pathogenic staphylococcus, under 20 ℃～30 ℃ environment, place the long period again, then very easily cause poisoning; Milk and milk preparation and cold drink (ice cream, ice lolly) and the cream pastries made of milk often are the food that causes poisoning; Fry egg, smoked fish, canned fish in oil etc. contain the more food of grease, and pathogenic staphylococcus also can produce toxin later on polluting.Streptococcus aureus more easily produces enterotoxin when the oxygen branch is low in air, therefore has the food of this bacterium, places under the high temperature of improper ventilation, and extremely favourable this bacteria growing also produces toxin.When food contamination staphylococcus and when having polluted other microorganisms simultaneously, staphylococcic breeding then is suppressed.Through heating, original various microorganisms are killed as food, and antagonistic microbe has not existed yet, and are polluted by staphylococcus again after ripe, then will promote staphylococcic growth and form toxin.The food poisoning that streptococcus aureus causes all has discovery all over the world.Often betide summer and autumn, this is because temperature is higher, helps bacterial reproduction.But in the winter time, as the food that is polluted is in the higher indoor preservation of temperature, and staphylococcus also can breed and produce toxin.When this bacterial count＞10
6Individual/when restraining, the enterotoxin of generation can cause illnesss such as food poisoning.
Therefore, set up 12 kinds of toxin genes of streptococcus aureus, promptly quick, accurate, the high-sensitive detection method of ETA, SEB, SEC, SEE, SEI, SEA, SEJ, ETB, TSST, SED, SEH and SEG is just particularly necessary.
Summary of the invention
The present invention at first is provided for detecting 12 kinds of toxin genes of streptococcus aureus, the i.e. test kit of ETA, SEB, SEC, SEE, SEI, SEA, SEJ, ETB, TSST, SED, SEH and SEG.
Comprise in the detection kit of staphylococcus aureus toxin gene in the food of the present invention and detect solution A and detect solution B:
Detect in the solution A and contain 10mM TrisCl, 50mM KCl, 25mM MgCl
2, each 2.5mM of dNTP, Taq archaeal dna polymerase 5U/ μ L and ETA, SEB, SEC, SEE, SEI and six kinds of toxin gene primers of SEA are to each 10 μ M; The specific primer sequence of these 6 kinds of toxin genes is as follows:
Detect in the solution B and contain 10mM TrisCl, 50mM KCl, 25mM MgCl
2, each 2.5mM of dNTP, Taq archaeal dna polymerase 5U/ μ L and SEJ, ETB, TSST, SED, SEH and six kinds of toxin gene primers of SEG are to each 10 μ M; The specific primer sequence that detects these 6 kinds of toxin genes in the solution B is as follows:
The present invention also provides the method for utilizing the mentioned reagent box to detect staphylococcus aureus toxin gene in the food, comprises the steps:
1. PCR reaction: get 1ul testing sample dna solution, add detection solution A and 14ul sterilization ultrapure water in the 10ul test kit, cumulative volume 25ul; Other gets 1ul testing sample dna solution, adds detection solution B and 14ul sterilization ultrapure water in the 10ul test kit, cumulative volume 25ul; Two reaction volumes carry out pcr amplification by following parameter then respectively at the centrifugal 10s of 5000r/min:
Pre-sex change: 94 ℃, 3min;
Enter circulation: 94 ℃ of sex change 60s, 60 ℃ of annealing 60s, 72 ℃ are extended 60s, 35 circulations;
Stop extending: 72 ℃, 7min;
2. pcr amplification product being carried out DHPLC analyzes:
Chromatographic column: PS-DVB﹠amp; The C18DNASep chromatographic column, 4.6mm * 50mm, granularity 3 μ m;
Column temperature: 50 ℃;
Moving phase (volume ratio): 0.0min, 55.0% buffered soln A, 45.0% buffered soln B;
0.5min, 50.2% buffered soln A, 49.8% buffered soln B;
2.8min, 40.9% buffered soln A, 59.1% buffered soln B;
5.0min, 37.3% buffered soln A, 62.7% buffered soln B;
7.3min, 35.5% buffered soln A, 64.5% buffered soln B;
9.5min, 34.3% buffered soln A, 65.7% buffered soln B;
Wherein, buffered soln A is the TEAA aqueous solution of concentration 0.1mM; Buffered soln B is that concentration is the 0.1M TEAA aqueous solution and 3: 1 by volume mixing solutions of acetonitrile;
Flow velocity: 0.9mL/min;
Detector: fluorimetric detector, light source: 150W Xenon lamp; Excitation spectrum bandwidth: 15nm; Emission spectrum bandwidth: 15.3nm; Detection sensitivity: at wavelength 350nm integration 2s;
Applied sample amount: PCR product 5 μ L.
After detect finishing, according to DHPLC detect feature chromatographic peak in the collection of illustrative plates peak shape, retention time and with the contrast of positive control spectrogram, determine the microbiological contamination situation of sample.
The present invention adopts the mPCR-DHPLC technology, sets up sensitivity detection method easily at staphylococcus aureus toxin gene in the food, and sets up the quick detection kit that is applied to this method.Use detection kit of the present invention and detection method, can detect 12 kinds of staphylococcus aureus toxin genes in the food in high sensitivity, and detect weak point consuming time, simple to operation, can save a large amount of labours and financial resources, be fit to the requirement of rapid detection.
Description of drawings
Accompanying drawing 12 width of cloth of the present invention are respectively:
Fig. 1 is the single separately PCR electrophorogram of ETA, SEB, SEC, SEE, SEI and SEA gene;
Fig. 2 is ETA, SEB, SEC, SEE, SEI and SEA gene mPCR-electrophorogram;
Fig. 3 is that ETA, SEB, SEC, SEE, SEI and SEA gene mPCR-DHPLC detect spectrogram;
Fig. 4 is the single separately PCR electrophorogram of SEJ, ETB, TSST, SED, SEH and SEG gene;
Fig. 5 is SEJ, ETB, TSST, SED, SEH, SEG gene mPCR-electrophorogram;
Fig. 6 is that SEJ, ETB, TSST, SED, SEH and SEG gene mPCR-DHPLC detect spectrogram;
Fig. 7 is ETA, SEB, SEC, SEE, SEI and SEA gene mPCR-DHPLC detection sensitivity test-results;
Fig. 8 is ETA, SEB, SEC, SEE, SEI and SEA gene mPCR-detected through gel electrophoresis sensitivity test result, wherein: 1,100bp Ladder DNA Marker; 2,10
4The bacteria concentration of the order of magnitude; 3,10
3The bacteria concentration of the order of magnitude;
Fig. 9 is the mPCR-DHPLC detection sensitivity test-results of SEJ, ETB, TSST, SED, SEH and SEG;
Figure 10 is SEJ, ETB, TSST, SED, SEH and SEG gene mPCR-detected through gel electrophoresis sensitivity test result, 1, and 100bp Ladder DNA Marker; 2,10
4The bacteria concentration of the order of magnitude; 3,10
3The bacteria concentration of the order of magnitude;
Figure 11 is that A simulating pollution sample mPCR-DHPLC detects collection of illustrative plates;
Figure 12 is that B simulating pollution sample mPCR-DHPLC detects collection of illustrative plates.
In the above-mentioned accompanying drawing, X-coordinate is retention time, and (unit: minute min), ordinate zou is represented absorption peak strength of signal (unit: millivolt mV).
Embodiment
Be specific embodiments of the invention below, its foundation and application thereof to present method is further described, but does not limit content of the present invention in any form.
If no specified otherwise, the employed main agents in this part, instrument and merchandise resources thereof are: the nutrient broth nutrient solution, the plain agar culture plate, and employed each bacterial strain in this part increase bacterium, separation and biochemical identification substratum etc. all according to the prescription in relevant national standard (GB), inspection and quarantine industry standard (SN) or the internal authority standard method, purchase in U.S. company BD.
Reagent such as bacterial genomes DNA extraction reagent (TakaRa MiniBEST Bacterial Genomic DNAExtraction kit) and Ex Taq are purchased white precious biotechnology (Dalian) company limited; Triethylamine acetyl salt (TEAA, chromatographically pure) is available from Transgenomic company; Acetonitrile (chromatographically pure) is available from Fisher company.
The employed reference culture in this part divides respectively available from U.S. ATCC standard biological product collecting center and Chinese medicine microbial strains preservation administrative center (CMCC).
The test strain that this part is related all adopts DNA of bacteria to extract test kit and extracts DNA of bacteria, be stored in-20 ℃ standby with to be detected.
The foundation of the detection kit of staphylococcus aureus toxin gene and detection method in embodiment 1, the food
(1) assembling of primer design, synthetic and test kit, described primer is as described below:
On this basis, be designed for the test kit that mPCR-DHPLC detects:
Comprise in the test kit and detect solution A and detect solution B:
Detect in the solution A and contain 10mM TrisCl, 50mM KCl, 25mM MgCl
2, each 2.5mM of dNTP, Taq archaeal dna polymerase 5U/ μ L and above-mentioned ETA, SEB, SEC, SEE, SEI and six kinds of toxin gene primers of SEA are to each 10 μ M;
Detect in the solution B and contain 10mM TrisCl, 50mM KCl, 25mM MgCl
2, each 2.5mM of dNTP, Taq archaeal dna polymerase 5U/ μ L and above-mentioned SEJ, ETB, TSST, SED, SEH and six kinds of toxin gene primers of SEG are to each 10 μ M.
(2) foundation of mPCR-DHPLC detection method
The test kit that the mPCR-DHPLC that this detection method uses present embodiment to set up detects comprises the steps:
1. the preparation of sample to be checked:
A. normal food sample: get food samples 25g, add aseptic 0.2mol/L pH7.5 phosphate buffered saline buffer 25mL, homogeneous becomes homogenate.Put into the 200mL enterotoxin toxin producing medium of sterilization, cultivate 48h for 36 ℃.
B. the preparation of bacterial strain sample: inoculation to be measured is cultivated 24h for 36 ℃ on nutrient agar medium.Wash lawn with about 3mL sterile saline, put into the enterotoxin toxin producing medium, 36 ℃ of shaking culture 48h.
Get the nutrient solution 1mL in the corresponding intestines toxin producing substratum of cultivation, extract test kit with DNA of bacteria and extract DNA of bacteria, be stored in-20 ℃ standby with to be detected.
2. pcr amplification:
Present embodiment is set up two groups of composite PCR systems, first group of composite PCR system one-time detection staphylotoxin ETA, SEB, SEC, SEE, SEI, SEA gene; Second group of composite PCR system one-time detection staphylotoxin SEJ, ETB, TSST, SED, SEH, SEG gene.
Get 1ul testing sample dna solution, add detection solution A and 14ul sterilization ultrapure water in the 10ul test kit, cumulative volume 25ul; Other gets 1ul testing sample dna solution, adds detection solution B and 14ul sterilization ultrapure water in the 10ul test kit, cumulative volume 25ul; Two reaction volumes carry out pcr amplification by following parameter then respectively at the centrifugal 10s of 5000r/min:
Pre-sex change: 94 ℃, 3min;
Enter circulation: 94 ℃ of sex change 60s, 60 ℃ of annealing 60s, 72 ℃ are extended 60s, 35 circulations;
Stop extending: 72 ℃, 7min;
2. pcr amplification product being carried out DHPLC analyzes:
Chromatographic column: PS-DVB﹠amp; The C18DNASep chromatographic column, 4.6mm * 50mm, granularity 3 μ m;
Column temperature: 50 ℃;
Moving phase (volume ratio): 0.0min, 55.0% buffered soln A, 45.0% buffered soln B;
0.5min, 50.2% buffered soln A, 49.8% buffered soln B;
2.8min, 40.9% buffered soln A, 59.1% buffered soln B;
5.0min, 37.3% buffered soln A, 62.7% buffered soln B;
7.3min, 35.5% buffered soln A, 64.5% buffered soln B;
9.5min, 34.3% buffered soln A, 65.7% buffered soln B;
Wherein, buffered soln A is the TEAA aqueous solution of concentration 0.1mM; Buffered soln B is that concentration is the 0.1M TEAA aqueous solution and 3: 1 by volume mixing solutions of acetonitrile;
Flow velocity: 0.9mL/min;
Detector: fluorimetric detector, light source: 150W Xenon lamp; Excitation spectrum bandwidth: 15nm; Emission spectrum bandwidth: 15.3nm; Detection sensitivity: at wavelength 350nm integration 2s;
Applied sample amount: PCR product 5 μ L.
The PCR-DHPLC method that adopts PCR-electrophoresis and embodiment 1 to set up respectively detects and contrasts.
Present embodiment is set up two groups of composite PCR systems, and the first composite PCR system detected object comprises ETA, SEB, SEC, SEE, SEI, SEA gene; The second composite PCR system detected object comprises ETASEJ, ETB, TSST, SED, SEH, SEG gene.
The PCR expection amplified fragments of the first composite PCR system ETA, SEB, SEC, SEE, SEI, six kinds of toxin genes of SEA is respectively: 119bp, 257bp, 317bp, 350bp, 375bp, 478bp; Its independent PCR-electrophoresis detection the results are shown in shown in the accompanying drawing 1, and the mPCR-electrophoresis detection the results are shown in shown in the accompanying drawing 2; The mPCR-DHPLC detected result is seen shown in the accompanying drawing 3.
From as seen, when these 6 kinds of toxin genes are made single pcr amplification and electrophoresis detection, all can detect single separately band significantly by Fig. 1 and Fig. 2; And when these 6 kinds of toxin genes were made compound pcr amplification and electrophoresis detection, the amplified band of 317bp, 350bp, 375bp but can't separate; As can be seen from Figure 3, the mPCR-DHPLC fignal center of 6 kinds of toxin genes is clear and legible separately, demonstrates than the better detected result of mPCR-electrophoresis.
The PCR expection amplified fragments of the second composite PCR system SEJ, ETB, TSST, SED, SEH, six kinds of toxin genes of SEG is respectively: 142bp, 200bp, 350bp, 482bp, 576bp, 642bp; Its independent PCR-electrophoresis detection the results are shown in shown in the accompanying drawing 4, and the mPCR-electrophoresis detection the results are shown in shown in the accompanying drawing 5; The mPCR-DHPLC detected result is seen shown in the accompanying drawing 6.
From as seen, when these 6 kinds of toxin genes are made single pcr amplification and electrophoresis detection, all can detect single separately band significantly by Fig. 4 and Fig. 5; And when these 6 kinds of toxin genes were made compound pcr amplification and electrophoresis detection, the amplified band of 576bp, 642bp but can't separate; As can be seen from Figure 6, the mPCR-DHPLC fignal center of 6 kinds of toxin genes is clear and legible separately, demonstrates than the better detected result of mPCR-electrophoresis.
Get reference strain listed in the table 1, the method for being set up according to embodiment 1 detects.
Table 1
Get listed test strain in the table 1, after cultivating, extract genomic dna respectively, set up template base.Be template with these genomic dnas then, adopting six kinds of toxin genes of ETA, SEB, SEC, SEE, SEI, SEA and the six kinds of toxin genes of SEJ, ETB, TSST, SED, SEH, SEG in the second composite PCR system in the first composite PCR system respectively is that the condition that goal gene adopts embodiment 1 to be set up is carried out the mPCR-DHPLC analyzing and testing.The result shows that in the first composite PCR reaction system, the staphylococcus aureus strains that only produces ETA the ETA absorption peak occurs at 2min, and other bacterial strains are negative findings; The staphylococcus aureus strains that produces SEB the SEB absorption peak occurs at 3.6min, and other bacterial strains are negative findings; The staphylococcus aureus strains that produces SEC the SEC absorption peak occurs at 4.7min, and other bacterial strains are negative findings; The staphylococcus aureus strains that produces SEE the SEE absorption peak occurs at 5.4min, and other bacterial strains are negative findings; The staphylococcus aureus strains that produces SEI the SEI absorption peak occurs at 5.7min, and other bacterial strains are negative findings; The staphylococcus aureus strains that produces SEA the SEA absorption peak occurs at 7.8min, and other bacterial strains are negative findings.In the second composite PCR reaction system, the staphylococcus aureus strains that produces SEJ the SEJ absorption peak occurs at 2.9min, and other bacterial strains are negative findings; The staphylococcus aureus strains that produces ETB the ETB absorption peak occurs at 3.9min, and other bacterial strains are negative findings; The staphylococcus aureus strains that produces TSST the TSST absorption peak occurs at 6.6min, and other bacterial strains are negative findings; The staphylococcus aureus strains that produces SED the SED absorption peak occurs at 7.7min, and other bacterial strains are negative findings; The staphylococcus aureus strains that produces SEH the SEH absorption peak occurs at 8.5min, and other bacterial strains are negative findings; The staphylococcus aureus strains that produces SEG the SEG absorption peak occurs at 8.9min, and other bacterial strains are negative findings.
Embodiment 4, sensitivity test
The various streptococcus aureuses of concentration known are produced strains, i.e. table of determining with the reduced turbidity method 2 and the L in the table 3
01-L
08The compound enrichment liquid of concentration, the method for setting up according to claim 1 detects, and determines the sensitivity that various product strain enterotoxins detect.
Table 2, first group of gradient dilution bacterial count scale
Second group of gradient dilution bacterial count of table 3 scale
Sensitivity test result is shown in accompanying drawing 7～10:
As shown in Figure 7, first group mPCR-DHPLC can detect L in the table 2
04Gradient: produce ETA bacterial strain 170CFU/mL, produce SEB bacterial strain 240CFU/mL, produce SEC bacterial strain 190CFU/mL, produce SEE bacterial strain 210CFU/mL, produce SEI bacterial strain 710CFU/mL, produce SEA bacterial strain 430CFU/mL, promptly can detect 10
2The bacteria concentration of the order of magnitude; And the PCR-gel electrophoresis can only detect L
02Gradient (10
4The order of magnitude) bacteria concentration, as shown in Figure 8.
As shown in Figure 9, second group mPCR-DHPLC can detect L in the table 2
08Gradient: produce SEJ bacterial strain 340CFU/mL, produce ETB bacterial strain 610CFU/mL, produce TSST bacterial strain 570CFU/mL, produce SED bacterial strain 370CFU/mL, produce SEH bacterial strain 290CFU/mL, produce SEG bacterial strain 320CFU/mL, promptly can detect 10
2The bacteria concentration of the order of magnitude; And the PCR-gel electrophoresis can only detect L
06Gradient (10
4The order of magnitude) bacteria concentration, as shown in Figure 10.
When carrying out above-mentioned sensitivity and detect, the detection sensitivity of several method is compared, comprise the mPCR-DHPLC method that PCR-gel electrophoresis, colloid gold immune test strip method, VIDAS method and embodiment 1 are set up.Several detection methods result relatively is shown in table 4 and table 5.
The comparison of table 4, four kinds of different methods detected results of first group of staphylotoxin
The comparison of table 5, four kinds of different methods detected results of second group of staphylotoxin
From the result of above-mentioned comparison as seen: height is all wanted in the sensitivity of other four kinds of methods of remolding sensitivity that PCR-DHPLC detects.
Embodiment 5, the test of simulating pollution sample detection
Add streptococcus aureus and produce malicious reference culture in the food samples that choosing supplies to test, the A analog sample is to have added streptococcus aureus to produce ETA, SEB, SEC, SEE, SEI, the strain of SEA toxin in the fish gruel; The B analog sample is to have added streptococcus aureus to produce SEJ, ETB, TSST, SED, SEH, the strain of SEG toxin in the fish gruel, and analog sample directly increases bacterium 18～24h, extracts its genome according to the method that embodiment 1 is set up, and detects.
A group simulating pollution sample is compound, and to increase the inoculum size of bacterium gold Portugal bacterium as shown in table 6:
Table 6, the compound inoculum size that increases bacterium gold Portugal bacterium of A group simulating pollution sample
B group simulating pollution sample is compound, and to increase the inoculum size of bacterium gold Portugal bacterium as shown in table 7:
Table 7, the compound inoculum size that increases bacterium gold Portugal bacterium of B group simulating pollution sample
Accompanying drawing 11 increases bacterium test DHPLC detected result for the A analog sample is compound, experimental result shows and detects this six kinds of toxin producing strains.
Figure 12 increases bacterium test DHPLC detected result for the B analog sample is compound, experimental result shows and detects this six kinds of toxin producing strains.
Embodiment 6, test kit of the present invention and detection method are used in actual sample detects
Test kit and multiplex PCR-DHPLC method that embodiment 1 is set up are used for actual survey work, and compare with existing other detection methods, the practicality and the reliability of checking detection method.During 7 months of year June in December, 2007 to 2008, multiplex PCR-DHPLC method the rapid screening that adopts this research to set up, adopt culture identification method (GB/T 4789.10-2003), Radioactive colloidal gold and the VIDAS method of national standard to verify comparison simultaneously, detect actual sample simultaneously, detect 3840 duplicate samples altogether.The result is from 349 detected strain streptococcus aureuses, detect the 54 strain streptococcus aureus toxin producing strain positives (with toxin serotype statistics) with multiplex PCR-DHPLC method screening, adopt Radioactive colloidal gold and VIDAS method to verify, the checking comparative result sees Table 8.
At present commercially available Radioactive colloidal gold only has staphylococcus aureus toxin A, three kinds of immunoassay bars of B, C, and the VIDAS method also only can detect staphylococcus aureus toxin A, B, C, five kinds of enterotoxins of D, E, and concrete somatotype.
From the checking comparative result of three kinds of methods, the kind that the PCR-DHPLC method that this institute sets up has evaluation is complete, can carry out the advantage that somatotype detects simultaneously, shows that this method has suitability preferably.
The different detection method checkings of table 8. are to the actual sample detected result
To 54 parts of streptococcus aureus toxin producing strain positive sample that go out with PCR-DHPLC method institute separation detection, having carried out the toxin somatotype by the PCR-DHPLC method detects, found that: isolated 287 strain streptococcus aureuses detect 33 strains that have of toxin gene from freeze the cod sample, account for 11.5%, the bacterium of carrying 2 kinds and above toxin gene simultaneously has 4 strains, accounts for 1.4%; Isolating 37 strain streptococcus aureuses detect 14 strains that have of toxin gene from meat product, account for 37.8%; Isolating 25 strain streptococcus aureuses detect 7 strains that have of toxin gene from other food samples, account for 28.0%.The bacterium of carrying 2 kinds and above toxin gene simultaneously has 4 strains, accounts for 10.8%.Wherein, the SEA toxin gene at most, 29 strains are arranged, account for 53.7%, and the bacterial strain that contains other traditional toxin gene type SEB, SEC, SED gene has only 6 strains, account for 11.1% (SEB, SEC, SED account for 3.7%, 1.8%, 5.6% respectively), do not detect the bacterial strain that carries SEE, ETB, TSST gene.The bacterial strain that carries newfound toxin gene SEG, SEH, SEI, SEJ and ETA is more, 19 strains are arranged, account for 35.2%, wherein bacterial strain 3 strains of SEG gene, bacterial strain 7 strains of SEH gene, bacterial strain 3 strains of SEI gene, bacterial strain 5 strains of SEJ gene, bacterial strain 1 strain of ETA gene accounts for 5.6%, 13.0%, 5.5%, 9.3% and 1.8% respectively.
Claims (2)
1, the detection kit of staphylococcus aureus toxin gene in the food is characterized in that comprising and detects solution A and detect solution B:
Detect in the solution A and contain 10mM TrisCl, 50mM KCl, 25mM MgCl
2, each 2.5mM of dNTP, Taq archaeal dna polymerase 5U/ μ L and ETA, SEB, SEC, SEE, SEI and six kinds of toxin gene primers of SEA are to each 10 μ M;
Detect in the solution B and contain 10mM TrisCl, 50mM KCl, 25mM MgCl
2, each 2.5mM of dNTP, Taq archaeal dna polymerase 5U/ μ L and SEJ, ETB, TSST, SED, SEH and six kinds of toxin gene primers of SEG are to each 10 μ M;
Wherein, the specific primer sequence of 6 kinds of toxin genes is as follows in the detection solution A:
The specific primer sequence that detects 6 kinds of toxin genes in the solution B is as follows:
2, the detection method of staphylococcus aureus toxin gene in the food is characterized in that using the described test kit of claim 1, comprises the steps:
1. PCR reaction: get 1ul testing sample dna solution, add detection solution A and 14ul sterilization ultrapure water in the 10ul test kit, cumulative volume 25ul; Other gets 1ul testing sample dna solution, adds detection solution B and 14ul sterilization ultrapure water in the 10ul test kit, cumulative volume 25ul; Two reaction volumes carry out pcr amplification by following parameter then respectively at the centrifugal 10s of 5000r/min:
Pre-sex change: 94 ℃, 3min;
Enter circulation: 94 ℃ of sex change 60s, 60 ℃ of annealing 60s, 72 ℃ are extended 60s, 35 circulations;
Stop extending: 72 ℃, 7min;
2. pcr amplification product being carried out DHPLC analyzes:
Chromatographic column: PS-DVB ﹠amp; C18 DNASep chromatographic column, 4.6mm * 50mm, granularity 3 μ m;
Column temperature: 50 ℃;
Moving phase (volume ratio): 0.0min, 55.0% buffered soln A, 45.0% buffered soln B;
0.5min, 50.2% buffered soln A, 49.8% buffered soln B;
2.8min, 40.9% buffered soln A, 59.1% buffered soln B;
5.0min, 37.3% buffered soln A, 62.7% buffered soln B;
7.3min, 35.5% buffered soln A, 64.5% buffered soln B;
9.5min, 34.3% buffered soln A, 65.7% buffered soln B;
Wherein, buffered soln A is the TEAA aqueous solution of concentration 0.1mM; Buffered soln B is that concentration is the 0.1M TEAA aqueous solution and 3: 1 by volume mixing solutions of acetonitrile;
Flow velocity: 0.9mL/min;
Detector: fluorimetric detector, light source: 150W Xenon lamp; Excitation spectrum bandwidth: 15nm; Emission spectrum bandwidth: 15.3nm; Detection sensitivity: at wavelength 350nm integration 2s;
Applied sample amount: PCR product 5 μ L.
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| CN105483133A (en) * | 2015-11-26 | 2016-04-13 | 中国人民解放军南京军区福州总医院 | Staphylococcus aureus toxic shock syndrome toxin-1(TSST-1) aptamer T-7 and preparation method and application thereof |
| CN106501394A (en) * | 2016-10-11 | 2017-03-15 | 西北农林科技大学 | The LC-MS detection method detected simultaneously by staphylococcus aureus IV kinds AIPs signaling molecule |
| CN108342497A (en) * | 2018-03-13 | 2018-07-31 | 芜湖市食品药品检验中心(市药品不良反应监测中心) | A kind of Staphylococcus aureus enterotoxin genotype rapid detection method in dining food |
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| CN105483133B (en) * | 2015-11-26 | 2018-07-06 | 中国人民解放军南京军区福州总医院 | - 1 aptamer T-7 of Staphylococcus aureus toxic shock toxin and its preparation method and application |
| CN106501394A (en) * | 2016-10-11 | 2017-03-15 | 西北农林科技大学 | The LC-MS detection method detected simultaneously by staphylococcus aureus IV kinds AIPs signaling molecule |
| CN108342497A (en) * | 2018-03-13 | 2018-07-31 | 芜湖市食品药品检验中心(市药品不良反应监测中心) | A kind of Staphylococcus aureus enterotoxin genotype rapid detection method in dining food |
| CN110412168A (en) * | 2019-08-12 | 2019-11-05 | 西南民族大学 | Method based on metabolic marker analyte detection Staphylococcus aureus in food viable bacteria |
| CN111004855A (en) * | 2020-01-03 | 2020-04-14 | 上海海关动植物与食品检验检疫技术中心 | Primer combination and kit for detecting staphylococcus aureus |
| CN111378727A (en) * | 2020-03-19 | 2020-07-07 | 上海市动物疫病预防控制中心(上海市兽药饲料检测所、上海市畜牧技术推广中心) | Triple fluorescent quantitative PCR kit for detecting staphylococcus aureus enterotoxin sea, seb and sec genes |
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Application publication date: 20091118 |