CN103725771B - Quick and precisely detect the product vomitoxin of living in food simultaneously and do not produce vomitoxin bacillus cereus test kit and detection method thereof - Google Patents
Quick and precisely detect the product vomitoxin of living in food simultaneously and do not produce vomitoxin bacillus cereus test kit and detection method thereof Download PDFInfo
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- 241000193755 Bacillus cereus Species 0.000 title claims abstract description 53
- LINOMUASTDIRTM-QGRHZQQGSA-N deoxynivalenol Chemical compound C([C@@]12[C@@]3(C[C@@H](O)[C@H]1O[C@@H]1C=C(C([C@@H](O)[C@@]13CO)=O)C)C)O2 LINOMUASTDIRTM-QGRHZQQGSA-N 0.000 title claims abstract description 50
- LINOMUASTDIRTM-UHFFFAOYSA-N vomitoxin hydrate Natural products OCC12C(O)C(=O)C(C)=CC1OC1C(O)CC2(C)C11CO1 LINOMUASTDIRTM-UHFFFAOYSA-N 0.000 title claims abstract description 50
- 235000013305 food Nutrition 0.000 title claims abstract description 28
- 238000001514 detection method Methods 0.000 title claims abstract description 18
- 238000012360 testing method Methods 0.000 title claims description 13
- 238000000034 method Methods 0.000 claims abstract description 29
- 241000894006 Bacteria Species 0.000 claims abstract description 28
- 238000007403 mPCR Methods 0.000 claims abstract description 18
- 230000003321 amplification Effects 0.000 claims abstract description 13
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 13
- TZRXHJWUDPFEEY-UHFFFAOYSA-N Pentaerythritol Tetranitrate Chemical compound [O-][N+](=O)OCC(CO[N+]([O-])=O)(CO[N+]([O-])=O)CO[N+]([O-])=O TZRXHJWUDPFEEY-UHFFFAOYSA-N 0.000 claims abstract description 9
- 230000031709 bromination Effects 0.000 claims abstract description 9
- 238000005893 bromination reaction Methods 0.000 claims abstract description 9
- 238000009835 boiling Methods 0.000 claims abstract description 4
- 239000000284 extract Substances 0.000 claims abstract description 4
- 238000006243 chemical reaction Methods 0.000 claims description 19
- 239000000047 product Substances 0.000 claims description 13
- 239000002953 phosphate buffered saline Substances 0.000 claims description 12
- 239000000243 solution Substances 0.000 claims description 12
- 239000000203 mixture Substances 0.000 claims description 8
- 239000000725 suspension Substances 0.000 claims description 8
- 239000000872 buffer Substances 0.000 claims description 6
- 239000006228 supernatant Substances 0.000 claims description 6
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 claims description 5
- 238000004519 manufacturing process Methods 0.000 claims description 5
- 238000012408 PCR amplification Methods 0.000 claims description 3
- 238000000246 agarose gel electrophoresis Methods 0.000 claims description 3
- 230000004087 circulation Effects 0.000 claims description 3
- 238000013016 damping Methods 0.000 claims description 3
- 239000012530 fluid Substances 0.000 claims description 3
- 238000005286 illumination Methods 0.000 claims description 3
- 239000003112 inhibitor Substances 0.000 claims description 3
- 238000001556 precipitation Methods 0.000 claims description 3
- 238000004458 analytical method Methods 0.000 claims description 2
- 238000002156 mixing Methods 0.000 claims description 2
- 230000001351 cycling effect Effects 0.000 claims 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 abstract description 5
- 108020004465 16S ribosomal RNA Proteins 0.000 abstract description 3
- 244000005700 microbiome Species 0.000 abstract description 3
- 230000036963 noncompetitive effect Effects 0.000 abstract description 3
- 230000009182 swimming Effects 0.000 description 8
- 241000209094 Oryza Species 0.000 description 6
- 235000007164 Oryza sativa Nutrition 0.000 description 6
- 235000009566 rice Nutrition 0.000 description 6
- 208000005374 Poisoning Diseases 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 238000001962 electrophoresis Methods 0.000 description 3
- 235000012149 noodles Nutrition 0.000 description 3
- 231100000572 poisoning Toxicity 0.000 description 3
- 230000000607 poisoning effect Effects 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 235000013580 sausages Nutrition 0.000 description 3
- 206010012735 Diarrhoea Diseases 0.000 description 2
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 2
- 206010016952 Food poisoning Diseases 0.000 description 2
- 208000019331 Foodborne disease Diseases 0.000 description 2
- 206010047700 Vomiting Diseases 0.000 description 2
- 238000000137 annealing Methods 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 229910052736 halogen Inorganic materials 0.000 description 2
- 150000002367 halogens Chemical class 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
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- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 241000607142 Salmonella Species 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- 239000000877 Sex Attractant Substances 0.000 description 1
- 241000191967 Staphylococcus aureus Species 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 230000006806 disease prevention Effects 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000003651 drinking water Substances 0.000 description 1
- 235000020188 drinking water Nutrition 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000000147 enterotoxin Substances 0.000 description 1
- 231100000655 enterotoxin Toxicity 0.000 description 1
- 244000078673 foodborn pathogen Species 0.000 description 1
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- 239000007788 liquid Substances 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000002203 pretreatment Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000010865 sewage Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
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Abstract
The invention belongs to microorganism detection field, disclose a kind of method quick and precisely detecting product vomitoxin alive in food and do not produce vomitoxin bacillus cereus.Auele Specific Primer is designed for the cesB of bacillus cereus coding vomitoxin and the 16S rRNA of bacillus cereus, nitrine bromination third ingot (PMA) is adopted to eliminate the interference of the dead bacterium of bacillus cereus in food, boiling water bath method extracts viable bacteria DNA in food, carries out multiplex PCR detection.Add noncompetitive amplification interior label (IAC) and indicate the false negative result that may exist in PCR system.The false negative that present method is avoided molecular biology method to detect often having and false positive results interference, make detected result more accurate, present method is compared with classical culture protocols simultaneously, and detection time is shorter.
Description
Technical field
The invention belongs to microorganism detection field, particularly relate in a kind of quick food of detection simultaneously the method for producing vomitoxin and not producing vomitoxin bacillus cereus viable bacteria.
Technical background
Bacillus cereus is the sporiferous gram-positive microorganism of a kind of energy, is distributed widely in air, sewage and all kinds of raw and cooked foods.At present, from numerous food, isolate this bacterium, comprise parched rice meal, meat, newborn class and fish etc., be common food-borne pathogens, very likely endanger the health of people.The poisoning caused China bacillus cereus is relatively more serious, and 1993 ~ 2003 during the decade, bacillus cereus poisoner is up to 1758 people.The toxicity symptom that bacillus cereus causes mainly contains vomiting and diarrhoea, the food origin disease of diarrhoea is caused to study by enterotoxin more, the ripe also commercialization of Comparison between detecting methods, but it is relatively less about the research of bacillus cereus vomitoxin, but the food poisoning that vomiting type bacillus cereus causes, especially the poisoning of rice class happens occasionally.In the U.S., poisoning major cause occurs edible parched rice meal is wherein pollute the bacillus cereus producing vomitoxin.At present, bacillus cereus is regarded as by Norway and Holland the sex pheromone the most easily detected in food.Bacillus cereus mainly causes food poisoning with the antihygienic crowd of drinking-water, wherein uses contaminated food to be the main path causing bacillus cereus to infect.
The method of current detection bacillus cereus is mainly by traditional cultural method.Traditional method has the shortcomings such as labour intensity is high, consuming time, expense is high, and poor specificity, easily cause a large amount of wrong identification.Therefore the method detecting bacillus cereus is quickly and accurately most important.
The cesB of bacillus cereus is a subunit of vomitoxin synthetic enzyme structure gene, the vomitoxin of bacillus cereus of can encoding.The present invention adopts cesB to detect as target gene design primer, and high specificity, can be used for accurately detecting bacillus cereus.
Summary of the invention
The object of the present invention is to provide and a kind ofly can eliminate false negative and false positive and the method for producing vomitoxin and not producing vomitoxin bacillus cereus can be detected simultaneously.The method can be used for the product vomitoxin of living in food samples and the primary dcreening operation detection of not producing vomitoxin bacillus cereus, and its operation is quick, result is objective and accurate, avoids existing method complex operation, wastes time and energy and shortcoming with high costs.
The present invention is achieved by the following technical solutions.
While, is quick and precisely detected the product vomitoxin of work in food and is not produced a test kit for vomitoxin bacillus cereus, it is characterized in that being respectively containing primer: from 5 '-3 ' be
cesB-F:ACCCATCTTGCGTCATT
cesB-R:CAGCCAAGTGAAGAATACC
16S-F:GCGGCGTGCCTAATACATGC
16S-R:CTCAGGTCGGCTACGCATCG
IAC-F:CCTACGGGAGGCAGCAGT
IAC-R:CGTTTACGGCGTGGACTAC。
Described test kit also contains nitrine bromination third ingot PMA, PBS phosphate buffered saline buffer, Taqmix.
The present invention relates to the process of a kind of nitrine bromination third ingot and detect the product vomitoxin of living in food and the detection method of not producing vomitoxin bacillus cereus test kit fast in conjunction with multiplex PCR simultaneously, comprise the steps:
(1) take food sample, add PBS damping fluid by mass volume ratio 1:9, fully mix, centrifugal, get supernatant and obtain bacteria suspension;
(2) get the bacteria suspension prepared and add nitrine bromination third ingot solution, make the whole mass concentration of PMA be 5 μ g/mL, after mixing, room temperature lucifuge is cultivated, halogen lamp exposes, when illumination is cross-linked, sample is placed on ice, and the suspension after crosslinked is centrifugal, and gained precipitation Boiling bath method extracts DNA;
(3) gained DNA carries out mPCR, and system comprises 20 μ L reaction solutions altogether, wherein, template DNA solution prepared by 3 μ L, 10 μ L2 × Taq mix, the concentration of cesB primer is 0.4 μM, the concentration of 16S rRNA primer is 0.1 μM, and the concentration of amplification interior label is 0.25 μM; Reaction conditions is: 95 DEG C of 10min, then 40 circulations (94 DEG C of 30s, 51 DEG C of 30s, 72 DEG C of 30s), and last 72 DEG C extend 10min;
(4), after mPCR reaction terminates, carry out object bacterium and detect analysis.
Damping fluid described in step (1) is PBS phosphate buffered saline buffer.
Step (3) described primer is respectively: from 5 '-3 ' be
cesB-F:ACCCATCTTGCGTCATT
cesB-R:CAGCCAAGTGAAGAATACC
16S-F:GCGGCGTGCCTAATACATGC
16S-R:CTCAGGTCGGCTACGCATCG
IAC-F:CCTACGGGAGGCAGCAGT
IAC-R:CGTTTACGGCGTGGACTAC
The described object bacterium of step (4) detects to be analyzed as detecting pcr amplification result with agarose gel electrophoresis, if there is 475bp band, then Indicator Reaction system is normal; If there is not 475bp band, then reaction system has PCR inhibitor or PCR operation to occur mistake, needs to re-start PCR.Under amplified production has the prerequisite of 475bp band, if having 267bp band and 154bp band, then illustrate to have and produce vomitoxin bacillus cereus; If only there is 267bp band, then illustrates to have and do not produce vomitoxin bacillus cereus.
Compared with prior art, the present invention has following beneficial effect:
Auele Specific Primer is designed for the cesB of bacillus cereus coding vomitoxin and the 16SrRNA of bacillus cereus, nitrine bromination third ingot (PMA) is adopted to eliminate the interference of the dead bacterium of bacillus cereus in food, boiling water bath method extracts viable bacteria DNA in food, carries out multiplex PCR detection.Add noncompetitive amplification interior label (IAC) and indicate the false negative result that may exist in PCR system.The false negative that present method is avoided molecular biology method to detect often having and false positive results interference, make detected result more accurate, present method is compared with classical culture protocols simultaneously, and detection time is shorter.Specific as follows:
1, can detect product vomitoxin simultaneously and not produce vomitoxin bacillus cereus, speed is fast, can obtain result in 4 hours.
2, only detect viable bacteria, eliminate false positive results, efficiency is higher.
3, the false negative result because the reasons such as food substrate cause can be eliminated.
4, the Auele Specific Primer that the present invention relates to, highly sensitive, testing goal bacterium, easy and simple to handle efficiently, and result judges simple, reduces testing cost.
Accompanying drawing explanation
Fig. 1 multiplex PCR detects dead product vomitoxin bacillus cereus (numbering JDZ102Y) electrophoresis result of PMA process.M:DL2000DNA Marker; I ~ VI swimming lane is the untreated dead bacterium amplified band of PMA, and dead bacteria concentration is 4.0 × 10
6~ 4.0 × 10
2cFU/mL; 1 ~ 6 swimming lane is the amplification situation after PMA handling dead bacterium.
Fig. 2 multiplex PCR detects the product vomitoxin bacillus cereus electrophoresis result of the work of PMA process.M:DL2000DNA Marker; I ~ VIII swimming lane is the untreated viable bacteria amplified band of PMA, and viable bacteria concentration is 7.5 × 10
7~ 7.5 × 10
0cFU/mL; 1 ~ 8 swimming lane is the amplification situation after PMA process viable bacteria, Ⅸ and 9 swimming lane be negative control.
Fig. 3 multiplex PCR detects in contaminated food products and produces vomitoxin and do not produce vomitoxin bacillus cereus (numbering NC0084LY) and detectability after obtaining PMA process.It is 4.3 × 10 that bacillus cereus pollutes noodles, rice and sausage concentration
6~ 4.3 × 10
0cFU/g; The concentration of producing the pollution of vomitoxin bacillus cereus is 3.6 × 10
6~ 3.6 × 10
0cFU/g.
Fig. 4 multiplex PCR detects the product vomitoxin in contaminated food products under miscellaneous bacteria existence condition and does not produce vomitoxin bacillus cereus.In noodles, rice and sausage, the concentration of miscellaneous bacteria (streptococcus aureus PSAV021, Salmonellas ATCC) is 1.0 × 10
5cFU/g, the concentration of bacillus cereus is 10
3cFU/g.Swimming lane 1 and I is noodles sample, and swimming lane 2 and II is rice sample, and swimming lane 3 and III is sausage sample.
Embodiment
Below in conjunction with specific embodiment, explain the present invention further, should be understood that these embodiments are only not used in for illustration of the present invention and limit the scope of the invention.In the following example, unreceipted actual conditions is experimental technique, condition in usual conveniently condition, according to the suggestion condition of manufacturer, the bacterial strain related in embodiment all belongs to prior art, and those skilled in the art can obtain from open commercial channel easily.
Embodiment 1
While, is quick and precisely detected the product vomitoxin of work in food and is not produced a test kit for vomitoxin bacillus cereus, it is characterized in that being respectively containing primer: from 5 '-3 ' be
cesB-F:ACCCATCTTGCGTCATT
cesB-R:CAGCCAAGTGAAGAATACC
16S-F:GCGGCGTGCCTAATACATGC
16S-R:CTCAGGTCGGCTACGCATCG
IAC-F:CCTACGGGAGGCAGCAGT
IAC-R:CGTTTACGGCGTGGACTAC。
Described test kit is also purchased from Biotium company of the U.S. containing nitrine bromination third ingot PMA(), PBS phosphate buffered saline buffer, Taq mix(is purchased from Japanese TaKaRa company).
Concrete detection method is as follows:
Step 1, food sample pre-treatment
Take the food sample of 1g, add the PBS of 9mL, fully mix, the centrifugal 5min of 900g, get supernatant (the necessary aseptic technique of this process).
Step 2, PMA process
1g PMA is dissolved in 1mL methyl-sulphoxide (DMSO), and be mixed with the PMA solution of 1mg/mL ,-20 DEG C keep in Dark Place.Get 400 μ L step 1 supernatant liquors and be placed in 1.5mL Eppendorf tube, add the PMA solution of 2 μ L1mg/mL, make the whole mass concentration of PMA be 5 μ g/mL; After PMA mixes with bacteria suspension, lucifuge cultivates 5min at ambient temperature, utilize the halogen lamp exposure 5min of 500W, when illumination is cross-linked, sample is placed on ice (it is overheated to avoid), and at distance light source 20cm place, suspension after crosslinked is in the centrifugal 5min of 9000g, and gained precipitation is used for the extraction of DNA.
Step 3, the extraction of genomic dna
Through step 2 obtain sample with 9000g, 4 DEG C of centrifugal 5min, abandon supernatant, and carefully siphon away residual liquid, add the aseptic deionized water of 30 μ L 100 DEG C and boil 10min, 12000g after cooling, centrifugal 5min under 4 DEG C of conditions, get supernatant as mPCR reaction template, the template of preparation should detect for PCR immediately.
Step 4, chooses target spot and design primer
Multiplex PCR why do by difficulty, and the key of problem is that multiple target spot amplification condition is incompatible.Each target spot needs the primer on both sides to coordinate simultaneously.Therefore, the present invention chooses respectively and produces vomitoxin and do not produce vomitoxin bacillus cereus specific gene, adopt Oligo7 software design multiple PCR primer, adjust the annealing temperature between each pair of primer, and regulate the amplification cooperate degree between primer pair, make the annealing temperature of each pair of primer as far as possible consistent, amplification rate is as far as possible equal.And the expanding effect come by experiment with producing vomitoxin bacillus cereus and to verify as template multiplex PCR, detection primer below the primer pair choosing condition optimum is used as.And the bacterial strain adopted table 1 and provide carries out primer specificity checking, the numbering of bacterial strain and source are in table 1.
Table 1 strains tested and detected result
ajX-CDC, disease prevention and control center, Jiangxi Province, China;
bnCTC, national Culture Collection, Britain;
caTCC, American Type Culture preservation center, the U.S.;
dcMCC, Chinese medicine DSMZ, China.
Step 5, mPCR reaction system and reaction parameter
This multiplex PCR system comprises 20 μ L reaction solutions altogether, wherein, template DNA solution prepared by 3 μ L, 10 μ L2 × Taq mix, the concentration of cesB primer is 0.4 μM, and the concentration of 16S rRNA primer is 0.1 μM, and the concentration of amplification interior label is 0.25 μM; Reaction conditions is: 95 DEG C of 10min, then 40 circulations (94 DEG C of 30s, 51 DEG C of 30s, 72 DEG C of 30s), and last 72 DEG C extend 10min;
Wherein primer is respectively: from 5 '-3 ' be
cesB-F:ACCCATCTTGCGTCATT
cesB-R:CAGCCAAGTGAAGAATACC
16S-F:GCGGCGTGCCTAATACATGC
16S-R:CTCAGGTCGGCTACGCATCG
IAC-F:CCTACGGGAGGCAGCAGT
IAC-R:CGTTTACGGCGTGGACTAC。
Step 6, mPCR amplification detects
After mPCR reaction terminates, draw 5 μ L reaction solutions with rifle head, be added in the loading hole of 2% sepharose, 85V electrophoresis 30min, take out gel piece, in ultraviolet imagery system, observe electrophoretic band, and Taking Pictures recording.Determine that the specific standards whether having object bacterium in sample is: detect pcr amplification result with agarose gel electrophoresis, if there is 475bp band, then Indicator Reaction system is normal, can determine whether object bacterium by other band; If there is not 475bp band, then reaction system has PCR inhibitor or PCR operation to occur mistake, needs to re-start PCR.Under amplified production has the prerequisite of 475bp band, if having 267bp band and 154bp band, then illustrate to have and produce vomitoxin bacillus cereus; If only there is 267bp band, then illustrates to have and do not produce vomitoxin bacillus cereus.
Discussion of results
Traditional detection method complex operation, cycle length and specificity are strong, and round pcr is considered to bacillus cereus detection method the most accurately.The present invention utilizes round pcr to eliminate dead bacterium to the impact detected in conjunction with PMA, effectively have detected bacillus cereus alive; Meanwhile, a pair noncompetitive amplification interior label that the present invention adds in PCR system eliminates the impact that supressor and misoperation etc. in amplification procedure cause effectively.Therefore, the present invention utilizes specific primer can detect bacillus cereus first, and can eliminate false negative and false-positive result simultaneously, detected result high specificity, highly sensitive.
SEQUENCE LISTING
<110> Wuxi Zhongde Bore Bioisystech Co., Ltd
Quick and precisely detect product vomitoxin alive in food while of <120> mono-kind and do not produce vomitoxin bacillus cereus test kit and detection side thereof
<130>
<160> 6
<170> PatentIn version 3.3
<210> 1
<211> 17
<212> DNA
<213> cesB-F
<400> 1
acccatcttg cgtcatt 17
<210> 2
<211> 19
<212> DNA
<213> cesB-R
<400> 2
cagccaagtg aagaatacc 19
<210> 3
<211> 20
<212> DNA
<213> 16S-F
<400> 3
gcggcgtgcc taatacatgc 20
<210> 4
<211> 20
<212> DNA
<213> 16S-R
<400> 4
ctcaggtcgg ctacgcatcg 20
<210> 5
<211> 18
<212> DNA
<213> IAC-F
<400> 5
cctacgggag gcagcagt 18
<210> 6
<211> 19
<212> DNA
<213> IAC-R
<400> 6
cgtttacggc gtggactac 19
Claims (5)
1. quick and precisely detect the product vomitoxin of living in food and the test kit not producing vomitoxin bacillus cereus while, it is characterized in that being respectively containing primer: from 5 '-3 ' be
cesB-F: ACCCATCTTGCGTCATT
cesB-R: CAGCCAAGTGAAGAATACC
16S-F: GCGGCGTGCCTAATACATGC
16S-R: CTCAGGTCGGCTACGCATCG
IAC-F: CCTACGGGAGGCAGCAGT
IAC-R: CGTTTACGGCGTGGACTAC 。
2. according to claim 1 a kind of while quick and precisely detect the product vomitoxin of living in food and do not produce the test kit of vomitoxin bacillus cereus, it is characterized in that also containing nitrine bromination third ingot PMA, PBS phosphate buffered saline buffer,
taqmix.
3. according to claim 1 and 2 a kind of while quick and precisely detect the product vomitoxin of living in food and do not produce the detection method of vomitoxin bacillus cereus test kit, it is characterized in that comprising the following step:
(1) take food sample, to add sterile buffer by mass volume ratio 1:9, fully mix, centrifugal, get supernatant and obtain bacteria suspension;
(2) get the bacteria suspension prepared and add nitrine bromination third ingot solution, the whole mass concentration of nitrine bromination third ingot PMA is made to be 5 μ g/mL, room temperature lucifuge 5 min after mixing, shake up once every 30 s, exposure, when illumination is cross-linked, sample is placed on ice, and the suspension after crosslinked is centrifugal, and gained precipitation Boiling bath method extracts DNA;
(3) gained DNA carries out mPCR, and system comprises 20 μ L reaction solutions altogether, wherein, template DNA solution prepared by 3 μ L, 10 μ L 2 ×
taqmix,
cesBthe concentration of primer is 0.4 μM,
16S rRNAthe concentration of primer is 0.1 μM, and the concentration of amplification interior label is 0.25 μM; Reaction conditions is: 95 DEG C of 10 min, then 40 circulations, and its cycling condition is 94 DEG C of 30 s, 51 DEG C of 30 s, 72 DEG C of 30 s, and last 72 DEG C extend 10 min;
(4), after mPCR reaction terminates, carry out object bacterium and detect analysis.
4. method as claimed in claim 3, is characterized in that the damping fluid described in step (1) is PBS phosphate buffered saline buffer.
5. method as claimed in claim 3, it is characterized in that the described object bacterium of step (4) detects and analyze as detecting pcr amplification result with agarose gel electrophoresis, if there are 475 bp bands, then Indicator Reaction system is normal; If do not have appearance 475 bp band, then reaction system has PCR inhibitor or PCR operation to occur mistake, needs to re-start PCR;
Under amplified production has the prerequisite of 475 bp bands, if having 267 bp bands and 154 bp bands, then illustrate to have and produce vomitoxin bacillus cereus; If only there are 267 bp bands, then illustrate to have and do not produce vomitoxin bacillus cereus.
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| CN102844433A (en) * | 2010-04-14 | 2012-12-26 | 东洋制罐株式会社 | Primer set for pcr, reaction liquid for pcr, and method for detecting food poisoning bacteria |
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| CN102844433A (en) * | 2010-04-14 | 2012-12-26 | 东洋制罐株式会社 | Primer set for pcr, reaction liquid for pcr, and method for detecting food poisoning bacteria |
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| 张志鸿等.基于不同靶基因的荧光定量PCR快速检测蜡样芽孢杆菌的研究.《食品工业科技》.2013,http://www.cnki.net/kcms/detail/11.1759.TS.20130626.1047.006.html. * |
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