CN107739749A - The method of DNA probe Colony Hybridization In Situ For Screening technology screening paralytic shellfish poison's producing bacterial strain - Google Patents
The method of DNA probe Colony Hybridization In Situ For Screening technology screening paralytic shellfish poison's producing bacterial strain Download PDFInfo
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- 238000012216 screening Methods 0.000 title claims abstract description 44
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- 238000000034 method Methods 0.000 title claims abstract description 34
- 108020003215 DNA Probes Proteins 0.000 title claims abstract description 21
- 239000003298 DNA probe Substances 0.000 title claims abstract description 21
- 230000001769 paralizing effect Effects 0.000 title claims abstract description 21
- 239000000238 shellfish toxin Substances 0.000 title claims abstract description 20
- 238000011065 in-situ storage Methods 0.000 title claims abstract description 18
- 238000005516 engineering process Methods 0.000 title claims abstract description 16
- 108020004414 DNA Proteins 0.000 claims abstract description 38
- 241000894006 Bacteria Species 0.000 claims abstract description 16
- SHIBSTMRCDJXLN-UHFFFAOYSA-N Digoxigenin Natural products C1CC(C2C(C3(C)CCC(O)CC3CC2)CC2O)(O)C2(C)C1C1=CC(=O)OC1 SHIBSTMRCDJXLN-UHFFFAOYSA-N 0.000 claims abstract description 11
- QONQRTHLHBTMGP-UHFFFAOYSA-N digitoxigenin Natural products CC12CCC(C3(CCC(O)CC3CC3)C)C3C11OC1CC2C1=CC(=O)OC1 QONQRTHLHBTMGP-UHFFFAOYSA-N 0.000 claims abstract description 11
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Abstract
The invention discloses a kind of method of DNA probe Colony Hybridization In Situ For Screening technology screening paralytic shellfish poison's producing bacterial strain, including：Pretreatment, hybridization, screening, pass through specific forward primer and reverse primer, the ssDNA probe with digoxigenin labeled has been synthesized through aggregated PCR amplification, the DNA of bacterial strain to be tested and DNA probe are subjected to hybridization, test hybridization matching result filters out toxigenic bacterium strain by developing the color.Have the beneficial effect that：The inventive method can complete the screening of 90 samples in 8 10 hours, method rapidly and efficiently, high specificity, and accuracy is high；The inventive method operating procedure is simple, and workload is small, and the cycle is shorter, and agents useful for same and material are biochemical test common agents, and harmless, environmentally friendly, analysis cost is cheap, has higher market prospects and economic value.
Description
Technical field
The invention belongs to field of marine biotechnology, more particularly, to a kind of DNA probe-Colony Hybridization In Situ For Screening technology screening
The method of paralytic shellfish poison's producing bacterial strain.
Technical background
Paralytic shellfish poisoning (PSP)（Paralytic shellfish posoning, PSP）In known red-tide toxin Poisoning
Most strong, distribution is most extensively, menace is maximum, triggers murder by poisoning event most frequent.It is mainly by saxitoxin（Saxitoxin, STX）
And its derivative composition, belong to guanamines toxoid, be typical Na-ion channel blocker.The PSP homologues having now been found that
Up to 58 kinds.Its biosynthesis gene issxtGene cluster.STX examines in red tide detection, molecular biology, Neurobiology, medical science
There is important application in the researchs such as disconnected, drug development, military chemical and biological weapons war agent.In terms of medical diagnosis and drug development, STX
Unique chemical constitution and toxicological effect mechanism, turn into research cell Na+The important tool medicine of passage.It has antitumor, anti-
The multiple efficacies such as virus, analgesia, anesthesia, spasmolysis, decompression, Zhichuan.Its local anaesthetic effect is stronger than procaine or cocaine extremely
It is few 100,000 times.
At present, PSP acquisition is mainly prepared by pilot scale culture and the product extraction of dinoflagellate.This method cycle
Long, cost height；And need special algae culture device.Compared with dinoflagellate, bacterium has small genome, easy culture, genetic manipulation
The characteristic such as simple, it is an effective alternative using toxigenic bacterium strain fermentation and separating product for PSP shellfish poisons therefore
Method.Its key is the quick screening of toxigenic bacterium strain.It can by microbial fermentation, metabolite extract, product chemistry analysis come
Complete.But workload is big, cycle length, and toxin standard items used in chemical analysis are expensive, analysis cost is high.And it there is no at present
Produce the Rapid screening techniques of paralytic shellfish poison's bacterial strain.
The content of the invention
The purpose of this method is to provide a kind of DNA probe-Colony Hybridization In Situ For Screening technology screening paralytic shellfish poison's producing strains
The method of strain, used probe, which has, to be specifically bound with target dna and hybridizes rapidity, and detection method of the invention has
Detection speed is fast, accuracy is high, the characteristic of high sensitivity.
Sulfurous acidfast bacilli used in the inventive method（Sulfitobactersp.）（CCTCC AB 2016296）, it is conspicuous
Husband strangles Salmonella（Hoefleasp.）（CCTCC AB 2016294）Buy from China typical culture collection center；The present invention
Escherichia coli used in method（Escherichia coli）（ATCC 25922）, Song Shi Shigellas（Shigella
sonnet）（ATCC 29930）, staphylococcus aureus（Staphylococcus aureus）（ATCC 6538）, withered grass gemma
Bacillus（Bacillus subtilis）（ATCC 23857）, pseudomonas aeruginosa（Pseudomonas aeruginosa）
（ATCC 27853）, Vibrio harveyi（Vibrio harveyi）（ATCC BB120）, Vibrio salmonicida（Vibrio
salmonicida）（ATCC 43839）, lacuna catarrhalis（Moraxella lacunata）（ATCC 17952）, class will congratulate
Plesiomonas（Plesiomonas shigelloides）（ATCC 14029）This nine kinds of bacterial strains are bought from US mode culture
Thing collection warehousing (American type culture collection).
To obtain the specificity DNA probing needle that paralytic shellfish poison produces bacterium, the present invention is used to expand paralytic shellfish poisoning (PSP)
Biosynthesis genesxtThe specific primer of the DNA probe of gene is：Forward primer 17a, sequence 2：
TAGTAGGAGTAGCACGCTGCA；Reverse primer 17b, sequence 3：TCCTTCCTRGACCACGA.Pass through the He of specific primer sequence 2
Primer described in sequence 3, aggregated PCR（PCR）Amplification has synthesized the ssDNA probe with digoxigenin labeled, should
DNA probe has 254 bases, and its sequence is sequence 1：
GATGTCTGGACTACGTCCCTCCTACTCGCGCTGTCGAACCTGAATATCGATCCGAGGACGCCGGCTGGGGCTTCGCC
GACGCGGGCTCGGCGCGCATGGGCGGCACGTGACTCTCAACGCACGCTGAACGTCGGCAGGACCCTGCAGCACCAAG
TCCAGCACCGCACGTTCGTGCAGCAGATCAGCGCCGTTTGAGGGCGAGTTCGCCTCGCATCGCGTCGTGGCAGACGG
GATTAGGCAGATCTACAGAAGTC。
To establish the Colony Hybridization In Situ For Screening method based on above-mentioned DNA probe, the present invention uses following technical scheme：DNA is visited
The method of pin-Colony Hybridization In Situ For Screening technology screening paralytic shellfish poison's producing bacterial strain, including：Pretreatment, hybridization, screening, specific bag
Include following steps：
Pretreatment：10cm-12cm × 10cm-12cm nitrocellulose filter is made, takes bacterial strain single bacterium drop point to be tested in nitric acid
Cellulose membrane, 0.50-0.52MNaOH solution 8-10 minutes are dipped in, take out and be clipped in after spontaneously drying between two dry filter paper, placed
In vacuum oven, 78-80 DEG C of baking 2-2.5 hour；Lysozyme is added in TE buffer solutions, is put into DNA films, in 36-37 DEG C of temperature
Degree is rinsed after lower effect 30-32 minutes with TE buffer solutions；DNA films are placed in 10-12mL hybridization buffers, 40-42 DEG C of incubation
1.5-2.0 hour；Pretreatment can fix the DNA of bacterial strain to be tested, and remove bacterial cell residue, facilitate follow-up miscellaneous
Hand over operation；
Hybridization：It is denatured 10-15 minutes are boiled in boiling water with the DNA of digoxigenin labeled, puts in ice bath, take 5.0-6.0mL to mark
DNA is added in 2.5-3.0mL hybridization buffers, and film is placed on plastic sheeting by being taken out in hybridization buffer, added containing ground
In the hybridization buffer of Gaoxin labeled DNA probe, 2.0-2.5 hours are acted at 40-42 DEG C；By film put to fill 2.0-3.0 ×
In the plate of SSC solution, wash 2-3 times, each 5-6 minutes；
Screening：Film is put 1-2 minutes are washed into the plate for fill buffer solution, discard cleaning solution；Add 20-22mL1.0-1.2%'s
Confining liquid, room temperature place 60-70 minutes, discard solution；20-25mL alkali phosphatase enzyme mark anti digoxin antibody solution is added,
36-37 DEG C of incubation 30-35 minute, antibody conjugates are discarded, are washed 3-4 times with flushing liquor, each 40-50mL；Buffered in colour developing
After balancing 2-3 minutes in liquid, topple over colorbuffer, film is placed in polybag；Add in BCIP/NBT nitrite ions, be placed in dark
Place, TE solution terminating reactions are added after developing the color, spottiness occurs, that is, represents that bacterial strain production poison is positive；Immaculate, that is, represent bacterial strain
Production poison is negative；Screening technique is efficient rapidly, high specificity, and the degree of accuracy is high.
Preferably, the pH of TE buffer solutions used is 8.0.
Preferably, the dosage of lysozyme used, which is every milliliter of TE buffer solution, adds 4.8-5.2mg lysozymes.
Preferably, hybridization buffer used is：5 × SSC solution, 50-52mM phosphate buffers, 1.0-
1.1mMEDTA, 10-12% dextran sulfate, 48-50% deionized formamides, pH7.0-7.1.
Preferably, buffer solution used is：0.10-0.11M maleic acids, 0.14-0.15MNaCl, pH7.4-
7.5。
Preferably, confining liquid used is：0.10-0.11MTris-HCl, 0.15-0.16MNaCl, 0.10-
0.11MMgCl2, pH7.9-8.0.
Preferably, flushing liquor used is：0.10-0.11MTris-HCl, 0.15-0.16MNaCl, pH7.4-7.5.
Preferably, colorbuffer used is：0.10-0.11MTris, 0.10-0.11MNaCl, 50-
52mMMgCl2, pH9.3-9.5.
Preferably, nitrite ion used is：0.30-0.32mg/mLNBT, 0.15-0.16mg/mLBCIP, pH9.0-
9.1。
Compared with prior art, the advantage of the invention is that：1）By Bacterial Colony In Situ spot molecular hybridization, DNA probe can
Whether there is paralytic shellfish poison's synthesis in specific detection strain to be tested DNA samplesxtThe presence of A gene nucleic acids, can be small in 8-10
When it is interior complete the screening of 90 samples, the inventive method rapidly and efficiently, high specificity, and accuracy is high；2）The inventive method is grasped
It is simple to make step, workload is small, and the cycle is shorter, and agents useful for same and material are biochemical test common agents, harmless, right
Environment-friendly, analysis cost is cheap, has higher market prospects and economic value.
Embodiment
The present invention program is described further below by embodiment：
Embodiment 1：
The method of DNA probe-Colony Hybridization In Situ For Screening technology screening paralytic shellfish poison's producing bacterial strain, the sensitivity assays bag of this method
Include following steps：
1）Selecting bacteria He Fule Salmonellas（Hoefleasp.）（CCTCC AB 2016294）For sample to be tested, it is metabolized production
Thing chemical analysis confirms production PSP toxin；
2）The flat board single bacterium colony of picking He Fule Salmonella bacterial strains, its DNA sample is extracted according to molecular cloning conventional method, its is dilute
Release 2-10 times；
3）2 μ L dilute samples points are taken after being dipped in 0.5MNaOH solution 10 minutes, to be clipped in nitrocellulose filter after natural drying
Between two dry filter paper, it is positioned in vacuum oven, 80 DEG C are dried 2 hours, fixed dna；Lysozyme is placed in by 5mg/mL amount
In TE buffer solutions, it is put into after DNA films act on 30 minutes at 37 DEG C and is rinsed with TE buffer solutions, removes bacterial cell residue；
4）DNA films are placed in 10mL hybridization buffers, 42 DEG C incubate 1.5 hours；The DNA of digoxigenin labeled is boiled in boiling water
It is denatured within 10 minutes, puts in ice bath, takes 5mL marker DNAs to add in 2.5mL hybridization buffers, by film by being taken in pre-hybridization buffer
Go out to be placed on plastic sheeting, add the hybridization buffer containing Digoxigenin labeled DNA probe, acted on 2 hours at 42 DEG C.By film put to
In the plate for filling 2xSSC solution, wash 2 times, every time 5 minutes；
5）Film is put and washed into the plate for fill buffer solution 1 minute, discards cleaning solution, adds 20mL1% confining liquids, room temperature is placed
60 minutes, discard, add 20mL alkali phosphatase enzyme mark anti digoxin antibody solution（5000 times of dilutions are carried out using confining liquid）,
37 DEG C incubate 30 minutes, discard antibody conjugates, are washed 3 times with flushing liquor, each 50mL.2 points are balanced in colorbuffer
Zhong Hou, topple over nitrite ion.Film is placed in polybag.Add in 20mLBCIP/NBT nitrite ions, be placed in dark place, add after developing the color
Enter TE solution terminating reactions.Observation has immaculate appearance.
Test result shows that all DNA sample dilution samples are positive in hybridization；The inventive method has higher standard
True property and sensitiveness, can be as the screening of paralytic shellfish poison's producing bacterial strain.
Embodiment 2：
The method of DNA probe-Colony Hybridization In Situ For Screening technology screening paralytic shellfish poison's producing bacterial strain, specimen in use are：2 plants of production poison
Bacterium：Including sulfurous acidfast bacilli, He Fule Salmonellas；9 plants of non-toxigenic bacteriums：Including Escherichia coli, Song Shi Shigellas, golden yellow grape
Coccus, bacillus subtilis, pseudomonas aeruginosa, Vibrio harveyi, Vibrio salmonicida, lacuna catarrhalis, class will congratulate adjacent unit cell
Bacterium.The specific assay of this method comprises the following steps：
The single bacterium drop point of strain to be tested is taken after 0.5MNaOH solution on nitrocellulose filter, is dipped in 10 minutes, is spontaneously dried
After be clipped between two dry filter paper, be positioned in vacuum oven, 80 DEG C dry 2 hours, fixed dna；Will be molten according to 5mg/mL amount
Bacterium enzyme is added in TE buffer solutions, is put into DNA films and is acted on 30 minutes at 37 DEG C, is rinsed with TE buffer solutions, and it is residual to remove bacterial cell
Stay thing.
The above-mentioned DNA films prepared are placed in 10mL hybridization buffers（5 × SSC solution, 50mM phosphate buffers,
1mMEDTA, 10% dextran sulfate, 50% deionized formamide, pH7.0）In, 42 DEG C incubate 1.5 hours.By digoxigenin labeled
DNA is boiled 10 minutes in boiling water and is denatured, and is put in ice bath, takes 5mL marker DNAs to add in 2.5mL hybridization buffers, by film by pre- miscellaneous
Hand over to take out in buffer solution and be placed on plastic sheeting, add the hybridization buffer containing Digoxigenin labeled DNA probe, 2 are acted at 42 DEG C
Hour.Film is put to the plate for filling 2 × SSC solution, 2 of washing 5 minutes every time.
Film is put to filling buffer solution（0.1M maleic acids, 0.15MNaC1, pH7.5）Plate in wash 1 minute.Abandon
Cleaning solution is removed, adds 20mL1% confining liquids（0.1MTris-HCl、0.15MNaCl、0.1MMgCl2, pH8.0), room temperature places 60
Minute.Discard, add 20mL alkaline phosphatases（AP）Mark anti digoxin antibody solution（It is dilute using 5000 times of confining liquid progress
Release）, 37 DEG C incubate 30 minutes, discard antibody conjugates, use flushing liquor（0.1MTris-HCl, 0.15MNaCl, pH7.5）Washing 3
It is secondary, each 50mL.In colorbuffer（0.1MTris, 0.1MNaCl, 50mMMgCl, pH9.5）Middle balance is inclined after 2 minutes
Nitrite ion.Film is placed in polybag, adds 20mLBCIP/NBT nitrite ions（0.3mg/mLNBT, 0.16mg/mLBCIP,
pH9.0）In, dark place is placed in, TE solution terminating reactions are added after developing the color, observation has immaculate appearance.Specific assay result
As shown in table 1：
The specific assay of table 1.
| Bacterial strain | Numbering | Results of hybridization |
| Sulfurous acidfast bacilli（Sulfitobactersp.） | CCTCC AB 2016296 | + |
| He Fule Salmonellas（Hoefleasp.） | CCTCC AB 2016294 | + |
| Escherichia coli（Escherichia coli） | ATCC 25922 | - |
| Song Shi Shigellas（Shigella sonnet） | ATCC 29930 | - |
| Staphylococcus aureus（Staphylococcus aureus） | ATCC 6538 | - |
| Bacillus subtilis（Bacillus subtilis） | ATCC 23857 | - |
| Pseudomonas aeruginosa（Pseudomonas aeruginosa） | ATCC 27853 | - |
| Vibrio harveyi（Vibrio harveyi） | ATCC BB120 | - |
| Vibrio salmonicida（Vibrio salmonicida） | ATCC 43839 | - |
| Lacuna catarrhalis（Moraxella lacunata） | ATCC 17952 | - |
| Plesiomonas shigelloides（Plesiomonas shigelloides） | ATCC 14029 | - |
As can be seen from Table 1, including the equal spottiness of this 2 plants of toxigenic bacterium strains of sulfurous acidfast bacilli, He Fule Salmonellas reacts, other 9 plants
The equal immaculate of bacterial strain occurs, and the above is shown as positive bacterial strain through dot hybridization detection, passes through the change of its fermentating metabolism product
Credit is analysed, and confirms that it produces PSP toxin；And the bacterial strain of feminine gender is detected as through dot hybridization, then its fermentating metabolism product chemical analysis
Confirm that it does not produce PSP toxin, show the high specificity of this method, testing result is accurately, reliably.
Routine operation in the operating procedure of the present invention is well known to those skilled in the art, herein without repeating.
Technical scheme is described in detail embodiment described above, it should be understood that it is described above only
For the specific embodiment of the present invention, it is not intended to limit the invention, all any modifications made in the spirit of the present invention,
Supplement or similar fashion replacement etc., should be included in the scope of the protection.
Claims (8)
- The method of 1.DNA probes-Colony Hybridization In Situ For Screening technology screening paralytic shellfish poison's producing bacterial strain, it is characterised in that described To contain the ssDNA probe described in 254 base sequences 1 through digoxigenin labeled, described sequence 1 is DNA probe：GATGTCTGGACTACGTCCCTCCTACTCGCGCTGTCGAACCTGAATATCGATCCGAGGACGCCGGCTGGGGCTT CGCCGACGCGGGCTCGGCGCGCATGGGCGGCACGTGACTCTCAACGCACGCTGAACGTCGGCAGGACCCTGCAGCAC CAAGTCCAGCACCGCACGTTCGTGCAGCAGATCAGCGCCGTTTGAGGGCGAGTTCGCCTCGCATCGCGTCGTGGCAG ACGGGATTAGGCAGATCTACAGAAGTC。
- 2. the side of DNA probe according to claim 1-Colony Hybridization In Situ For Screening technology screening paralytic shellfish poison's producing bacterial strain Method, it is characterised in that：Described DNA probe is to pass through specific forward primer 17a and reverse primer 17b, aggregated enzyme chain type Reaction（PCR）Amplification has synthesized the ssDNA probe with digoxigenin labeled.
- 3. the side of DNA probe according to claim 2-Colony Hybridization In Situ For Screening technology screening paralytic shellfish poison's producing bacterial strain Method, it is characterised in that：Described forward primer 17a, sequence are：TAGTAGGAGTAGCACGCTGCA；Described reverse primer 17b, sequence are：TCCTTCCTRGACCACGA.
- 4. the side of DNA probe according to claim 1-Colony Hybridization In Situ For Screening technology screening paralytic shellfish poison's producing bacterial strain Method, it is characterised in that：Described method includes：1）Pretreatment：Make and fix the DNA of bacterial strain to be tested；2）Hybridization：The DNA of bacterial strain to be tested and DNA probe are subjected to hybridization；3）Screening：Hybridization matching result is tested by developing the color.
- 5. the side of DNA probe according to claim 4-Colony Hybridization In Situ For Screening technology screening paralytic shellfish poison's producing bacterial strain Method, it is characterised in that：Described pre-treatment step is：10cm-12cm × 10cm-12cm nitrocellulose filter is made, takes and treats Test strain single bacterium drop point is dipped in 0.50-0.52MNaOH solution 8-10 minutes, taken out after spontaneously drying in nitrocellulose filter It is clipped between two dry filter paper, is positioned in vacuum oven, 78-80 DEG C of baking 2-2.5 hour；Bacteriolyze is added in TE buffer solutions Enzyme, DNA films are put into, are rinsed after 30-32 minutes are acted at a temperature of 36-37 DEG C with TE buffer solutions；DNA films are placed in 10-12mL In hybridization buffer, 40-42 DEG C of incubation 1.5-2.0 hour.
- 6. the side of DNA probe according to claim 4-Colony Hybridization In Situ For Screening technology screening paralytic shellfish poison's producing bacterial strain Method, it is characterised in that：Described hybridization step is：It is denatured, puts by 10-15 minutes are boiled in boiling water with the DNA of digoxigenin labeled In ice bath, take 5.0-6.0mL marker DNAs to be added in 2.5-3.0mL hybridization buffers, film is put by being taken out in hybridization buffer In on plastic sheeting, add in the hybridization buffer containing Digoxigenin labeled DNA probe, 2.0-2.5 hours are acted at 40-42 DEG C； Film is put to the plate for filling 2.0-3.0 × SSC solution, washed 2-3 times, each 5-6 minutes.
- 7. the side of DNA probe according to claim 4-Colony Hybridization In Situ For Screening technology screening paralytic shellfish poison's producing bacterial strain Method, it is characterised in that：Described screening step is：Film is put 1-2 minutes are washed into the plate for fill buffer solution, discard washing Liquid；20-22mL1.0-1.2% confining liquid is added, room temperature places 60-70 minutes, discards solution；Add 20-25mL alkaline phosphatases Enzyme marks anti digoxin antibody solution, 36-37 DEG C of incubation 30-35 minute, discards antibody conjugates, is washed 3-4 times with flushing liquor, Each 40-50mL；After balancing 2-3 minutes in colorbuffer, topple over colorbuffer, film is placed in polybag；Add In BCIP/NBT nitrite ions, dark place is placed in, TE solution terminating reactions are added after developing the color, spottiness occurs, that is, represents bacterial strain production It is malicious positive；Immaculate, that is, represent that bacterial strain production poison is negative.
- 8. the side of DNA probe according to claim 5-Colony Hybridization In Situ For Screening technology screening paralytic shellfish poison's producing bacterial strain Method, it is characterised in that：The dosage of lysozyme in described pre-treatment step is that every milliliter of TE buffer solutions addition 4.8-5.2mg is molten Bacterium enzyme；Hybridization buffer is：5 × SSC solution, 50-52mM phosphate buffers, 1.0-1.1mMEDTA, 10-12% sulfuric acid Portugal gather Sugar, 48-50% deionized formamides, pH7.0-7.1.
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