CN107418951A - A kind of low stain and high sensitive inexpensive general strain method for identifying molecules - Google Patents
A kind of low stain and high sensitive inexpensive general strain method for identifying molecules Download PDFInfo
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- CN107418951A CN107418951A CN201710686441.5A CN201710686441A CN107418951A CN 107418951 A CN107418951 A CN 107418951A CN 201710686441 A CN201710686441 A CN 201710686441A CN 107418951 A CN107418951 A CN 107418951A
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- identifying molecules
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
- C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
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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
- 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
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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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
- 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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- 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
- 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
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
Abstract
A kind of low stain and high sensitive inexpensive general strain method for identifying molecules, belong to biological technical field.It comprises the following steps：1）Strain pre-processes；2）Prepare the pillar of high absorption affinity；3）Bacterium cracks and nucleic acid absorption；4）Cleaning；5）Elution；6）First round PCR is expanded；7）Second wheel PCR amplifications；8）Electrophoresis detection and purifying；9）It is sequenced and is compared with primer M13F, it is determined that category and kind.The present invention utilizes simple and efficient broken wall and extractive technique, in conjunction with efficient nucleic acid absorption technology, and PCR enlarge-effect twice, the initial stage that can be formed in microbe colony, bacterium can accurately be identified, and the primer that the present invention is used to identify has taken into account the uniformity in fungi and bacterium site, not only bacterium can be identified in kind of a level, the category even level of kind can be also reached to fungi, identified while thus facilitating some bacteriums and fungi, efficiency is substantially increased, has saved experimental cost.
Description
Technical field
The invention belongs to biological technical field, and in particular to a kind of low stain and high sensitive inexpensive general strain molecule
Authentication method.
Background technology
It is the important hand that current microflora is evolved with taxonomic identification that applied molecular biology means, which carry out strain idenfication,
Section, such as utilize bacterial 16 S rDNA/16S-23S rDNA sequence of intervals and the 18S rDNA/ ITS1-5.8S-ITS2 of fungi
When the strain that sequence pair is separated to is measured, mostly carry out DNA extraction first, recycle the DNA sample to enter performing PCR expansion
Increase, sequencing and sequence alignment finally by PCR primer, determine the classification position of the bacterium.Existing detection means is in extraction sample
During product DNA, it usually needs the separation and culture of bacterium, the time is long, and efficiency comparison is low, along with some bacterium can only be specific
Under the conditions of cultivate, cellar culture is not easy to, and causes the difficulty of sample collection.If a small amount of extraction is carried out to sample, but it is past
Sensitiveness toward authentication method is not enough difficult to satisfaction and required.If it is sensitive to improve detection simply by PCR amplification mechanisms
Property, PCR after directly being cracked to sample is feasible in theory, and in actual mechanical process, but often it is not easy into
Work(, particularly most grampostive bacteria, actinomyces and fungi, because cell membrane is thicker, cause target dna to be difficult to receive
Collect, and the denier pollution that some are easily expanded easily is mixed into reaction, so that the result finally expanded is often big
The false positive of amount.Further, since the Molecular Identification region of bacterium and fungi difference, causes bacterium and fungi to separate and reflect
It is fixed, trouble experimentally is brought, particularly some single celled fungies, can produce and obscure with bacterium sometimes, cause to repeat to test
Card, greatly reduces the efficiency of qualification process, improves testing cost.
The content of the invention
The problem of existing for prior art, it is an object of the invention to design to provide a kind of low stain and high sensitive low
The technical scheme of the general strain method for identifying molecules of cost.
A kind of described low stain and high sensitive inexpensive general strain method for identifying molecules, it is characterised in that including with
Lower step：
1）Strain pre-processes
The picking bacterium to be measured from flat board, more than the bacterium diameter 2mm to be measured, adds into aaerosol solution A, then carry out mechanical crushing
Broken wall；
2）Prepare the pillar of high absorption affinity
The centrifuge tube with Filter column is taken, adsorbent suspension S is added, centrifugal treating, adsorbent is formed inclined-plane in Filter column, is given up
Abandon filtrate；
3）Bacterium cracks and nucleic acid absorption
Lysate L is added to step 1）In pretreated bacterium solution, acutely concussion, stands up and down, centrifugation, then takes supernatant to be added to
Step 2）In the adsorption column made, room temperature is placed, and centrifugation, gives up filtrate, collecting pipe places back in；
4）Cleaning
Take step 3）Obtained supernatant adds cleaning fluid W, centrifuges, and cleaning, outwells the filtrate of collecting pipe every time, gets rid of for the last time
After 15s, adsorption column no longer puts back to collecting pipe, and described cleaning fluid W is the alcoholic solution of concentration 70%；
5）Elution
Adsorption column is transferred in new centrifuge tube, eluent E 10-15ul in drop, covers tightly lid, keep 60 DEG C, 5-10min, from
The heart, the filtrate being collected into are the trace dna solution extracted, and described eluent E is sterilized PH8.5 Tris solution；
6）First round PCR is expanded
Take step 5）Obtained trace dna solution is template, enters performing PCR amplification, primer SF1 nucleosides with primer SF1 and SR1
Acid sequence is as shown in SEQ No 1, and primer SR1 nucleotide sequence is as shown in SEQ No 1；
7）Second wheel PCR amplifications
Take step 6）The 1/1000 of obtained reaction solution is template, enters performing PCR amplification, primer with primer MSF1, SR1 and M13F
MSF1 nucleotide sequence is as shown in SEQ No 3, and primer M13F nucleotide sequence is as shown in SEQ No 4；
8）Electrophoresis detection and purifying；
9）It is sequenced and is compared with primer M13F, it is determined that category and kind.
A kind of described low stain and high sensitive inexpensive general strain method for identifying molecules, it is characterised in that described
Step 1）Middle aaerosol solution A is：100 mM Tris-HCl, 25 mM EDTA, 2.0 M NaC1, RnaseA 10ug/ml, pH
8.0。
A kind of described low stain and high sensitive inexpensive general strain method for identifying molecules, it is characterised in that described
Step 1）Middle mechanical crushing broken wall treatment method for it is following any one：
A. added in bacterium solution it is sterilized after 3-5mm tungsten carbides ball 1, be placed in boiling water bath after 5min, in liquid nitrogen flash freezer
Under, ground with reciprocating pulverizer, it is to be restored to normal temperature after grinding；
B. the bead for the 0.1mm-1.5mm that 20ul volumes were anticipated is added in bacterium solution, as 5min in boiling water bath, is treated
Recover to room temperature, whirlpool concussion 1min, bead processing method is first cleans bead 1-2 times with clear water, then with 12N HCl
Soaked overnight, subsequent rinsed clean, last autoclaving, drying.
A kind of described low stain and high sensitive inexpensive general strain method for identifying molecules, it is characterised in that described
Step 2）The middle centrifuge tube with Filter column is that the hydrophilic nanoporous filter membrane that 1-3 layers aperture 1-10um is spread by centrifugal column compresses to obtain.
A kind of described low stain and high sensitive inexpensive general strain method for identifying molecules, it is characterised in that described
Step 2）Middle adsorbent suspension S is made by following steps：3g tripoli is weighed into 50ml triangular flasks, adds 20-30ml distilled waters,
Shake well, a moment is stood, remove supernatant, cleaned 3 times, add distilled water to 2 times of tripoli volumes, cumulative volume about 20ml, through high pressure
Normal temperature storage is stand-by after sterilizing.
A kind of described low stain and high sensitive inexpensive general strain method for identifying molecules, it is characterised in that described
Step 3）Middle lysate L is mixed to get by following components：Guanidinium isothiocyanate 30g, sterilized water 40ml, PH5.2 sterilizing NaAc
3ml, 10% SDS 2.5ml of sterilizing, sterile glycerol 2.5ml, cumulative volume 50ml.
A kind of described low stain and high sensitive inexpensive general strain method for identifying molecules, it is characterised in that described
Step 3）Inclined-plane is on one side outwardly during the centrifugation of middle adsorption column.
A kind of described low stain and high sensitive inexpensive general strain method for identifying molecules, it is characterised in that described
Step 6）Middle first round PCR reaction volumes are 15ul, 10X Taq Buffer1.5ul, 2.5 mM dNTP mixture
1.2ul, Taq 1u, 10uM each 0.6ul of primer, template volume are 5ul, and residue is supplied with pure water；PCR response procedures are：
95°C 300s；94 DEG C of 15s, 52 °C of 45s, 72 °C of 45s, common 35-40 wheels circulation；72℃ 300s.
A kind of described low stain and high sensitive inexpensive general strain method for identifying molecules, it is characterised in that described
Step 7）Middle reaction system is 50-100ul, and template is the 1/1000 of first round reaction product, and its PCR is TP-PCR, wherein
Primer MSF1 working concentration is 0.1uM, is the 1/4 of normal PCR concentration, primer SR1 working concentration is 0.4 uM and primer
M13F working concentration is 0.4 uM；PCR response procedures are：95°C 300s；94 DEG C of 15s, 52 °C of 45s, 72 °C of 45s, altogether
35-40 wheel circulations；72℃ 300s.
The present invention is using simple and efficient broken wall and extractive technique, in conjunction with efficient nucleic acid absorption technology, and PCR
Enlarge-effect twice, can microbe colony formed initial stage, can accurately identify bacterium, and the present invention is used for
The primer of identification has taken into account the uniformity in fungi and bacterium site, not only bacterium can be identified in kind of a level, to fungi
The category even level of kind can be reached, is identified while thus facilitating some bacteriums and fungi, substantially increases efficiency, saved
Experimental cost.For many years, laboratory utilizes more than 800 individual strains of the technical appraisement, effective percentage more than 90%.The behaviour of the present invention
Make be substantially stopped pipe operation, and involved reagent can stringent sterilization, can not only ensure to be not easy dirt between sample well
Dye, also cause sample to be not easy to have an impact operator, therefore be also particularly suitable for company and carry out strain idenfication side using the technology
The service in face.
Compared with prior art, the invention has the advantages that：
（1）Only need a small amount of bacterium colony sample to carry out minim DNA extraction, so as to realize precise Identification, avoid the fiber crops of strain preparation
It is tired；
（2）Using two kinds of easy crushing wall breaking technologies, operated in the case of stopped pipe and sterilizing, not only greatly reduce dirt
Dye, to operator and good protection；
（3）Using the combination of adsorbent suspension and adsorption column, yield or concentration that DNA is extracted are improved so that micro bacterium sample
Identification be possibly realized；
（4）Degenerate primer during amplification can precise Identification bacterium to kind level, category or the level planted can also arrive for fungi,
Eliminate the trouble that fungi and bacterium expand respectively；
（5）Using TRAP twice, the sensitiveness of identification had both been substantially increased, has caused amplified production conveniently to utilize universal primer again
Direct Sequencing.
Brief description of the drawings
Fig. 1 expands principle and flow chart for PCR in the present invention；
Fig. 2 is electrophoretogram in embodiment 1.
In Fig. 2：From left to right：DL2000 marker, blank control, BA1, BA2 ... BA19, wherein BA4 and BA11 expand
Increase failure.
Embodiment
Further illustrate the present invention with reference to embodiments.
Embodiment 1
Trace dna extracts and purifying
1.1 strains pre-process
The sterile centrifugation tube for taking 2.0ml to thicken or reinforce（Pipe break when preventing smudge cells）, 20ul solution As are previously added,
With the bacterium of transfer needle or toothpick the picking 5-10ul volumes from flat board into 2.0ml centrifuge tubes, then using in two methods
It is a kind of：
A. 3-5mm tungsten carbides ball 1 is added, is placed in boiling water bath after 5min, under liquid nitrogen flash freezer, is ground with reciprocating pulverizer
Mill, it is to be restored to normal temperature after grinding；
B. the bead for the 0.1mm-1.5mm that 20ul volumes were anticipated is added, it is to be restored to room as 5min in boiling water bath
Temperature, whirlpool concussion 1min.
1.2 prepare the pillar of high absorption affinity in advance
Prepare the 2ml centrifuge tubes with Filter column, add the adsorbent suspension S 50-100ul prepared in advance, 12,000g angles turn
1min is centrifuged in son, adsorbent is formed inclined-plane in Filter column, gives up filtrate, centrifugal process moulds the outer of adsorbent in the step
Shape, and optimum contact area and depth when inclined-plane ensures nucleic acid absorption outwardly on one side when allowing the adsorption column to centrifuge, ensure separation effect
Rate.
1.3 bacterium crack and nucleic acid absorption
Lysate L 500ul are added to containing above-mentioned（1.1）In pretreated bacterium, up and down acutely concussion 10s, stand 3min with
On, 12000g centrifugation 1min, take supernatant 400ul to be added to above-mentioned（1.2）In the adsorption column made, room temperature places more than 5min,
1min is centrifuged with 6000g-8000g rotating speed, gives up filtrate, collecting pipe places back in.Pay special attention to, inclined-plane one side court during centrifugation
Outside（Similarly hereinafter）, to ensure filtrate farthest by adsorbent, this is also one of importance of the present invention, is avoided general
Absorption membrane plane to place absorption when causing centrifugation uneven.
1.4 cleaning
500ul cleaning fluids W, 12000g centrifugation 15s is added, is cleaned 2 times, is outwelled the filtrate of collecting pipe every time, get rid of 15s for the last time
Afterwards, adsorption column no longer puts back to collecting pipe.
1.5 elution
Adsorption column is transferred in new 1.5ml centrifuge tubes, and 20-30ul eluents E, covers tightly lid in drop, is held in 60 DEG C of 5-
10min, then centrifuges 1min with 12000g, and the filtrate being collected into is the trace dna solution extracted.The solution need not enter
Row Concentration Testing, it can be directly used for 2-4 PCR.
The composition and formula of reagent：
Aaerosol solution A：100 mM Tris-HCl, 25 mM EDTA, 2.0 M NaC1, RnaseA 10ug/ml (pH 8.0).
Bead processing method：First a collection of bead is cleaned 1-2 times with clear water, then with 12N HCl soaked overnights, then
Rinsed clean, last autoclaving are standby after drying.
Preparation with Filter column centrifuge tube：Centrifugal column is spread into the hydrophilic miillpore filter of 1-3 layers（Aperture 1-10um）And press
Tightly, this film need not adsorb the function of nucleic acid in itself, but can carry out autoclaving, and to prevent to pollute, this is also difference city
The importance of other all products on field.
Adsorbent suspension S：3g tripoli is weighed into 50ml triangular flasks, adds 30ml distilled waters, shake well, stands a moment,
Supernatant is removed, so cleaning 3 times, add distilled water to 2 times of tripoli volumes（Cumulative volume about 20ml）, normal temperature is deposited after autoclaving
It is stand-by.1.5ml centrifuge tubes can be used to dispense.
Lysate L：First sterilize 50ml triangular flasks 1,1 bottle of sterilized water, sterile medicine spoon, is sequentially added on super-clean bench：
Guanidinium isothiocyanate 30g；
Sterilized water is sufficiently stirred dissolving as far as possible to about 40ml；
Sterilize NaAc (PH5.2) 3ml；
Sterilize the SDS 2.5ml of SDS 10%；
Sterile glycerol 2.5ml；
Cumulative volume is about 50ml, and after a few houres fully dissolve, normal temperature storage is stand-by.
Cleaning fluid W：First prepare 1 bottle of sterilized water, 70% solution is made into absolute alcohol.Alcohol 35ml can first be added during preparation,
Again plus sterilized water is stand-by to 50ml, normal temperature.
Eluent E：Predominantly sterilized Tris solution（PH8.5）.
The characteristics of embodiment is that the diatomite of absorption can spread out naturally to a certain extent, is easy to adsorb and washes
It is de-, natural inclined-plane is formed in centrifugation, is easy to form optimal adsorption conditionses, even therefore common diatomite adsorbant
Also good effect can be reached, substantially increase the extraction efficiency to denier sample.In addition, above-mentioned material can integrally be carried out
Autoclaving, in general adsorbed film is prevented and has been unable to autoclaving, so as to easily cause the pollution being widely present.
Embodiment 2
PCR is expanded：Expanded using nested primer, avoid the generation of primer dimer.Reaction is made up of twice PCR, principle
As shown in Figure 1.
1.1 prepare the preparation of PCR reaction solutions
For first time PCR, reaction volume is 15ul, 10X Taq Buffer1.5ul, 2.5 mM dNTP mixture
1.2ul, Taq 1u, 10uM each 0.6ul of primer, template volume are 5ul, and residue is supplied with pure water；All PCR reactions exist
Genepro Thermal Cycle（Bioer, Hangzhou）Carried out in PCR instrument, program is：95°C 300s；94 DEG C of 15s,
52 °C of 45s, 72 °C of 45s, common 35-40 wheels circulation；72℃ 300s.
1.2 first round PCR
First round the primer sense primer is SF1：CWYCYGGRAGGCAGCAG（As shown in SEQ No 1）, anti-sense primer
It is SR1：AATTCCTTTRAGTTTCARMCTTGCG（As shown in SEQ No 2）.
2.3 second wheel PCR
Reaction solution is same as above, and template is the 1/1000 of above-mentioned reaction solution.React for TP-PCR, wherein one is M13F and upstream
The combination of primer, i.e. primer MSF1：5'-CACGACGTTGTAAAACGACCWYCYGGRAGGCAGCAG-3'（Such as the institutes of SEQ No 3
Show）, its working concentration is 0.1uM；Anti-sense primer is still primer SR1：AATTCCTTTRAGTTTCARMCTTGCG（Such as SEQ No
Shown in 2）, its working concentration is 0.4uM；Sense primer is primer M13F 5'-CACGACGTTGTAAAACGAC-3'（Such as SEQ
Shown in No.4）, its working concentration is also 0.4uM.
3. electrophoresis detection and purifying
With 1.2% plain agar sugar electrophoresis, in 1 × TAE of buffer solution（pH8.0）In the presence of, under 10v/cm electric-field intensity, electricity
Swim half an hour, take pictures, electrophoretogram, as shown in Figure 2.In Fig. 2 from left to right：DL2000 marker, blank control, BA1,
BA2 ... BA19.Wherein BA4 and BA11 amplification failures.
4.PCR product purifications are used for direct Sequencing
Sequencing primer is M13F
Picking band, it is sequenced with M13F sequencing primers, is compared by Blast with foregoing purification process after purification
It can determine to belong to and plant（Table 1）.From the results, it was seen that fungi and bacterium are different in the size distribution of amplified band.
The strain that table 1. speculates according to BLAST comparison results
Note：BLAST comparison result bracket numerals represent respectively（Measured sequence is used for the available sequences ratio compared, the ratio
To sequence with speculating similarity of the strain in sequence）.
By accumulating accreditation strain nearly thousand of the invention, success rate reaches more than 90%.
SEQUENCE LISTING
<110>Zhejiang Prov. Forest Science Inst
<120>A kind of low stain and high sensitive inexpensive general strain method for identifying molecules
<130> 1
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 17
<212> DNA
<213>It is artificial synthesized
<400> 1
cwycyggrag gcagcag 17
<210> 2
<211> 25
<212> DNA
<213>It is artificial synthesized
<400> 2
aattcctttr agtttcarmc ttgcg 25
<210> 3
<211> 36
<212> DNA
<213>It is artificial synthesized
<400> 3
cacgacgttg taaaacgacc wycyggragg cagcag 36
<210> 4
<211> 19
<212> DNA
<213>It is artificial synthesized
<400> 4
cacgacgttg taaaacgac 19
Claims (9)
1. a kind of low stain and high sensitive inexpensive general strain method for identifying molecules, it is characterised in that comprise the following steps：
1）Strain pre-processes
The picking bacterium to be measured from flat board, more than the bacterium diameter 2mm to be measured, adds into aaerosol solution A, then carry out mechanical crushing
Broken wall；
2）Prepare the pillar of high absorption affinity
The centrifuge tube with Filter column is taken, adsorbent suspension S is added, centrifugal treating, adsorbent is formed inclined-plane in Filter column, is given up
Abandon filtrate；
3）Bacterium cracks and nucleic acid absorption
Lysate L is added to step 1）In pretreated bacterium solution, acutely concussion, stands up and down, centrifugation, then takes supernatant to be added to
Step 2）In the adsorption column made, room temperature is placed, and centrifugation, gives up filtrate, collecting pipe places back in；
4）Cleaning
Take step 3）Obtained supernatant adds cleaning fluid W, centrifuges, and cleaning, outwells the filtrate of collecting pipe every time, gets rid of for the last time
After 15s, adsorption column no longer puts back to collecting pipe, and described cleaning fluid W is the alcoholic solution of concentration 70%；
5）Elution
Adsorption column is transferred in new centrifuge tube, eluent E 10-15ul in drop, covers tightly lid, keep 60 DEG C, 5-10min, from
The heart, the filtrate being collected into are the trace dna solution extracted, and described eluent E is sterilized PH8.5 Tris solution；
6）First round PCR is expanded
Take step 5）Obtained trace dna solution is template, enters performing PCR amplification, primer SF1 nucleosides with primer SF1 and SR1
Acid sequence is as shown in SEQ No 1, and primer SR1 nucleotide sequence is as shown in SEQ No 1；
7）Second wheel PCR amplifications
Take step 6）The 1/1000 of obtained reaction solution is template, enters performing PCR amplification, primer with primer MSF1, SR1 and M13F
MSF1 nucleotide sequence is as shown in SEQ No 3, and primer M13F nucleotide sequence is as shown in SEQ No 4；
8）Electrophoresis detection and purifying；
9）It is sequenced and is compared with primer M13F, it is determined that category and kind.
2. a kind of low stain as claimed in claim 1 and high sensitive inexpensive general strain method for identifying molecules, its feature
It is described step 1）Middle aaerosol solution A is：100 mM Tris-HCl, 25 mM EDTA, 2.0 M NaC1, RnaseA
10ug/ml, pH 8.0.
3. a kind of low stain as claimed in claim 1 and high sensitive inexpensive general strain method for identifying molecules, its feature
It is described step 1）Middle mechanical crushing broken wall treatment method for it is following any one：
A. added in bacterium solution it is sterilized after 3-5mm tungsten carbides ball 1, be placed in boiling water bath after 5min, in liquid nitrogen flash freezer
Under, ground with reciprocating pulverizer, it is to be restored to normal temperature after grinding；
B. the bead for the 0.1mm-1.5mm that 20ul volumes were anticipated is added in bacterium solution, as 5min in boiling water bath, is treated
Recover to room temperature, whirlpool concussion 1min, bead processing method is first cleans bead 1-2 times with clear water, then with 12N HCl
Soaked overnight, subsequent rinsed clean, last autoclaving, drying.
4. a kind of low stain as claimed in claim 1 and high sensitive inexpensive general strain method for identifying molecules, its feature
It is described step 2）The middle centrifuge tube with Filter column is the hydrophilic nanoporous filter membrane that 1-3 layers aperture 1-10um is spread by centrifugal column
Compression obtains.
5. a kind of low stain as claimed in claim 1 and high sensitive inexpensive general strain method for identifying molecules, its feature
It is described step 2）Middle adsorbent suspension S is made by following steps：3g tripoli is weighed into 50ml triangular flasks, adds 20-
30ml distilled waters, shake well, a moment is stood, remove supernatant, cleaned 3 times, add distilled water to 2 times of tripoli volumes, cumulative volume is about
20ml, normal temperature storage is stand-by after autoclaving.
6. a kind of low stain as claimed in claim 1 and high sensitive inexpensive general strain method for identifying molecules, its feature
It is described step 3）Middle lysate L is mixed to get by following components：Guanidinium isothiocyanate 30g, sterilized water 40ml, PH5.2
Sterilize NaAc 3ml, 10% SDS 2.5ml of sterilizing, sterile glycerol 2.5ml, cumulative volume 50ml.
7. a kind of low stain as claimed in claim 1 and high sensitive inexpensive general strain method for identifying molecules, its feature
It is described step 3）Inclined-plane is on one side outwardly during the centrifugation of middle adsorption column.
8. a kind of low stain as claimed in claim 1 and high sensitive inexpensive general strain method for identifying molecules, its feature
It is described step 6）Middle first round PCR reaction volumes are 15ul, 10X Taq Buffer1.5ul, 2.5 mM dNTP
Mixture 1.2ul, Taq 1u, 10uM each 0.6ul of primer, template volume are 5ul, and residue is supplied with pure water；PCR reacts
Program is：95°C 300s；94 DEG C of 15s, 52 °C of 45s, 72 °C of 45s, common 35-40 wheels circulation；72℃ 300s.
9. a kind of low stain as claimed in claim 1 and high sensitive inexpensive general strain method for identifying molecules, its feature
It is described step 7）Middle reaction system is 50-100ul, and template is the 1/1000 of first round reaction product, and its PCR draws for three
Thing PCR, wherein primer MSF1 working concentration are 0.1uM, are the 1/4 of normal PCR concentration, and primer SR1 working concentration is 0.4
UM and primer M13F working concentration are 0.4 uM；PCR response procedures are：95°C 300s；94 DEG C of 15s, 52 °C of 45s, 72 °C
45s, common 35-40 wheels circulation；72℃ 300s.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710686441.5A CN107418951B (en) | 2017-08-11 | 2017-08-11 | Low-pollution and high-sensitivity low-cost general strain molecular identification method |
Applications Claiming Priority (1)
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