CN108410971A - A kind of bacterial nucleic acid sequencing identification method and bacterial identification kits based on DNA characteristics sequence - Google Patents
A kind of bacterial nucleic acid sequencing identification method and bacterial identification kits based on DNA characteristics sequence Download PDFInfo
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Abstract
The present invention relates to a kind of, and identification method and kit is sequenced in the bacterial nucleic acid based on DNA characteristics sequence, and the method comprising the steps of：Bacterium bacterial strain to be identified is chosen, and extracts and purify its genomic DNA；The characteristic sequence fragment of the genomic DNA of PCR amplification after purification；Extraction and purifying pcr amplification product；The sequence of pcr amplification product after purification is measured, and uses versatility sequence assembly software, guiding region is removed, obtains the DNA sequence dna of respective segments；The DNA sequence dna of respective segments is imported into standard nucleic acid sequence library and carries out sequence alignment, carries out the identification of bacterium bacterial strain；Wherein, the extraction and purifying of the genomic DNA of bacterial strain carry out bacterial cell disruption using lysozyme, lauryl sodium sulfate, cetyl trimethylammonium bromide；Using phenol chloroform isoamyl alcohol, ethanol precipitation nucleic acid, the genomic DNA for meeting quality control requirement is obtained.The present invention can be sequenced identification method for bacterial nucleic acid and provide objective, accurate judgment basis, realize quick, the integrated identification of bacterial pollutant.
Description
Technical field
The present invention relates to nucleic acid sequencing identification method more particularly to a kind of bacterial nucleic acid sequencings based on DNA characteristics sequence
Identification method and bacterial identification kits.
Background technology
Using Protocols in Molecular Biology to the dirt in medicine material, auxiliary material, water for pharmaceutical purposes, intermediate, finished product and environment
Dye microorganism is identified, classified and is traced, be reinforce drug production process quality control, Improving The Quality of Products and safety,
Reduce the effective means of drug risk.Differentiate with traditional morphological observation, microscope inspection, physics and chemistry and the technologies phase such as biochemical analysis
It, can be from the inhereditary material level of essence to bacterial strain than, Protocols in Molecular Biology using biotinylated nucleic acid as target detection thing
Kind and source are made explanations, and testing result is more accurate, sensitivity higher, and specificity is stronger, have been increasingly becoming state, interior at present
The inexorable trend of outer drug standard development.Currently,《United States Pharmacopeia》(USP 40<1113>)、《European Pharmacopoeia》(EP 9.0<
5.1.6>)、《Japanese Pharmacopoeia》(JP 17<General Information G4>)、《Chinese Pharmacopoeia》It is received in (general rule 9204)
Related Sections of the Protocols in Molecular Biology for the control of drug microbial quality are carried.
DNA characteristics sequence (DNA bar code) Molecular Identification utilizes one section of generally acknowledged, relatively short DNA in genome
Sequence come carry out species identification molecular biology classify method.This method is logical by screening based on Nucleic acid sequencing techniques
With DNA characteristics sequence, database and identification platform are established, with bioinformatics method analyses and comparison DNA data, and then to object
Kind is identified, is to effective supplement of traditional biological identification method and important breakthrough, in recent years by domestic and international drug quality
The extensive concern of standards setting organizations, and it is increasingly becoming species identification and the research hotspot of classification.
16S rRNA full length genes cover most comprehensive bacterial strain genetic evolution information, but are operated by method for nucleic acid sequencing
Property, sequencing quality, sequencing cost, nucleic acid sequencing reaction length, particularly high throughput nucleic acid sequencing reaction read long limitation, it is most
Document report is still studied using partial nucleic acid sequence (Partial Sequence), has the core of identification and category significance
Heart district domain waits to discuss, such as：USP40<1113>In think that the ends gene 5' 16S rRNA have more the identification of bacterium " kind " level
It is significant；When the researchs such as Chan are reported in the age identification for being clinically separated microorganism, the areas V1~V2 have more category significance；
Kataoka etc. is in streptomysin taxonomic identification using the areas V1~V3 as identification target spot；Becker etc. classifies in staphylococcus to be reflected
Using the comparative approach in the areas V1~V3 and the areas V1~V8 in fixed；Engelbrektson etc. utilizes Roche454 nucleic acid sequencing platforms
Research finds that the area V1, V2 and V8 has more specific positions；Kim etc. is by OTUs (Operational taxonomic
Units) the theory analyzed, it is believed that more there is category significance in the areas V1~V3 and the areas V1~V4 in microorganism species identification；
The researchs such as Chakravorty find that most bacteriums " kind " levels can be achieved in nucleic acid sequence series connection in the area V2, V3, V6 in 16S rRNA
Identification and classification；Lu etc. is classified identification using the microorganism species that PCR and RFLP methods detach clinical cerebrospinal fluid,
Think that it is more low to distinguish resolution by V4~V9 in 16S rRNA.
Currently, the microorganism identification method based on 16S rRNA Nucleic acid sequencing techniques is it has been established that and be applied to face
The research of bed isolated strains, but its identification for being used for functions on common pollutant bacteria in drug quality control and pharmaceutical production environment also rarely has report
Road；In the document for carrying out microbial identification with 16S rRNA method for nucleic acid sequencing, selected sequencing target spot and sequencing primer are each
Different, the DNA characteristics sequence fragment with identification and category significance for meeting drug quality control requirement is still unintelligible clear；It is existing
Nucleic acid sequencing result majority in research is compared by Blast analytic approach and the nucleic acid sequence in Genebank databases
Right, the accuracy attention rate of sequence information is smaller in the quality verification and Genebank databases for sequencing result, sequencing knot
The unification still lack of standardization of the judgment basis of fruit.
Invention content
The purpose of the present invention is to overcome the defects in the prior art, variable by measuring the ends 16S ribosomal RNA gene 5'
The nucleic acid sequence in area carries out bacterial strain identification, provides a kind of easy, quick, standardization bacterial nucleic acid based on DNA characteristics sequence
Identification method is sequenced, and a kind of bacterial identification kits based on DNA characteristics sequence are provided on this basis.
To achieve the goals above, the technical solution that the present invention takes includes：
First purpose of the present invention is a kind of bacterial nucleic acid sequencing identification method based on DNA characteristics sequence comprising
Following steps：
Step 1: choosing bacterium bacterial strain to be identified, and extract and purify the genomic DNA of the bacterial strain；
Step 2: using the characteristic sequence fragment of the genomic DNA in amplimer PCR amplification step 1 after purification；
Step 3: the pcr amplification product obtained in extraction and purification step two；
Step 4: the sequence of the pcr amplification product in determination step two after purification, and it is soft using versatility sequence assembly
Part removes guiding region, obtains the DNA sequence dna of respective segments；
Step 5: the DNA sequence dna of the respective segments described in step 4, which is imported standard nucleic acid sequence library, carries out sequence
It compares, carries out the identification of bacterium bacterial strain；
Wherein, in the step 1, the extraction and purifying of the genomic DNA of bacterial strain are using lysozyme (Lysozyme), ten
Sodium dialkyl sulfate (Sodium dodecyl sulfate, SDS), cetyl trimethylammonium bromide
(Cetyltriethylammnonium Bromide, CTAB) carries out bacterial cell disruption；It is heavy using phenol chloroform-isoamyl alcohol, ethyl alcohol
Shallow lake nucleic acid obtains the genomic DNA for meeting quality control requirement.
Identification method is sequenced in order to advanced optimize above-mentioned bacterial nucleic acid, the technical measures that the present invention takes further include：
Further, a concentration of 10mg/mL~20mg/ml lysozymes of each reagent during the bacterial cell disruption, 5%~
10% (w/v) sodium dodecyl sulfate solution, 5%~10% (w/v) cetyl trimethylammonium bromide solution.Further
Ground, a concentration of 10mg/ml lysozymes of each reagent during the bacterial cell disruption, 5% (w/v) sodium dodecyl sulfate solution,
5% (w/v) cetyl trimethylammonium bromide solution；Or 10mg/ml lysozymes, 10% (w/v) lauryl sodium sulfate are molten
Liquid, 10% (w/v) cetyl trimethylammonium bromide solution；Or 20mg/ml lysozymes, 5% (w/v) lauryl sodium sulfate
Solution, 5% (w/v) cetyl trimethylammonium bromide solution.Most preferably, during the bacterial cell disruption each reagent concentration
It is molten for 10mg/ml lysozymes, 10% (w/v) sodium dodecyl sulfate solution, 10% (w/v) cetyl trimethylammonium bromide
Liquid.
Further, the extraction and purifying of the genomic DNA of the bacterial strain include the following steps：After taking appropriate isolate and purify
Thalline be placed in centrifuge tube, suitable buffer solution, lysozyme, mixing, heating water bath is added；It is molten that lauryl sodium sulfate is added
Liquid, heating water bath；Sodium chloride solution, cetyl trimethylammonium bromide solution, heating water bath is added；Saturation phenol-chlorine is added
Imitative-isoamyl alcohol acutely shakes, is stored at room temperature, centrifuges, supernatant is taken to be placed in new centrifuge tube, repetitive operation 1 time；Add
Enter absolute ethyl alcohol, stood under cryogenic conditions, centrifuges, discards supernatant liquid；The washing of 75% ethanol solution is added, centrifuges, discard supernatant
Liquid, room temperature air-dry complete to ethyl alcohol volatilization；Suitable buffer solution is added, nucleic acid extraction solution is used as after mixing.In this step,
The suitable buffer solution can be conventional any suitable buffer solution that this field uses, preferably TE buffer solutions.
Further, the extraction and purifying (CTAB direct extraction methods) of the genomic DNA of the bacterial strain include following step
Suddenly：It takes the thalline after appropriate isolate and purify to be placed in centrifuge tube, 450 μ l of TE buffer solutions, lysozyme (10mg/ml~20mg/ is added
Ml) 25 μ l, mixing, 37 DEG C of heating water baths 30 minutes；It is added 5%~10% (w/v) sodium dodecyl sulfate solution 50 μ l, 37 DEG C
Heating water bath 15 minutes；1% sodium chloride solution, 80 μ l, 5%~10% (w/v) CTAB solution, 70 μ l, 65 DEG C of heating water baths are added
15 minutes；Saturation phenol chloroform-isoamyl alcohol (25 is added:24:1, v/v/v) 350 μ l of solution acutely shake, are stored at room temperature 5 points
Clock, centrifugation (rotating speed is 12000 turns per minute) 10 minutes, takes supernatant to be placed in new centrifuge tube, repetitive operation 1 time；It is added 2
Times volume absolute ethyl alcohol, -20 DEG C stand no less than 30 minutes, and centrifugation (rotating speed is 12000 turns per minute) 10 minutes discards supernatant
Liquid；Appropriate 75% ethyl alcohol (v/v) solution washing is added, centrifugation (rotating speed is 12000 turns per minute) 10 minutes discards supernatant liquid,
Room temperature air-dries complete to ethyl alcohol volatilization；The TE buffer solutions of appropriate volume are added, nucleic acid extraction solution (template is used as after mixing
DNA), set spare in 4 DEG C of refrigerators.
Further, the extraction and purifying of the genomic DNA of the bacterial strain also can be replaced following steps：Take appropriate separation
Thalline after purification is placed in centrifuge tube, and lysozyme soln, mixing, heating water bath is added；Sodium dodecyl sulfate solution is added,
Heating water bath；Sodium chloride solution, cetyl trimethylammonium bromide solution, heating water bath is added；Cellular lysate liquid is transferred to
On suitable carrier, thalline and lysate are removed by way of centrifugation, nucleic acid is trapped on suitable carrier, and saturation is added
Phenol chloroform-isoamyl alcohol, the washings such as ethanol solution, centrifuges, discards cleaning solution；Room temperature is air-dried to ethyl alcohol and is evaporated completely
Entirely；Suitable buffer solution nucleic acid is added, centrifugation elutes it from suitable carrier, as nucleic acid extraction solution.
In this step, the suitable carrier is any suitable carrier commonly used in the art, preferably adsorption column；It is described suitable
Suitable buffer solution can be conventional any suitable buffer solution that this field uses, preferably TE buffer solutions.In this step, respectively
The dosage and concentration of substance are identical as above-mentioned CTAB direct extraction methods.
It will be understood that, it is possible to use the extraction and purification of suitable commercial kit progress genomic DNA, including but
It is not limited to：Genomic DNA purification kit (DNAiso Reagent)；Bacterial genomes DNA small scale purification kits (TaKaRa
MiniBEST Bacteria Genomic DNA Extraction Kit Ver.3.0)；Genome DNA extracting reagent kit
(Qiagen DNeasy Blood&Tissue Kit).It should operate to specifications, and meet the quality in kit specification
Control requires.
Further, using the concentration and purity of template DNA in acid extraction solution described in determined by ultraviolet spectrophotometry core；
Wherein, nucleic acid concentration is not less than 10ng/ μ l, A260/A280 ratios preferably between 1.8~2.0.
Further, in the step 3, the extraction of pcr amplification product and purification process include the following steps：
Step 1) takes suitable amplified production to be blended in 1.5% Ago-Gel with sample-loading buffer and carries out electrophoresis；
Step 2) cuts the nucleic acid amplification product in the Ago-Gel for terminating electrophoresis in step 1), and suitable delay is added
Fliud flushing, water-bath are completely dissolved；Sodium acetate solution and sodium ethylene diamine tetracetate solution, mixing is added；Absolute ethyl alcohol is added, stand,
It centrifuges, abandon supernatant；The washing of 75% ethanol solution is added, centrifuges, abandon supernatant, ethyl alcohol volatilizees completely；Suitable delay is added
Rush solution, the purification solution after mixing as nucleic acid amplification product.In this step, the suitable buffer solution can be this field
The conventional any suitable buffer solution used, preferably TE buffer solutions
Further, the process of the electrophoresis in the step 1) is：5 μ l of pcr amplification product, 1 μ l of sample-loading buffer are taken, is mixed
Loading after even, the electrophoresis under 100~150V voltages, bromophenol blue band, which is moved at the 1/2~2/3 of gel film, terminates electrophoresis.
Further, the concrete operations of the step 2) are as follows：Nucleic acid amplification product in Ago-Gel is cut, is set
In centrifuge tube, the TE buffer solutions of appropriate volume are added, 65 DEG C of water-bath to gel pieces are completely dissolved；It is separately added into 1/10 volume second
Acid sodium solution (3mol/L, pH5.2) and sodium ethylene diamine tetracetate solution (125mmol/L, pH 8.0), mixing；2 times of volumes are added
Absolute ethyl alcohol, -20 DEG C stand 30 minutes；Centrifugation (rotating speed is 12000 turns per minute) 10 minutes, discards supernatant liquid；It is added appropriate
75% ethyl alcohol (v/v) solution washs, and centrifugation (rotating speed is 12000 turns per minute) 10 minutes discards supernatant liquid, room temperature is air-dried to second
Alcohol volatilization is complete；The TE buffer solutions of appropriate volume are added, the purification solution after mixing as nucleic acid amplification product sets 4 DEG C of ice
It is spare in case
Further, the step 2) can be replaced following steps：It will be in the Ago-Gel that terminate electrophoresis in step 1)
Nucleic acid amplification product cut, be added sodium acetate, mixing；Absolute ethyl alcohol is added, mixing centrifuges, abandons supernatant；70% second is added
Alcoholic solution centrifuges, abandons supernatant；Sterile water dissolving DNA, the purifying as nucleic acid amplification product is added after ethyl alcohol volatilization completely
Solution.
Further, the concrete operations of replaced step 2) are as follows：1/10 volume is added into nucleic acid amplification product
Sodium acetate, mixing；The absolute ethyl alcohol of 2 times of volumes, mixing are added into system；5min is centrifuged with 12000rpm/min, is discarded
Supernatant；Appropriate 70% ethyl alcohol is added, 1min is centrifuged with 12000rpm/min, is discarded supernatant；Wait for that the ethyl alcohol in centrifuge tube is evaporated completely
Appropriate amounts of sterilized water dissolving DNA is added after complete to get to the PCR product of purifying.
It will be understood that, it is possible to use suitable commercial kit carries out the extraction and purification of amplified production DNA fragmentation,
Including but not limited to：Purification kit (TaKaRa MiniBEST DNA Fragment Purification Kit
Ver.4.0), operation carries out to specifications.
Further, the amplimer sequence is as follows：
Forward primer：5'-AGAGTTTGATCCTGGCTCAG-3'；
Reverse primer：5'-GTATTACCGCGGCTGCTGGC-3'；
Wherein, the nucleic acid sequence of the areas the V1~V3 segment in the amplimer amplification 16S rRNA.
Further, in the step 2 pcr amplification reaction system using 25 μ l as reference, including：10 × PCR buffer solutions,
2.5 μ l, 2.5mmol/L dNTPs of 25mmol/L magnesium ion solutions 2 μ l, 10 μm of ol/L primer pairs each 0.5 μ l, 0.5 μ of template DNA
0.2 μ l of l, 5U/ μ l Taq archaeal dna polymerases, add aseptic double-distilled water to 25 μ l；Pcr amplification reaction program is：94 DEG C, pre-degeneration 3
~5 minutes；It 94 DEG C, is denaturalized 30 seconds；It 55~60 DEG C, anneals 30 seconds；72 DEG C, extend 30~60 seconds, 28~32 cycles；72 DEG C, 3
~5 minutes.
Further, when sequence assembly, sequencing quality need to be assessed, the low quality portion at removal sequencing result both ends
Point, and sequence direction should be consistent with PCR amplification forward primer direction.
Second object of the present invention is to provide a kind of bacterium mirror based on DNA characteristics sequence applied in the above method
Determine kit, wherein the kit includes：The nucleic acid extracting reagents such as lysozyme, lauryl sodium sulfate, CTAB, PCR draw
Object, pcr amplification reaction reagent, PCR product purified reagent etc..
Further, the bacterial identification kits, including box body, box cover and liner, the interior setting nucleic acid of the box body carry
Take reagent storage chamber, PCR reaction systems storage chamber and PCR product purified reagent storage chamber, the box body and each storage chamber it
Between fill the liner.
Further, the several test tubes of setting in the nucleic acid extracting reagent storage chamber, the test tube include at least lysozyme
Pipe, sodium dodecyl sulfate solution pipe, cetyl trimethylammonium bromide pipe.
Further, the several test tubes of setting in the PCR reaction systems storage chamber, the test tube include at least primer pipe,
Archaeal dna polymerase pipe, PCR reaction solution pipe；Wherein, PCR buffer solutions, magnesium ion solution and deoxidation are contained in the PCR reaction solution pipe
Ribonucleoside triphosphote.
Further, the several test tubes of setting in the PCR product purified reagent storage chamber, the test tube are slow including at least TE
Fliud flushing pipe, sodium acetate solution pipe and sodium ethylene diamine tetracetate solution conduit.
Further, the off-duty storage chamber at least placing sample-loading buffer test tube is also set up in the box body.
Further, refrigeration chamber is also set up in the box body, the refrigeration chamber is detachable installation with the box body.
Further, the refrigeration storage chamber includes refrigeration exocoel and refrigeration inner cavity chamber, and the refrigeration inner cavity chamber is used for
The test tube containing the reagent that need to be stored under cryogenic is placed, is a sealing between the refrigeration exocoel and refrigeration inner cavity chamber
Hollow structure, built-in cold-retaining agent.
Further, a fastener is fixedly installed on the box body, the box cover setting one makes with fastener cooperation
Convex block.
Further, the shape and size of the box body and each storage chamber are matched according to the shape and size of each test tube
Setting.
Further, liner is fixedly installed in the inside of the box cover.
The present invention is had the following technical effect that compared with prior art using above-mentioned technical proposal：
The present invention establishes the nucleic acid sequencing identification method of common bacteria pollutant in pharmaceutical production and quality control, and bright
Really there is in 16S rRNA the range and length of the DNA characteristics sequence of identification and category significance；Nucleic acid sequencing result is carried out
Quality verification establishes bacterial nucleic acid sequencing identification of dna characteristic sequence in such a way that universal sequencing primer object positions；It establishes at this
Standard nucleic acid sequencing data storehouse on the basis of invention can provide reliable judgment basis for the inspection result of drug microorganism.
The present invention is using the standard nucleic acid sequence built, according to unified generalized description and canonical code typing bacterial nucleic acid
Standard of perfection GenBank is sequenced, identification method can be sequenced for bacterial nucleic acid and objective, accurate judgment basis is provided；
Kit of the present invention can be used for bacterial pollutant from all operationss process to before nucleic acid sequencing that isolates and purifies, and realize thin
Quick, the integrated identification of bacterium pollutant.
Description of the drawings
Fig. 1 is genomic DNA Ago-Gel (1.5% Ago-Gel) electrophoresis in one embodiment of the invention after purification
Figure；Wherein, 1-6 is staphylococcus, and 7-12 is bacillus, and the method for extraction and purification used from left to right in figure is respectively：
Marker, RNA isolation kit 1, RNA isolation kit 2, RNA isolation kit 3, alkaline lysis 1, alkaline lysis 2, alkaline lysis 3, RNA isolation kit 1,
RNA isolation kit 2, RNA isolation kit 3, alkaline lysis 1, alkaline lysis 2, alkaline lysis 3.
Fig. 2 is staphylococcus PCR product Ago-Gel (1.5% Ago-Gel) electrophoresis in one embodiment of the invention
Figure；Wherein, it is respectively from left to right in figure：Marker；1-4：S.aureus (CMCC26003), S.aureus (ATCC6538),
S.aureus (CICC23656), S.aureus isolates are all made of 55 DEG C of annealing；5-8：S.aureus (CMCC26003),
S.aureus (ATCC6538), S.aureus (CICC23656), S.aureus isolates are all made of 58 DEG C of annealing；9-12：
S.aureus (CMCC26003), S.aureus (ATCC6538), S.aureus (CICC23656), S.aureus isolates,
It is all made of 60 DEG C of annealing.
Fig. 3 is pcr amplification product Ago-Gel (1.5% Ago-Gel) electrophoresis after purification in one embodiment of the invention
Figure；Wherein, the method for extraction and purification used from left to right is respectively：Marker；1-2：Method one；3-4：Method two；5-6：Side
Method three.
Fig. 4 and Fig. 5 is PCR product nucleic acid sequencing result splicing schematic diagram in one embodiment of the invention.
Fig. 6 is angstrom uncommon bacterium and enterobacteriaceae reference culture nucleic acid sequencing standard of perfection sequence evolution in one embodiment of the invention
Joining tree figure.
Fig. 7 is the cluster evolution NJ of bacillus reference culture nucleic acid sequencing standard of perfection sequence in one embodiment of the invention
Tree.
Specific implementation mode
The present invention provides a kind of bacterial nucleic acid sequencing identification method based on DNA characteristics sequence comprising following steps：
Step 1: choosing bacterium bacterial strain to be identified, and extract and purify the genomic DNA of the bacterial strain；
Step 2: using the characteristic sequence fragment of the genomic DNA in amplimer PCR amplification step 1 after purification；
Step 3: the pcr amplification product obtained in extraction and purification step two；
Step 4: the sequence of the pcr amplification product in determination step two after purification, and it is soft using versatility sequence assembly
Part removes guiding region, obtains the DNA sequence dna of respective segments；
Step 5: the DNA sequence dna of the respective segments described in step 4, which is imported standard nucleic acid sequence library, carries out sequence
It compares, carries out the identification of bacterium bacterial strain；
Wherein, in the step 1, the extraction and purifying of the genomic DNA of bacterial strain are using lysozyme, dodecyl sulphate
Sodium, cetyl trimethylammonium bromide carry out bacterial cell disruption；Using phenol chloroform-isoamyl alcohol, ethanol precipitation nucleic acid, accorded with
Close the genomic DNA of quality control requirement.
The invention further relates to a kind of bacterial identification kits based on DNA characteristics sequence applied in the above method.
With reference to the accompanying drawings and examples, the specific implementation mode of the present invention is further described.Following embodiment is only
For clearly illustrating technical scheme of the present invention, and not intended to limit the protection scope of the present invention.
The foundation of the standard nucleic acid sequence library of embodiment 1- bacterial nucleic acids sequencing identification
(1) screening of DNA of bacteria characteristic sequence standard of perfection nucleic acid sequence fragments, in the first patent of inventor
(CN201610984864.0) expand verification on the basis of, the scope of application for the sequence fragment that the present embodiment is screened is wider.
The present invention is respectively with staphylococcus (Staphylococcus), pseudomonad (Pseudomonas), angstrom uncommon bacterium
(Escherichia), detection of Salmonella (Salmonella), clostridium (Clostridium), bacillus (Bacillus), micrococcus luteus
(Micrococcus), Cook Salmonella (Kocuria), enterobacteria (Enterobacter), general bacterium (Pantoea), klebsiella
(Klebsiella), Cronobacter sakazakii (Cronobacter), citric acid bacillus (Citrobacter), bulkholderia cepasea
(Burkholderia), enterococcus (Enterococcus), streptococcus (Streptococcus), acinetobacter calcoaceticus
(Acinetobacter), the pharmaceutical production such as Legionella (Legionella) and common bacteria pollutant in inspection are research object,
Screening to sequence in the public nucleic acid databases of Genbank has been obtained and has covered 18 common kinds totally 458 sequences, bacterial strain
Classification information (is shown in Table 1), respectively with the section of constant region position, interception sequence length be about 500bp V1~V3, V2~V4,
V3~V5, V4~V6, the areas V7~V9 nucleic acid sequence, analyze different zones sequence fragment allelic and SNP site number
It measures (being shown in Table 2).Shown in statistical result：The sequence fragment of each group variable region combination allele and SNP containing different number
The length in site, wherein V1~V3 region sequences differs, and there are the insertion in constant site or missings between different kinds, and kind is presented
Specificity between category；In addition to the sequence in the areas V1~V3, the sequence fragment length of each region combination is similar, wherein the areas V3~V5
The sequence of (containing 275 allele, 287 SNP sites) and the areas V4~V6 (containing 284 allele, 249 SNP sites)
Row feature is similar；Compared with the above two, 301 allele, 242 SNP sites are contained in the areas V2~V4, prompt the region to include
More identification and classification information；271 allele are also contained in the areas V7~V9, and 177 SNP sites prompt base to occur prominent
The probability of change is more single.
Different from the sequence fragment of above-mentioned zone, the independent analysis sequence fragment in the areas V1~V3 in table 2, data statistics is aobvious
Show：The nucleic acid sequence of different Pseudomonas is larger in the areas V1~V3 difference, in addition to the allele containing different number and SNP site,
The sequence length of different Pseudomonas is also not exactly the same, wherein escherichia coli, detection of Salmonella, klebsiella, pseudomonad and legion
The sequence length of bacterium is similar, and 99 allele and 190 SNP sites are contained in the sequence of 119 analyzed；Grape ball
Bacterium (50 allele, 81 SNP sites), streptococcus (26 allele, 104 SNP sites), bacillus (22 etc.
Position gene 101 SNP sites) sequence length similar but each " category " between sequence difference it is larger, the sequence with previous group
Compared to the insertion for having small fragment；There is longer Insert Fragment in enterococcus (16 allele, 72 SNP sites), and microballoon
Bacterium (8 allele, 39 SNP sites) and clostridium (31 allele, 143 SNP sites) then have different degrees of sequence
Missing, as a result prompts：The areas V1~V3 include more identifications and classification information, the allele between different Pseudomonas and SNP
Point also more has specificity.The above result shows that：Each variable region fragment in 16S rRNA combines carry out bacterium that can be in various degree
The identification of strain " category " level, the wherein identification in the areas V1~V3 and category significance are more notable.Namely：16S rRNA gene orders expand
Primer：Forward primer (16SV1F)：5'-AGAGTTTGATCCTGGCTCAG-3'；Reverse primer (16SV3R)：5'-
The scope of application of GTATTACCGCGGCTGCTGGC-3' is more extensive.
The bacterial strain information table of 16S rRNA fragment analysis is used in 1 the present embodiment of table
Common bacteria pollutant 16S rRNA sequence-specific Locus Analysis in Shoots in 2 pharmaceutical production of table and inspection
(2) extraction and purification of bacterial genomes DNA
One, test method and operating procedure
1. alkaline lysis
According to the design feature of bacteria cell wall, using lysozyme (Lysozyme), lauryl sodium sulfate, cetyl
The reagents such as trimethylammonium bromide (Cetyltriethylammnonium Bromide, CTAB) carry out bacterial cell disruption；Using phenol-
The agent precipitates nucleic acid such as chloroform-isoamyl alcohol, ethyl alcohol obtains the genomic DNA for meeting quality control requirement.Including but not limited to
Following methods：
(1) it takes the thalline after appropriate isolate and purify to be placed in centrifuge tube, 450 μ l of TE buffer solutions, lysozyme (10mg/ is added
Ml) 25 μ l, mixing, 37 DEG C of heating water baths 30 minutes；5% sodium dodecyl sulfate solution, 50 μ l, 37 DEG C of heating water baths 15 are added
Minute；It is added 1% sodium chloride solution, 80 μ l, 5%CTAB solution 70 μ l, 65 DEG C of heating water baths 15 minutes；Saturation phenol-chlorine is added
Imitative-isoamyl alcohol (25:24:1, v/v/v) 350 μ l of solution acutely shake, are stored at room temperature 5 minutes, and (rotating speed is per minute for centrifugation
12000 turns) 10 minutes, take supernatant to be placed in new centrifuge tube, repetitive operation 1 time；2 times of volume absolute ethyl alcohols of addition, -20 DEG C
No less than 30 minutes are stood, centrifugation (rotating speed is 12000 turns per minute) 10 minutes discards supernatant liquid；Appropriate 75% ethyl alcohol is added
(v/v) solution washs, and centrifugation (rotating speed is 12000 turns per minute) 10 minutes discards supernatant liquid, room temperature is air-dried to ethyl alcohol and is evaporated completely
Entirely；The TE buffer solutions of appropriate volume are added, is used as nucleic acid extraction solution (template DNA) after mixing, sets spare in 4 DEG C of refrigerators.It adopts
With the concentration and purity of determined by ultraviolet spectrophotometry template DNA, the requirement of follow-up test should be able to be met, nucleic acid concentration is preferably not
Less than 10ng/ μ l, A260/A280 ratios preferably between 1.8~2.0；
(2) it takes the thalline after appropriate isolate and purify to be placed in centrifuge tube, 450 μ l of TE buffer solutions, lysozyme (10mg/ is added
Ml) 25 μ l, mixing, 37 DEG C of heating water baths 30 minutes；10% sodium dodecyl sulfate solution, 50 μ l, 37 DEG C of heating water baths 15 are added
Minute；It is added 1% sodium chloride solution, 80 μ l, 10%CTAB solution 70 μ l, 65 DEG C of heating water baths 15 minutes；Saturation phenol-is added
Chloroform-isoamyl alcohol (25:24:1, v/v/v) 350 μ l of solution acutely shake, are stored at room temperature 5 minutes, and (rotating speed is per minute for centrifugation
12000 turns) 10 minutes, take supernatant to be placed in new centrifuge tube, repetitive operation 1 time；2 times of volume absolute ethyl alcohols of addition, -20 DEG C
No less than 30 minutes are stood, centrifugation (rotating speed is 12000 turns per minute) 10 minutes discards supernatant liquid；Appropriate 75% ethyl alcohol is added
(v/v) solution washs, and centrifugation (rotating speed is 12000 turns per minute) 10 minutes discards supernatant liquid, room temperature is air-dried to ethyl alcohol and is evaporated completely
Entirely；The TE buffer solutions of appropriate volume are added, is used as nucleic acid extraction solution (template DNA) after mixing, sets spare in 4 DEG C of refrigerators.It adopts
With the concentration and purity of determined by ultraviolet spectrophotometry template DNA, the requirement of follow-up test should be able to be met, nucleic acid concentration is preferably not
Less than 10ng/ μ l, A260/A280 ratios preferably between 1.8~2.0；
(3) it takes the thalline after appropriate isolate and purify to be placed in centrifuge tube, 450 μ l of TE buffer solutions, lysozyme (20mg/ is added
Ml) 25 μ l, mixing, 37 DEG C of heating water baths 30 minutes；5% (w/v) sodium dodecyl sulfate solution, 50 μ l are added, 37 DEG C of water-baths add
Heat 15 minutes；It is added 1% sodium chloride solution, 80 μ l, 5% (w/v) CTAB solution 70 μ l, 65 DEG C of heating water baths 15 minutes；It is added full
With phenol chloroform-isoamyl alcohol (25:24:1, v/v/v) 350 μ l of solution acutely shake, are stored at room temperature 5 minutes, and (rotating speed is for centrifugation
12000 turns per minute) 10 minutes, take supernatant to be placed in new centrifuge tube, repetitive operation 1 time；2 times of anhydrous second of volume are added
Alcohol, -20 DEG C stand no less than 30 minutes, and centrifugation (rotating speed is 12000 turns per minute) 10 minutes discards supernatant liquid；It is added appropriate
75% ethyl alcohol (v/v) solution washs, and centrifugation (rotating speed is 12000 turns per minute) 10 minutes discards supernatant liquid, room temperature is air-dried to second
Alcohol volatilization is complete；The TE buffer solutions of appropriate volume are added, is used as nucleic acid extraction solution (template DNA) after mixing, sets in 4 DEG C of refrigerators
It is spare.Using the concentration and purity of determined by ultraviolet spectrophotometry template DNA, the requirement of follow-up test, nucleic acid should be able to be met
Concentration is preferably not less than 10ng/ μ l, A260/A280 ratios preferably between 1.8~2.0.
2. other methods
Suitable commercial kit can also be used to carry out the extraction and purification of genomic DNA, including but not limited to：Base
Because of a group DNA purified reagents (DNAiso Reagent)；Bacterial genomes DNA small scale purifications kit (TaKaRa MiniBEST
Bacteria Genomic DNA Extraction Kit Ver.3.0)；Genome DNA extracting reagent kit (Qiagen
DNeasy Blood&Tissue Kit).It should operate to specifications, and meet the quality control requirement in kit specification.
The methods of column centrifugation can also be used.
Using the concentration and purity of determined by ultraviolet spectrophotometry template DNA, the requirement of follow-up test, core should be able to be met
Acid concentration is preferably not less than 10ng/ μ l, A260/A280Ratio is preferably between 1.8~2.0；
Two, test result
1. extracting genome DNA results contrast
Above method is respectively adopted and carries out nucleic acid extraction, obtains bacterial genomes DNA, 5 μ l loadings is taken, with Ago-Gel
Electrophoresis detection result such as Fig. 1, the results showed that：Using alkaline lysis, 10mg/ml lysozymes, 10% lauryl sodium sulfate is added
When solution, 10%CTAB solution, the genomic DNA band of acquisition is single, and non-specific and degradation band is less.
2. extracting genome DNA concentration, purity and method applicability are investigated
The representative bacterial strain of gram-positive cocci, Grain-positive bacillus, gram negative bacilli etc. is selected, totally 10 kinds of 10 bacterium
Strain carries out the extraction (i.e. alkaline lysis 2) of genomic DNA using the alkaline lysis after optimization, obtains genomic DNA.Using purple
Outer spectrophotometry measures the concentration and purity of DNA after extraction, and nucleic acid concentration is not less than 10ng/ μ l, A260/A280Ratio exists
Between 1.8~2.0.Concrete outcome is as follows：
2 extracting genome DNA concentration of table, purity and method applicability are investigated
(3) amplification and detection of DNA of bacteria characteristic sequence fragment
One, test method and operating procedure
1.16S rRNA gene order amplimers：
Forward primer (16SV1F)：5'-AGAGTTTGATCCTGGCTCAG-3'；
Reverse primer (16SV3R)：5'-GTATTACCGCGGCTGCTGGC-3'.
2.PCR reaction systems：
Using 25 μ l as reference, including：10 × PCR buffer solutions (containing trishydroxymethylaminomethane (10mmol/L, pH 8.3~
8.8), 2.5 μ l of magnesium ion solution (25mmol/L), deoxynucleoside triphosphate (dNTPs, 2.5mmol/L) 2 μ l, primer pair (10 μ
Mol/L) each 0.5 μ l, 0.5 μ l of template DNA, 0.2 μ l of high-fidelity Taq archaeal dna polymerases (5U/ μ l), add aseptic double-distilled water to 25 μ
l.Suitable PCR amplification premixed liquid or kit operation can also be used.
3.16S rRNA gene order amplification programs：
94 DEG C 3~5 minutes (pre-degenerations)；94 DEG C 30 seconds (denaturation), 55~60 DEG C 30 seconds (annealing), 72 DEG C 30~60 seconds
(extension), 28~32 cycles；72 DEG C 3~5 minutes.By the way of grads PCR, select respectively 55 DEG C, 58 DEG C and 60 DEG C for
Annealing temperature verifies the specificity of PCR sequence amplifications.
Two, test result
Take 5 μ l of pcr amplification product, 1 μ l of sample-loading buffer, loading after mixing, the electrophoresis under 100~150V voltages, bromine phenol
Blue Streak band, which is moved at the 1/2~2/3 of gel film, terminates electrophoresis.Gel film is taken to be inspected in ultraviolet gel imager, nucleic acid expands
Increase production object and a purpose band occur in the position of about 500bp, such as Fig. 2 (by taking staphylococcus as an example), 60 DEG C of final choice is
The annealing temperature of end reaction system.
(4) purifying of DNA of bacteria characteristic sequence fragment
One, test method and operating procedure
The principle precipitated in acid condition using DNA, dissolved under alkaline condition carries out the PCR product of acquisition pure
Change.Including but not limited to following methods：
1. method one：Nucleic acid amplification product in Ago-Gel is cut, is placed in centrifuge tube, appropriate volume is added
TE buffer solutions, 65 DEG C of water-bath to gel pieces are completely dissolved；Be separately added into 1/10 volumes of acetic acid sodium solution (3mol/L, pH5.2) and
Sodium ethylene diamine tetracetate solution (125mmol/L, pH 8.0), mixing；2 times of volume absolute ethyl alcohols are added, -20 DEG C stand 30 points
Clock；Centrifugation (rotating speed is 12000 turns per minute) 10 minutes, discards supernatant liquid；Appropriate 75% ethyl alcohol (v/v) solution washing is added,
Centrifugation (rotating speed is 12000 turns per minute) 10 minutes, discards supernatant liquid, and room temperature air-dries complete to ethyl alcohol volatilization；It is added suitable for body
Long-pending TE buffer solutions, the purification solution after mixing as nucleic acid amplification product, set spare in 4 DEG C of refrigerators.
2. method two：The sodium acetate of 1/10 volume, mixing are added into PCR product；The nothing of 2 times of volumes is added into system
Water-ethanol, mixing；5min is centrifuged with 12000rpm/min, is discarded supernatant；Appropriate 70% ethyl alcohol is added, with 12000rpm/min from
Heart 1min, discards supernatant；Appropriate amounts of sterilized water dissolving DNA is added after the ethyl alcohol volatilization completely in centrifuge tube, remembers purifying
PCR product.
3. method three：Using DNA fragmentation purification kit (TaKaRa MiniBEST DNA Fragment
Purification Kit Ver.4.0), operation to specifications carries out.
Two, test result
Take 5 μ l of PCR amplification purified product, 1 μ l of sample-loading buffer, loading after mixing, the electrophoresis under 100~150V voltages,
Bromophenol blue band, which is moved at the 1/2~2/3 of gel film, terminates electrophoresis.Gel film is taken to be inspected in ultraviolet gel imager, core
There is a purpose band in the position of about 500bp in sour amplified production, and such as Fig. 3 (by taking staphylococcus as an example), purification effect is equal
Well.The reasonability and validity for considering PCR purification process, can most effective removal template DNA using " method one "
Interference, is determined as preferred method.
(5) foundation of nucleic acid sequencing and standard nucleic acid sequence
It is sequenced according to machine on the standard operating procedures of nucleic acid sequencing instrument, obtains nucleic acid sequence, carry out in accordance with the following methods
Sequence assembly, quality verification and result judgement.
1. sequence assembly and quality verification
The result of nucleic acid sequencing should carry out sequence assembly and quality evaluation, and when sequence assembly should use versatility sequence assembly
Software removes guiding region, obtains the DNA sequence dna of respective segments；To ensure the reliability of standard nucleic acid sequence, when sequence assembly,
Sequencing quality need to be assessed, the low quality part at removal sequencing result both ends.It sequence direction should be with PCR amplification forward primer
Direction is consistent.The average Q value of sequencing result should be greater than or be equal to 30.Nucleic acid sequencing result is spliced with the schematic diagram of quality verification such as
Fig. 4, Fig. 5.
2. result judgement
After sequence assembly and quality verification, standard nucleic acid sequence consistent in length is obtained, according to generalized description and uniformly
Coding, typing standard nucleic acid sequence library can provide objective, accurate judgment basis for the qualification result of this method.
The verification of the uncommon bacterium DNA characteristics Sequence Identification method of 2- angstroms of embodiment
Totally 10 plants of angstrom uncommon bacterium and the enterobacteriaceae other standards bacterial strain of domestic and international microbial strains collection are collected, including
2 plants of the sources ATCC, 2 plants of the sources CMCC, 6 plants of the sources CICC.Be respectively adopted Morphological Identification, biochemical identification method to bacterial strain into
Row hereditary information confirms；Then according to optimize in embodiment 1 method and step extraction genomic DNA, PCR, nucleic acid sequencing, to mark
Quasi- bacterial strain carries out nucleic acid sequencing, establishes standard nucleic acid sequence.
By hereditary information confirm angstrom uncommon bacterium and enterobacteriaceae other standards bacterial strain DNA characteristics Sequence clustering, structure NJ into
Change tree.The nucleic acid sequence of escherichia coli is polymerized to cluster, and it is preferable to illustrate that the level of escherichia coli dna characteristic sequence " kind " has
Identification and category significance；Other bacterial strains of Escherichia are accurately distinguished with enterobacteriaceae clostridium perfringen, Klebsiella Pneumoniae,
Illustrate that angstrom uncommon bacterium reference culture DNA characteristics sequence has preferable identification and category significance (such as Fig. 6) in the level of " category ".
The verification of embodiment 3- bacillus DNA characteristics Sequence Identification methods
Collect totally 5 plants of the bacillus reference culture of domestic and international microbial strains collection, including 2 plants of the sources ATCC,
3 plants of the sources CICC.Morphological Identification is respectively adopted, the method for biochemical identification carries out hereditary information confirmation to bacterial strain；Then according to
The method and step optimized in embodiment 1 extracts genomic DNA, PCR, nucleic acid sequencing, carries out nucleic acid sequencing to reference culture, establishes
Standard nucleic acid sequence.
The DNA characteristics sequence that qualification result is confirmed as to bacillus carries out sequence alignment analysis, and using clostridium as external source
Control group builds NJ chadograms, as a result shows：Bacillus subtilis DNA characteristics sequence can be distinguished clearly with other kinds (see figure
7)。
Bacterial identification kits of the embodiment 4- based on DNA characteristics sequence
To implement the test method after a kind of optimization as basic operation flow, it is fast to prepare the bacterium based on DNA characteristics sequence
Fast identification kit, kit include：The nucleic acid extracting reagents such as lysozyme, lauryl sodium sulfate, CTAB, PCR primer,
Pcr amplification reaction reagent, PCR product purified reagent etc..
Kit described in the present embodiment includes box body, box cover and liner, and the box body and box cover are realized at a side
It is collapsible hinged；It is corresponding respectively on box body and box cover that the fastener being used in conjunction with each other is fixedly installed in the hinged place opposite side
And convex block, to realize the closure of the kit, while liner is fixedly installed in the inside of box cover, to effectively avoid in kit
The big displacement movement of the test tube of its internal storage under closed state.
Setting nucleic acid extracting reagent storage chamber in the box bodys of above-mentioned bacterial identification kits, PCR reaction systems storage chamber,
PCR product purified reagent storage chamber fills the liner between the box body and each storage chamber；In wherein each storage chamber
Several test tubes are set, and nucleic acid extracting reagent storage chamber at least places TE bufferings liquid pipe, lysozyme pipe, cetyl trimethyl bromination
Ammonium pipe；PCR reaction systems storage chamber at least places primer pipe, archaeal dna polymerase pipe, PCR reaction solution pipe, and PCR reaction solution pipe includes
There are PCR buffer solutions, magnesium ion solution and deoxynucleoside triphosphate；PCR product purified reagent storage chamber at least places TE buffer solutions
Pipe, sodium acetate solution pipe and sodium ethylene diamine tetracetate solution conduit.
In above-mentioned storage chamber, it is molten that nucleic acid extracting reagent storage chamber also optionally places TE bufferings liquid pipe, sodium chloride
Liquid pipe, saturation phenol chloroform-isoamyl alcohol pipe, absolute ethyl alcohol pipe, 70% ethanol solution pipe；PCR product purified reagent is stored
Chamber also optionally places sodium acetate solution pipe, absolute ethyl alcohol pipe and 75% ethanol solution pipe.
Also optionally it is arranged in the box body of mentioned reagent box and at least places the reserve chamber of sample-loading buffer test tube and cold
Storage chamber is hidden, the centrifuge tube of all kinds of specifications, semi-commercial (semiworks) production storage pipe can be placed in the off-duty storage intracavitary.
Refrigeration storage chamber described in the present embodiment can be extracted out directly or is put into box body, between box body and refrigeration storage chamber
Cold insulation material is set, one waterproof layer is set on the surface of cold insulation material, to prevent the water vapour in air in the table of cold insulation material
Cold insulation material is drenched in face condensation, extends the service life of cold insulation material；It includes refrigeration exocoel and refrigeration inner cavity to refrigerate storage chamber
Room, refrigeration inner cavity chamber are used to place the test tube containing the reagent that need to be stored under cryogenic, low-temp storage reagent such as low temperature
Store the purification solution (4 DEG C of preservations) of reagent nucleic acid extraction solution (4 DEG C of preservations) and nucleic acid amplification product.Refrigerate exocoel and cold
The hollow structure sealed for one between inner cavity chamber is hidden, built-in cold-retaining agent refrigerates the inner wall of exocoel and refrigerates the outer of inner cavity chamber
Wall is respectively provided with waterproof sealing layer, and to prevent the leakage of cold-retaining agent, which includes gel or water and protect ice medium and heat-insulated
Particle, wherein it is methylcellulose or acrylate copolymer to protect ice medium, and insulation particle is Poly Foam material, using above-mentioned guarantor
Cryogen can effectively extend, meet the use demand of long-time reagent refrigerating.
In the present embodiment, the cross sectional shape of each storage chamber can be the conventional use of shape of ability, such as round, rectangle
Deng the placement mode of the arrangement mode of each storage chamber and each test tube can be selected according to actual conditions.
Kit described in the present embodiment is for bacterial pollutant from isolating and purifying all operationss mistake to before nucleic acid sequencing
Journey, realization bacterial pollutant is quick, integration is identified.
Specific embodiments of the present invention are described in detail above, but it is intended only as example, the present invention is simultaneously unlimited
It is formed on particular embodiments described above.To those skilled in the art, it is any to the equivalent modifications that carry out of the present invention and
It substitutes also all among scope of the invention.Therefore, without departing from the spirit and scope of the invention made by impartial conversion and
Modification, all should be contained within the scope of the invention.
Claims (10)
1. identification method is sequenced in a kind of bacterial nucleic acid based on DNA characteristics sequence, which is characterized in that include the following steps：
Step 1: choosing bacterium bacterial strain to be identified, and extract and purify the genomic DNA of the bacterial strain；
Step 2: using the characteristic sequence fragment of the genomic DNA in amplimer PCR amplification step 1 after purification；
Step 3: the pcr amplification product obtained in extraction and purification step two；
Step 4: the sequence of the pcr amplification product in determination step two after purification, and versatility sequence assembly software is used, it goes
Except guiding region, the DNA sequence dna of respective segments is obtained；
Step 5: the DNA sequence dna of the respective segments described in step 4, which is imported standard nucleic acid sequence library, carries out sequence alignment,
Carry out the identification of bacterium bacterial strain；
Wherein, in the step 1, the extraction and purifying of the genomic DNA of bacterial strain are using lysozyme, lauryl sodium sulfate, ten
Six alkyl trimethyl ammonium bromides carry out bacterial cell disruption；Using phenol chloroform-isoamyl alcohol, ethanol precipitation nucleic acid, acquisition meets quality
Control desired genomic DNA.
2. identification method is sequenced in a kind of bacterial nucleic acid based on DNA characteristics sequence according to claim 1, feature exists
In a concentration of 10mg/ml~20mg/ml lysozymes of each reagent, 5%~10% (w/v) 12 during the bacterial cell disruption
Sodium alkyl sulfate solution, 5%~10% (w/v) cetyl trimethylammonium bromide solution.
3. identification method is sequenced in a kind of bacterial nucleic acid based on DNA characteristics sequence according to claim 2, feature exists
In, a concentration of 10mg/ml lysozymes of each reagent during the bacterial cell disruption, 5% (w/v) sodium dodecyl sulfate solution,
5% (w/v) cetyl trimethylammonium bromide solution；Or 10mg/ml lysozymes, 10% (w/v) lauryl sodium sulfate are molten
Liquid, 10% (w/v) cetyl trimethylammonium bromide solution；Or 20mg/ml lysozymes, 5% (w/v) lauryl sodium sulfate
Solution, 5% (w/v) cetyl trimethylammonium bromide solution.
4. identification method is sequenced in a kind of bacterial nucleic acid based on DNA characteristics sequence according to claim 3, feature exists
In the extraction and purifying of the genomic DNA of the bacterial strain include the following steps：
It takes the thalline after appropriate isolate and purify to be placed in centrifuge tube, suitable buffer solution, lysozyme, mixing, heating water bath is added；
Sodium dodecyl sulfate solution, heating water bath is added；Sodium chloride solution, cetyl trimethylammonium bromide solution, water-bath is added
Heating；Saturation phenol chloroform-isoamyl alcohol is added, acutely shakes, is stored at room temperature, centrifuges, supernatant is taken to be placed in new centrifugation
Guan Zhong, repetitive operation 1 time；Absolute ethyl alcohol is added, is stood under cryogenic conditions, centrifuges, discards supernatant liquid；70% ethanol solution is added
Washing centrifuges, discards supernatant liquid, and room temperature air-dries complete to ethyl alcohol volatilization；Suitable buffer solution is added, is carried as nucleic acid after mixing
Take solution.
5. identification method is sequenced in a kind of bacterial nucleic acid based on DNA characteristics sequence according to claim 3, feature exists
In the extraction and purifying of the genomic DNA of the bacterial strain include the following steps：
It takes the thalline after appropriate isolate and purify to be placed in centrifuge tube, lysozyme soln, mixing, heating water bath is added；It is added 12
Sodium alkyl sulfate solution, heating water bath；Sodium chloride solution, cetyl trimethylammonium bromide solution, heating water bath is added；By bacterium
Body lysate is transferred on suitable carrier, and thalline and lysate are removed by way of centrifugation, and it is suitable that nucleic acid is trapped in
On carrier, saturation phenol chloroform-isoamyl alcohol is added, the washings such as ethanol solution centrifuge, discard cleaning solution；Room temperature wind
It does complete to ethyl alcohol volatilization；Suitable buffer solution nucleic acid is added, centrifugation elutes it from suitable carrier, as
Nucleic acid extraction solution.
6. identification method is sequenced in a kind of bacterial nucleic acid based on DNA characteristics sequence according to claim 1, feature exists
In in the step 3, the extraction of pcr amplification product and purification process include the following steps：
Step 1) takes suitable amplified production to be blended in 1.5% Ago-Gel with sample-loading buffer and carries out electrophoresis；
Step 2) cuts the nucleic acid amplification product in the Ago-Gel for terminating electrophoresis in step 1), and suitable buffering is added
Liquid, water-bath are completely dissolved；Sodium acetate solution and sodium ethylene diamine tetracetate solution, mixing is added；Be added absolute ethyl alcohol, stand, from
The heart abandons supernatant；The washing of 75% ethanol solution is added, centrifuges, abandon supernatant, ethyl alcohol volatilizees completely；Suitable buffering is added
Liquid, the purification solution after mixing as nucleic acid amplification product.
7. identification method is sequenced in a kind of bacterial nucleic acid based on DNA characteristics sequence according to claim 6, feature exists
In the step 2) can be replaced following steps：
Nucleic acid amplification product in the Ago-Gel for terminating electrophoresis in step 1) is cut, sodium acetate, mixing is added；Nothing is added
Water-ethanol, mixing centrifuge, abandon supernatant；70% ethanol solution is added, centrifuges, abandon supernatant；It is added after ethyl alcohol volatilization completely
Sterile water dissolving DNA, the purification solution as nucleic acid amplification product.
8. identification method is sequenced in a kind of bacterial nucleic acid based on DNA characteristics sequence according to claim 1, feature exists
In the amplimer sequence is as follows：
Forward primer：5'-AGAGTTTGATCCTGGCTCAG-3'；
Reverse primer：5'-GTATTACCGCGGCTGCTGGC-3'；
Wherein, the nucleic acid sequence of the areas the V1~V3 segment in the amplimer amplification 16S rRNA.
9. identification method is sequenced in a kind of bacterial nucleic acid based on DNA characteristics sequence according to claim 1, feature exists
In, in the step 2 pcr amplification reaction system using 25 μ l as reference, including：10 × PCR buffer solutions, 25mmol/L magnesium ions
2.5 μ l, 2.5mmol/L dNTPs of solution 2 μ l, each 0.5 μ l of 10 μm of ol/L primer pairs, 0.5 μ l, 5U/ μ l Taq of template DNA
0.2 μ l of archaeal dna polymerase, add aseptic double-distilled water to 25 μ l；Pcr amplification reaction program is：94 DEG C, pre-degeneration 3~5 minutes；94 DEG C,
Denaturation 30 seconds；It 55~60 DEG C, anneals 30 seconds；72 DEG C, extend 30~60 seconds, 28~32 cycles；72 DEG C, 3~5 minutes.
10. a kind of bacterial nucleic acid sequencing identification applied to according to any one of claims 1 to 9 based on DNA characteristics sequence
Bacterial identification kits in method.
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| TWI814499B (en) * | 2021-07-22 | 2023-09-01 | 中央研究院 | Device for continuously producing and analyzing rna, and methods of producing rna by using the device |
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