CN109722485A - A method of Rapid identification Human Fungi is sequenced based on sanger - Google Patents
A method of Rapid identification Human Fungi is sequenced based on sanger Download PDFInfo
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Classifications
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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
- C12Q1/6869—Methods for sequencing
-
- 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
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
Abstract
The invention discloses a kind of methods based on sanger sequencing Rapid identification Human Fungi, it is characterised in that it includes DNA is extracted and quality inspection step, design of primers and synthesis step, PCR amplification step, PCR product purification step, PCR product purification step, sequencing steps, data processing step.The beneficial effects of the present invention are: can by special primer the gene order of specific species amplify come, and by sequence verification compare, correspond, accuracy rate almost close to absolutely.And only need to extract after certain bacterium colony extracts DNA and save, it avoids and generates cross contamination during the cultivation process, cause to judge by accident.This method compares other methods, convenient, fast, accuracy is high.
Description
Technical field
The invention belongs to high throughput sequencing technologies fields, and in particular to one kind is true based on sanger sequencing Rapid identification human body
The method of bacterium.
Background technique
Nowadays common several two Methods for Fungi Detection have:
1. direct microscope inspection measures the tested material such as secretion and is applied on glass slide, in microscopic observation hypha form structure
To determine what bacterium bottom is.
2. being separately cultured sample to be inoculated in specific culture medium, bacterium colony is formed after incubated at room temperature, is by appearance judgement
What bacterium, while by bacterium colony smear for microscopic examination.
3. drug sensitivity test
Since the colonial morphology of present many bacterium is much like, easily causes and obscure, and with the evolution of species, many bacterium all have
There is drug resistance, the strain identified in a conventional way is not necessarily correct reliable, has defect.
Dideoxy chain termination (Sanger method) sequencing was invented by Frederick Sanger in 1977, was primarily useful for
DNA sequence analysis, it is nucleic acid-templated existing for nucleic acid polymerase, primer, the four kinds of monodeoxy bases under the conditions of replicate or when transcription,
4 pipes are divided to introduce with 4 kinds of double deoxidation bases of different fluorescence, DNA polymerase can be by 4 kinds of different dideoxyribonucleoside triphosphate raw materials
It is inserted into DNA chain, since 3 ' positions of dideoxyribonucleoside triphosphate lack-OH group, will not continue to extend, it is available in this way
Different length DNA fragmentation with different fluorescence.
Also someone starts the kind that this method is used to identify fungi, and based on the method it is further further investigation and
Development also becomes extremely important.
And the advantage of Sanger sequencing is:
(1) precisely: process is careful, and Quality Control link is more, and pollution is low, and visual result is visual, and spurious results are extremely low.
(2) personalized site primer can be carried out, arbitrarily individual event can be selected to be sequenced, personalized genetic test is sequenced in Sanger
There is price advantage.Since clinical sequencing feature is: " with clearly defined objective, result is accurate, flux is small ", Sanger sequencing are highly suitable for
It is clinical.
Summary of the invention
In order to overcome drawbacks described above present in the prior art, the purpose of the present invention is to provide one kind to be based on sanger
The method that Rapid identification Human Fungi is sequenced.
In order to achieve the object of the present invention, used technical solution is: Rapid identification people is sequenced based on sanger in one kind
The method of body fungi, includes the following steps:
DNA is extracted and quality inspection step: carrying out DNA extraction using secretion (fungi) DNA extraction kit；It uses
NanoDrop2000 test sample concentration and purity are considered as when the concentration > 10ng/uL and purity of sample are between 1.6~2.0
It is qualified；
Design of primers and synthesis step: according to the objective gene sequence to be expanded, the website NCBI (http: //
Www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi? LINK_LO C=BlastHome) on carry out
Design, the major parameter of setting are as follows: primer length is generally 18~24bp；55 DEG C~65 DEG C of theoretical annealing temperature Tm value；(G+C)
Content 40%~70%；Amplified fragments size < 800bp.The primer information designed is made a record, including Primer,
The size of sequence and product；Then the synthesis of primer is carried out；
PCR amplification step: it in the case that confirmation DNA profiling is up-to-standard, can arrange to carry out PCR amplification, reaction system
For 25ul；The super fidelity dna polymerase of New England Biolabs, Ltd Q5 is selected to carry out PCR expansion by template of genome
Increase；
PCR product purification step: the non-purification of samples after PCR amplification is carried out using 1.5% agarose gel electrophoresis pure
Change；
Sequencing steps: be put into after the pre-treatment being sequenced using the BDT3.1 sequencing kit of ABI company sequenator into
Row sequencing；
Data processing step: it is handled after being spliced using splicing software DNAStar using blast method.
In a preferred embodiment of the invention, the Human Fungi includes Candida glabrata, the Candida albicans/torrid zone
Candida albicans, aspergillus flavus, aspergillus fumigatus, any one or more in aspergillus niger.
In a preferred embodiment of the invention, the design of the primer includes Candida glabrata, Candida albicans/heat
It is specific as follows with the primer of any one or more in candida albicans, aspergillus flavus, aspergillus fumigatus, aspergillus niger:
The beneficial effects of the present invention are:
Can by special primer the gene order of specific species amplify come, and by sequence verification compare,
It corresponds, accuracy rate is almost close to absolutely.And only need to extract after certain bacterium colony extracts DNA and save, it avoids
Cross contamination is generated during the cultivation process, causes to judge by accident.This method compares other methods, convenient, fast, accuracy is high.
Detailed description of the invention
Fig. 1 is that data splice schematic diagram (1)；
Fig. 2 is that data splice schematic diagram (2)；
Fig. 3 is that data splice schematic diagram (3)；
Fig. 4 is that data splice schematic diagram (4)；
Fig. 5 is blast data processing schematic diagram (1)；
Fig. 6 is blast data processing schematic diagram (2)；
Fig. 7 is the comparison result schematic diagram of candida albicans；
Fig. 8 is the comparison result schematic diagram of Candida tropicalis；
Fig. 9 is the comparison result schematic diagram of Candida glabrata；
Figure 10 is the comparison result schematic diagram of aspergillus flavus；
Figure 11 is the comparison result schematic diagram of aspergillus fumigatus；
Figure 12 is the comparison result schematic diagram of aspergillus niger；
Specific embodiment
Referring to Fig. 1-6, specific embodiments of the present invention are as follows:
1, the extraction (Zhangjiagang indigo plant revive object Engineering Co., Ltd production) of bacterium or fungal chromosome DNA
1.1 secretion (fungi) DNA extraction kit
1.11 can directly carry out next step in room temperature or the sample saved at 2-8 DEG C in this way；In -20 DEG C or less ice
The sample saved in case, being first placed in room temperature environment melts sample completely, then carries out next step.
1.12, which take out whole suspension, is put into 1.5ml centrifuge tube, and 13000r/min is centrifuged 3 minutes, abandons supernatant.
1.13, which are added the concussion of 1ml sample cleaning solution, is resuspended, and 13000r/min is centrifuged 3 minutes, abandons supernatant.
1.14 are added 300 μ l sample treatment solution A, and precipitating is resuspended.Sample will be resuspended and be transferred completely into the extraction of solid containing DNA
In the centrifuge tube (enabling solid content combine in tube bottom using preceding pre- centrifugation) of object, shaken 10 minutes with strength oscillator high speed whirlpool, wink
When be centrifuged.200 μ l of sample treatment solution B is added, mixes, 13000rpm is centrifuged 10 minutes.Take supernatant (note: if any layering,
Not suck lower liquid), it is transferred in new 1.5ml centrifuge tube.The dehydrated alcohol of 0.5 times of volume is added, mixes.
Liquid transfers whole in centrifuge tube are no more than 600 μ l to DNA centrifugal purification pipe with well (hereinafter referred to as by 1.15
" purifying pipe ") in, 13000rpm is centrifuged 1 minute, discards the centrifugate in casing；This operation to whole liquid is repeated to be centrifuged
Finish.
1.16 purifying pipes are replaced in casing, flushing liquor 600 μ l, 13000rpm are added centrifugation 1 minute, discard casing
In centrifugate.Repeating step, this is primary.
1.17 purifying pipes are replaced in casing, and 13000rpm is centrifuged 3 minutes (this operation is to remove ethyl alcohol residual)；
Centrifugation finishes, and opening purifying pipe pipe lid and drying in the air 1-2 minutes is completely dried filter membrane.
1.18 abandon casing, and purifying pipe is put into new 1.5ml centrifuge tube, carefully drip above purifying bottom of the tube filter membrane
Add the 40 μ l of DNA eluent of 70 DEG C of preheatings, stands 3 minutes.
1.19 13000rpm are centrifuged 2 minutes (as centrifugation needs that centrifuge tube pipe lid, the DNA recovered liquid after centrifugation can be cut off
Separately centrifuge tube is taken to save).
1.20 centrifugation gained DNA recovered liquids can be used for subsequent experimental, and extracted fungal DNA should not be placed at room temperature for, answer
It is immediately placed at -20 DEG C of following temperature long-term preservations.
1.2 sputums (fungi) DNA extraction kit
1.21 can directly carry out next step in room temperature or the sample saved at 2-8 DEG C in this way；In -20 DEG C or less ice
The sample saved in case, being first placed in room temperature environment melts sample completely, then carries out next step.
1.22 are added 2-2.5ml sputum treatment fluid in 5ml centrifuge tube, and 1ml or so sputum, shaking liquefaction 15-30 is added
Minute (depending on sputum sample situation, can 37 DEG C of warm bath).
1.23 13000r/min are centrifuged 3 minutes, abandon supernatant, and the concussion of 1ml sample cleaning solution is added and is resuspended, 13000r/
Min is centrifuged 3 minutes, abandons supernatant, 300 μ l sample treatment solution A is added, precipitating is resuspended.
1.24 centrifuge tubes that the extract of solid containing DNA is transferred completely into sample is resuspended (enable solid content using preceding pre- centrifugation
Combine in tube bottom) in, it is shaken 10 minutes with strength oscillator high speed whirlpool, brief centrifugation.
1.25 are added 200 μ l of sample treatment solution B, mix, and 13000rpm is centrifuged 10 minutes.
1.26 take supernatant (note: if there is layering, not suck the liquid of lower layer), are transferred to new 1.5ml centrifugation
The dehydrated alcohol of 0.5 times of volume is added in Guan Zhong, mixes.
Liquid transfers whole in centrifuge tube are no more than 600 μ l to DNA centrifugal purification pipe with well (hereinafter referred to as by 1.27
" purifying pipe ") in, 13000rpm is centrifuged 1 minute, discards the centrifugate in casing；This operation to whole liquid is repeated to be centrifuged
Finish.
1.28 purifying pipes are replaced in casing, flushing liquor 600 μ l, 13000rpm are added centrifugation 1 minute, discard casing
In centrifugate.
1.29 repeat step (8) once.
1.30 purifying pipes are replaced in casing, and 13000rpm is centrifuged 3 minutes (this operation is to remove ethyl alcohol residual)；
Centrifugation finishes, and opening purifying pipe pipe lid and drying in the air 1-2 minutes is completely dried filter membrane.
1.31 abandon casing, and purifying pipe is put into new 1.5ml centrifuge tube, carefully drip above purifying bottom of the tube filter membrane
Add the 40 μ l of eluent of 70 DEG C of preheatings, stands 3 minutes.
1.32 13000rpm are centrifuged 2 minutes (as centrifugation needs that centrifuge tube pipe lid, the DNA recovered liquid after centrifugation can be cut off
Separately centrifuge tube is taken to save).
1.33 centrifugation gained DNA recovered liquids can be used for subsequent experimental, and extracted fungal DNA should not be placed at room temperature for, answer
It is immediately placed at -20 DEG C of following temperature long-term preservations.
2, DNA concentration and purity detecting
Using NanoDrop2000 test sample concentration and purity, detection scheme presses NanoDrop2000 standard operation stream
Journey.
3, design of primers
3.1 search corresponding genome sequence, following high-volume in database by the Classification systems of species on NCBI
Then the species sequence finds homologous conservative region by Clustalx analysis.
3.2 aim sequences expanded as needed, in the website NCBI (http://www.ncbi.nlm.nih.gov/
Tools/primer-blast/index.cgi? LINK_LO C=BlastHome) on be designed, the major parameter of setting
Are as follows: primer length is generally 18~24bp；55 DEG C~65 DEG C of theoretical annealing temperature Tm value；(G+C) content 40%~70%；Amplification
Clip size < 800bp.The primer information designed is made a record, including Primer, the size of sequence and product.
Record is referring to table 1:
Table 1
4, primer synthesizes
It is synthesized, is synthesized using 192 machine of Dr.Oligo, using being by the biotech inc upper Shanghai's style Sen Nuo
Solid phase phosphoramidite triester method.Drop DNA is fixed on synthesizing by 3 ' → 5 ' completion DNA chains, adjacent nucleotide on solid phase carrier
It is keyed by 3 ' → 5 ' di-phosphate esters.By alkaline high-temperature process, the primer being connected on carrier is scaled off, general PAGE
Primer is purified, finished product primer is concentrated with C18, desalination precipitating, and the primer aqueous suspension after precipitating measures OD260It is quantitative.
5, PCR amplification
After design of primers synthesis, in the case that confirmation DNA profiling is up-to-standard, it can arrange to carry out PCR amplification, instead
Answering system is 25ul.
The super fidelity dna polymerase of New England Biolabs, Ltd Q5 is selected to carry out PCR expansion by template of genome
Increase.
Pcr amplification reaction system:
Response parameter is referring to table 2:
Table 2
6, it purifies
(1) 1.5% agarose gel electrophoresis: taking the non-purification of samples of 25uL PCR, and Loading Buffer is added, and mixes
It adds in the hole of 1.5% Sepharose Purification glue, terminates in 120V electrophoresis about 30min afterwards.
(2) DNA gel recycles:
After electrophoresis, under ultraviolet lamp referring to DNA Marker according to clip size extract respective strap be put into 2mL from
In heart pipe.
400ul Buffer DE-A is added into centrifuge tube, in 75 DEG C of heating water baths until gel piece is completely molten after mixing
Change.
200ul Buffer DE-B is added into centrifuge tube, is uniformly mixed.
Mixed liquor is fully transferred to stand 5min on adsorption column, 3600r/min is centrifuged 1min, abandons waste liquid.
Adsorption column is put back into 2mL centrifuge tube, adds 500ul Buffer W1,3600r/min is centrifuged 1min after standing 3min,
Abandon waste liquid.
Adsorption column is put back into 2mL centrifuge tube, adds 700ul Buffer W2,3600r/min is centrifuged 1min after standing 3min,
Abandon waste liquid.It is primary to repeat the step.
Blank pipe is centrifuged 12000r/min, is centrifuged 2min, abandons waste liquid.
Adsorption column is shifted into new 1.5mL centrifuge tube, air spontaneously dries 8-10min.
Add 30ul ddH2O (70 DEG C of preheatings) to adsorption column filter membrane center, stands 5min, 12000r/min is centrifuged 1min.
Abandon adsorption column.
It is left that 1% agarose gel electrophoresis 5min is added to after taking 2ul DNA after the recovery and 1ul Loading Buffer to mix
The right side has seen whether band and band brightness under ultraviolet lamp.
Judge underproof sample, adjust system, carries out second of PCR amplification.
7, it is sequenced
(1) sequencing PCR reaction.The reagent of selection is the BDT3.1 sequencing kit of ABI company
(BigDyeTerminator v3.1), sequencing reaction are carried out according to BDT3.1 service manual.PCR reaction cycle program is sequenced
Are as follows: 95 DEG C of initial denaturation 2min → (95 DEG C of 30s, 50 DEG C of 1min, 60 DEG C of 4min) × 25 → 4 DEG C.
(2) purifying of PCR product is sequenced.Using the ethanol precipitation methods in BDT3.1 service manual, it is protected from light for 4 DEG C after drying
It saves, it can be up to one month as long as sealing the tight holding time.
(3) require: dispensing is checked after 1. expanding, and is removed undesirable.2. crossing column purification (not less than 300uL's
Binding)；Also the purifying of 96 orifice-plate type pillars can be used.3. PCR product recovering state is checked in secondary dispensing after elution.4. pure
Degree: OD260/OD280=1.6-2.0, dosage 30ng.
(4) PCR product sequencing reaction system is referring to table 3:
Table 3
(5) amplification program:
(6) it purifies:
1) 4ulEDTA, 95% ethyl alcohol of 45ul is added to every hole.Concussion is centrifuged 4500rpm, 25min after mixing.
2) remove supernatant, be slightly buckled on dustless blotting paper it is several under, then pad dustless blotting paper, 96 orifice plates be buckled to quick
Centrifugation is eliminated as much as remaining solution to 600rpm.
3) 75% ethyl alcohol after 100ul ice bath, 4500rpm after slight concussion mixes, centrifugation is added into 96 orifice plates again
5min。
4) remove supernatant, be slightly buckled on dustless blotting paper it is several under, then pad dustless blotting paper, 96 orifice plates be buckled to quick
Centrifugation is eliminated as much as remaining solution to 600rpm.
5) 37 DEG C of baking ovens are placed, are taken out after 20min.
6) 10ulFormamide Deionized is added into 96 orifice plates, after shaking 1-2min, rapid centrifugation is extremely
2000rpm。
7) be denaturalized: upper 95 DEG C of 4min of PCR instrument are quickly put into 4min in ice water.
8) it is put into sequenator, is sequenced after drying.
8, data processing
(1) splice:
Spliced using splicing software: DNAStar
SeqMan II not only can be by thousands of a sequence assemblies at contigs, and assembly can repair in early period
Whole ropy sequence and contamination data is removed from sequence, moreover it is possible to improved editor and output function.
Removal contradiction base and notch: incredible region delete is chosen.
Check covering surface and building result:
File is saved to be exported with sequence: after sequence modification, being clicked uppermost sequence, is clicked Ctrl A+Ctrl C duplication
Complete sequence is placed in sequence in corresponding txt text.
(2) blast:
The official website NCBI is opened, is looked on the right side of website and beats BLAST option and click to enter, find core in Basic Blast
Sour blast is simultaneously clicked to enter, and target sequence is squeezed into the dialog box of input inquiry, is clicked Blast and is compared.
Referring to Fig. 7-12, the present invention can by special primer the gene order of specific species amplify come, and
It is compared, is corresponded by sequence verification, accuracy rate is almost close to absolutely.
Sequence table
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<120>a kind of method based on sanger sequencing Rapid identification Human Fungi
<130> 20181120
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<211> 20
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ccacttcaga gcggagaatc 20
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<213> Artificial sequence
<400> 2
ctcgtgtccc acatactgat 20
<210> 3
<211> 20
<212> DNA
<213> Artificial sequence
<400> 3
cgtcgctact accgattgaa 20
<210> 4
<211> 20
<212> DNA
<213> Artificial sequence
<400> 4
gattctcacc ctctgtgacg 20
<210> 5
<211> 20
<212> DNA
<213> Artificial sequence
<400> 5
ctggaaaaag attgatttgc 20
<210> 6
<211> 20
<212> DNA
<213> Artificial sequence
<400> 6
agtgtagggt tcctagcgag 20
<210> 7
<211> 20
<212> DNA
<213> Artificial sequence
<400> 7
aaataaagtt gggtgtcggc 20
<210> 8
<211> 18
<212> DNA
<213> Artificial sequence
<400> 8
agtgagggcc ctctgggt 18
<210> 9
<211> 20
<212> DNA
<213> Artificial sequence
<400> 9
gaaagaatgg ttggaaaacg 20
<210> 10
<211> 18
<212> DNA
<213> Artificial sequence
<400> 10
gtcctttggg cccaacct 18
Claims (3)
1. a kind of method based on sanger sequencing Rapid identification Human Fungi, which comprises the steps of:
DNA is extracted and quality inspection step: carrying out DNA extraction using secretion (fungi) DNA extraction kit；It uses
NanoDrop2000 test sample concentration and purity are considered as when the concentration > 10ng/uL and purity of sample are between 1.6~2.0
It is qualified；
Design of primers and synthesis step: according to the objective gene sequence to be expanded, the website NCBI (http:// Www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi? LINK_LOC=BlastHome) on carry out
Design, the major parameter of setting are as follows: primer length is generally 18~24bp；55 DEG C~65 DEG C of theoretical annealing temperature Tm value；(G+C)
Content 40%~70%；Amplified fragments size < 800bp；The primer information designed is made a record, including Primer,
The size of sequence and product；Then the synthesis of primer is carried out；
PCR amplification step: it in the case that confirmation DNA profiling is up-to-standard, can arrange to carry out PCR amplification, reaction system is
25ul；The super fidelity dna polymerase of New England Biolabs, Ltd Q5 is selected to carry out PCR amplification by template of genome；
PCR product purification step: the non-purification of samples after PCR amplification is purified using 1.5% agarose gel electrophoresis；
Sequencing steps: sequenator is put into after the pre-treatment being sequenced using the BDT3.1 sequencing kit of ABI company and is surveyed
Sequence；
Data processing step: it is handled after being spliced using splicing software DNAStar using blast method.
2. a kind of method based on sanger sequencing Rapid identification Human Fungi as described in claim 1, which is characterized in that institute
State Human Fungi include Candida glabrata, it is Candida albicans/Candida tropicalis, aspergillus flavus, aspergillus fumigatus, any one in aspergillus niger
Kind is a variety of.
3. a kind of method based on sanger sequencing Rapid identification Human Fungi as described in claim 1, which is characterized in that institute
The design of primer is stated including any in Candida glabrata, Candida albicans/Candida tropicalis, aspergillus flavus, aspergillus fumigatus, aspergillus niger
One or more primers, specific as follows:
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| CN201811386212.2A CN109722485A (en) | 2018-11-20 | 2018-11-20 | A method of Rapid identification Human Fungi is sequenced based on sanger |
| PCT/CN2019/078350 WO2020103359A1 (en) | 2018-11-20 | 2019-03-15 | Rapid identification method for fungi in human body on basis of sanger sequencing |
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