CN106167820A - Identify or assist primer special and the application thereof identifying propionibacterium acnes - Google Patents
Identify or assist primer special and the application thereof identifying propionibacterium acnes Download PDFInfo
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- CN106167820A CN106167820A CN201610340522.5A CN201610340522A CN106167820A CN 106167820 A CN106167820 A CN 106167820A CN 201610340522 A CN201610340522 A CN 201610340522A CN 106167820 A CN106167820 A CN 106167820A
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- 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
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Abstract
The present invention relates in biological technical field, a kind of primer special and application thereof identified or assist qualification propionibacterium acnes.Primer is the primer pair of any two combined sequence in sequence shown in SEQ ID NO:1 6.The present invention is significant for species identification and its preventing and treating infecting the maxillofacial disease caused of propionibacterium acnes.
Description
Technical field
The present invention relates in biological technical field, a kind of qualification or auxiliary identify the special of propionibacterium acnes
With primer and application thereof.
Background technology
Propionibacterium acnes is a kind of actinomycetes addicted to fatty acid, and Gram-positive is distributed widely in the skin surface of people,
It is different because the vigorous degree of smegma is different in the distribution of skin surface.
Generally believe that propionibacterium acnes is the Main Pathogenic Bacteria causing acne at present.Additionally, at surgical operation such as heart
In valve surgery and orthopedic instrument operation, the case that complicated by postoperative propionibacterium acnes infects is the most rarely seen.
Human skin's microbe species is various, and only propionibacterium just has three kinds, and merely from cultivating and form
Learn and be almost difficult to it is made a distinction on observing.Now to the authentication method of propionibacterium acnes is loaded down with trivial details and the cycle is long, need elder generation
Cultivating sample with specific culture medium, then picking monoclonal carries out 16sPCR amplification and checks order, and just can be finally completed mirror
Fixed.Therefore, the preventing and treating that Species estimation and the initiation thereof of this bacterium are infected by the Rapid identification of propionibacterium acnes is significant.
Summary of the invention
Present invention aim at providing a kind of identify or assist primer special and the application thereof identifying propionibacterium acnes.
For achieving the above object, the present invention uses the technical scheme to be:
A kind of primer special identified or assist qualification propionibacterium acnes, primer is sequence shown in SEQ ID NO:1-6
In the primer pair of specific two combined sequence.
Described primer is to sequence shown in respectively SEQ ID NO:1 and SEQ ID NO:2 (primer to 12)；SEQ ID NO:
Sequence shown in 3 and SEQ ID NO:4 (primer to 34)；Or sequence (primer pair shown in SEQ ID NO:5 and SEQ ID NO:6
56)。
A kind of qualification or auxiliary are identified that the primer special of propionibacterium acnes is identified in preparation or assisted and are identified acne propanoic acid
Application in the test kit of bacillus.
A kind of qualification or auxiliary identify propionibacterium acnes test kit, including primer special described in claim 1.
A kind of detection is from the method whether carrying propionibacterium acnes in sample: comprise the steps:
(1) genomic DNA of testing sample is extracted；
(2) genomic DNA extracted with step (1) is as template, uses primer described in the claims 1 to carrying out
PCR expands；Amplified production meets at least one condition following, i.e. i.e. contains propionibacterium acnes genome in sample to be tested；
When using the primer pair of sequence shown in SEQ ID NO:1 and SEQ ID NO:2, obtain between 180bp and 200bp
Amplified production；
When using the primer pair of sequence shown in SEQ ID NO:3 and SEQ ID NO:4, obtain between 120bp and 140bp
Amplified production；
When using the primer pair of sequence shown in SEQ ID NO:5 and SEQ ID NO:6, obtain between 180bp and 200bp
Amplified production.
Described PCR amplification response procedures concretely: 94 DEG C of denaturations 3min；94 DEG C of degeneration 30s, 60 DEG C-65 DEG C move back
Fire 30s, 72 DEG C of extension 45s, 35 circulations.
Advantage for present invention:
The primer that the present invention provides, may be used for identifying whether sample to be tested contains propionibacterium acnes, its collection of illustrative plates is clear,
The DNA of the multiple samples such as speed is fast, highly sensitive, can be from skin surface wipe samples, bacterial cultures, bacterium colony monoclonal carries
Take detection propionibacterium acnes in product.The present invention is for the strain Rapid identification of propionibacterium acnes and causes the anti-of infection
Tool is significant.
Accompanying drawing explanation
Fig. 1 is the PCR result verification of primer specificity of the present invention.M:marker (D15000+2000)；N: without template pair
According to；A: propionibacterium acnes monoclonal；B: propionibacterium avidum monoclonal；C: propionibacterium granulosum monoclonal；D F: different next
The staphylococcus epidermidis monoclonal in source；G: staphylococcus aureus monoclonal；H: human facial skin's Sample culture；
Each sample 16sPCR in Fig. 1 is verified by Fig. 2 for what the embodiment of the present invention provided.M:marker (D15000+2000)；
N: no template control；A: propionibacterium acnes monoclonal；B: propionibacterium avidum monoclonal；C: propionibacterium granulosum monoclonal；
The staphylococcus epidermidis monoclonal of D F: separate sources；G: staphylococcus aureus monoclonal；H: human facial skin samples cultivation
Thing；
Fig. 3 for the embodiment of the present invention provide to A G sample 16s sequencing result in Fig. 2, after blast comparison, really
Recognizing sequencing result is: A: propionibacterium acnes；B: propionibacterium avidum；C: propionibacterium granulosum；D: staphylococcus epidermidis；E: table
Skin staphylococcus；F: staphylococcus epidermidis；G: staphylococcus aureus.
Fig. 4 Fig. 7, for the experimental result picture of the qPCR detection primer sensitivity of the embodiment of the present invention.
QPCR amplification curve under the template concentrations gradient condition that Fig. 4 provides for the embodiment of the present invention.
The qPCR product melting curve that Fig. 4 amplification curve that Fig. 5 provides for the embodiment of the present invention is corresponding.
QPCR amplification curve under the low template concentrations that Fig. 6 provides for the embodiment of the present invention.
The sepharose electrophoresis figure of the qPCR product that Fig. 7 provides for the embodiment of the present invention.
The testing result of the 6 example patients with acne face cotton swab samplings that Fig. 8 provides for the embodiment of the present invention, wherein M:
marker(D15000+2000)；N: no template control；A: propionibacterium acnes monoclonal (confirms through order-checking)；B D: coating training
Support sample；E G: uncoated culture sample.
The acne culture testing result that Fig. 9 provides for the embodiment of the present invention, wherein M:marker (500bp)；N: without mould
Plate compares；A: propionibacterium acnes monoclonal (confirms through order-checking)；B: be coated with the sample that spreads cultivation；C: protruding petite 1；D: protruding little
Bacterium colony 2；E: flat single bacterium colony.
Detailed description of the invention
Below example facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiment
Method, if no special instructions, is conventional method.Test material used in following embodiment, if no special instructions, is certainly
Routine biochemistry reagent shop is commercially available.
Embodiment 1
Testing sample: the rear culture on cotton swab after wiping the cotton swab after face or wiping face
Extract the DNA of testing sample, refer to the extracting method of gram positive bacteria.
Specifically, taking 6 example acne patient (order of severity 1-4 level) face wiping samples, wherein 3 examples use Colombia
Blood agar plate coating is cultivated, and culture PBS is washed from flat board lower recovery, and extracts culture STb gene.Remaining
3 examples directly use QIAamp DNA mini kit (QIAGEN) to extract sample total DNA；Stand-by.
PCR detects:
Using the DNA of above-mentioned acquisition as template, using primer to carry out PCR augmentation detection to 12, PCR reaction system is total
Volume 20 μ L, including: 0.5 μM of forward primer, 0.5 μM of downstream primer, 1 μ L template, (2 × PCR premixes the 2 × PCRmix of 10 μ L
Reactant liquor), deionized water；
Reaction condition is: 94 DEG C of denaturations 3min；94 DEG C of degeneration 30s, 60 DEG C of annealing 30s, 72 DEG C of extension 45s, 35 are followed
Ring；
Electrophoresis result as shown in Figure 8, wherein M:marker (D15000+2000)；N: no template control；A: acne propanoic acid bar
Bacterium monoclonal (confirms through order-checking, as positive control)；B D: patients with acne face samples coating culture sample；E G: acne
Patient facial region's sample.
As seen from Figure 8, N swimming lane does not has amplified production, and A G swimming lane all has amplified production, and product length is 180
Between 200bp, meet aforementioned amplified production length range.
According to aforementioned criterion, 6 example sample standard deviations can detect propionibacterium acnes.
Embodiment 2
Testing sample: sample culture detection propionibacterium acnes and bacterium monoclonal strain identification
Taking 1 example face has the cotton swab sample of open comedones (blackhead), coating Columbia Blood Agar flat board to cultivate.
(wherein 2 is to be dried protruding petite to 3 single bacterium colonies of picking, and doubtful propionibacterium acnes, another 1 is flat translucent bacterium
Fall) amplification culture, stand-by.
PCR detects:
Using the DAN of above-mentioned acquisition as template, use primer that 34 (seeing table 1) are carried out PCR augmentation detection, PCR
Reaction system cumulative volume 20 μ L, including: 0.5 μM of forward primer, 0.5 μM of downstream primer, 1 μ L template, the 2 × PCRmix (2 of 10 μ L
× PCR premixes reactant liquor), deionized water；
Reaction condition is: 94 DEG C of denaturations 3min；94 DEG C of degeneration 30s, 60 DEG C of annealing 30s, 72 DEG C of extension 45s, 35 are followed
Ring；
Electrophoresis result is as it is shown in figure 9, M:marker (500bp)；N: no template control；A: propionibacterium acnes monoclonal
(confirming through order-checking, as positive control)；B: be coated with the sample that spreads cultivation；C: protruding petite 1；D: protruding petite 2；E: flat list
Bacterium colony
As seen from Figure 9, N duct does not has amplified production；All there is amplified production in A D duct, and product length is 120
Between 140bp, meet aforementioned amplified production length range；E duct is without amplified production.
Judge according to aforesaid standards: containing propionibacterium acnes in cotton swab sample；This sample is dried the little of projection
Bacterium colony 1,2 is propionibacterium acnes；The non-propionibacterium acnes of flat colony in this sample
Embodiment 3
Testing sample: confirm as the genomic DNA of monoclonal culture of propionibacterium acnes as standard substance using order-checking,
Its concentration is diluted to 10 respectively7Individual/μ L；106Individual/μ L；105Individual/μ L；104Individual/μ L；103Individual/μ L；102Individual/μ L；101Individual/μ
L；Stand-by.
QPCR detects:
Using the DNA of the variable concentrations of above-mentioned acquisition as template, using primer to carry out PCR augmentation detection to 56, PCR is anti-
Answer system cumulative volume 20 μ L, including: 0.5 μM of forward primer, 0.5 μM of downstream primer, 1 μ L template, the 2 × qPCRmix (2 of 10 μ L
× qPCR premixes reactant liquor), deionized water；
Reaction condition is: 94 DEG C of denaturations 3min；94 DEG C of degeneration 30s, 60 DEG C of annealing 30s, 72 DEG C of extension 45s, 40 are followed
Ring, detects fluorescence (seeing Fig. 4-7) after each loop ends；
From fig. 4, it can be seen that 107For template concentrations 107The qPCR amplification curve of individual/μ L；106For template concentrations 106Individual/μ L's
QPCR amplification curve；105For template concentrations 105The qPCR amplification curve of individual/μ L；104For template concentrations 104The qPCR of individual/μ L expands
Increase curve；103For template concentrations 103The qPCR amplification curve of individual/μ L；102For template concentrations 102The qPCR amplification song of individual/μ L
Line；101For template concentrations 101The qPCR amplification curve of individual/μ L；N is no template control.It can be seen that primer amplification state is permissible
Reflect the change of template concentrations gradient.
As seen from Figure 5,10-7—10-1For template concentrations 107Individual/μ L 101The qPCR product melting curve of individual/μ L, N is
The qPCR product melting curve of no template control.From melting curve, under each template concentrations, q pcr amplification product is homogeneous；
There is not amplified production in no template control, illustrates that this primer has preferable specificity.
As seen from Figure 6, N is the qPCR amplification curve of no template control, and this reaction is repeated 3 times；101It is 10 for template concentrations1
Individual/μ L time, the amplification curve of qPCR, this reaction is repeated 3 times；102It is 10 for template concentrations2Individual/μ L time, the amplification curve of qPCR,
This reaction is repeated 3 times.When template concentrations is 102Individual/μ L time, can stably carry out qPCR amplification, when template concentrations is down to 101Individual/
μ L, still has certain probability to expand purpose fragment, illustrates that, when template copy numbers reaches 100, this primer sets can realize stable
Augmentation detection.
As seen from Figure 7, N is no template control, 101It is 10 for template concentrations1The qPCR amplified production of individual/μ L, 102For mould
Plate concentration is 102The qPCR amplified production of individual/μ L, 103It is 10 for template concentrations3The qPCR amplified production of individual/μ L, 104For template
Concentration is 104The qPCR amplified production of individual/μ L, 105It is 10 for template concentrations5The qPCR amplified production of individual/μ L, 106Dense for template
Degree is 106The qPCR amplified production of individual/μ L, 107It is 10 for template concentrations7The qPCR amplified production of individual/μ L.All amplified productions
Length all between 180-200bp, meet aforesaid amplified production length range.
Understand according to aforementioned criterion: when propionibacterium acnes DNA copy number is in 101Individual 107Individual graded
In, the primer sets of the present invention all can detect propionibacterium acnes.When in sample, the template DNA copy number of propionibacterium acnes is more than
When 100, stably can be detected by primer sets；When in sample, the template DNA copy number of propionibacterium acnes less than 100 and is more than
When 10, can be detected by primer sets probability.
Embodiment 4
Testing sample: propionibacterium acnes monoclonal；Propionibacterium avidum monoclonal；Propionibacterium granulosum monoclonal；3
The staphylococcus epidermidis monoclonal of separate sources；Staphylococcus aureus monoclonal；Human facial skin's Sample culture；Above institute
There is bacterial colony sample standard deviation to expand through the PCR of 16s total length, and confirm with generation order-checking；All above sample extraction STb gene
After, stand-by.
PCR detects:
Using the DNA of above-mentioned acquisition as template, using primer, 56 (seeing table 1) are carried out PCR augmentation detection (ginseng
See Fig. 1-3), PCR reaction system cumulative volume 20 μ L, including: 0.5 μM of forward primer, 0.5 μM of downstream primer, 1 μ L template, 10 μ L
2 × PCRmix (2 × PCR premixes reactant liquor), deionized water；
Reaction condition is: 94 DEG C of denaturations 3min；94 DEG C of degeneration 30s, 60 DEG C of annealing 30s, 72 DEG C of extension 45s, 35 are followed
Ring；
Electrophoresis result is as it is shown in figure 1, M:marker (D15000+2000)；N: no template control；A: propionibacterium acnes list
Clone；B: propionibacterium avidum monoclonal；C: propionibacterium granulosum monoclonal；The staphylococcus epidermidis list of D F: separate sources
Clone；G: staphylococcus aureus monoclonal；H: human facial skin's Sample culture.
As seen from Figure 1, only A swimming lane propionibacterium acnes and H swimming lane human facial skin's culture can detect amplified production, expand
Between volume increase a length of 180 200bp of thing, meet aforementioned amplified production length range.
Fig. 2 is the 16s amplification checking of all samples in Fig. 1,16s primer and amplification condition by " raw work biological engineering (on
Sea) limited company " provide.
From Figure 2 it can be seen that M:marker (D15000+2000)；N: no template control；A: propionibacterium acnes monoclonal；B:
Propionibacterium avidum monoclonal；C: propionibacterium granulosum monoclonal；The staphylococcus epidermidis monoclonal of D F: separate sources；G:
Staphylococcus aureus monoclonal；H: human facial skin's Sample culture.According to electrophoresis result, all sample DNAs in Fig. 1
It is the most no problem to extract.
Fig. 3 is the generation order-checking peak figure of monoclonal sample in Fig. 2, after blast comparison, confirms that sequencing result is: A:
Propionibacterium acnes；B: propionibacterium avidum；C: propionibacterium granulosum；D: staphylococcus epidermidis；E: staphylococcus epidermidis；F: table
Skin staphylococcus；G: staphylococcus aureus.
According to aforementioned criterion: in A H totally 8 parts of samples, only A propionibacterium acnes and H human facial skin adopt
Sample culture meets aforementioned criterion, can be detected propionibacterium acnes by primer sets of the present invention.This primer sets can effectively detect
Propionibacterium acnes DNA in sample, does not finds false positive situation.
Table 1
Claims (6)
1. identify or the primer special of auxiliary qualification propionibacterium acnes for one kind, it is characterised in that: primer is SEQ ID NO:1-6
The primer pair of specific two combined sequence in shown sequence.
2. identifying or the primer special of auxiliary qualification propionibacterium acnes as described in claim 1, it is characterised in that draw described in:
Thing is to sequence shown in respectively SEQ ID NO:1 and SEQ ID NO:2；Sequence shown in SEQ ID NO:3 and SEQ ID NO:4；
Or sequence shown in SEQ ID NO:5 and SEQ ID NO:6.
3. identifying or assisting the primer special identifying propionibacterium acnes to identify or auxiliary in preparation described in a claim 1
Identify the application in the test kit of propionibacterium acnes.
4. identify or auxiliary qualification propionibacterium acnes test kit for one kind, it is characterised in that: include special described in claim 1 drawing
Thing.
5. one kind is detected the method whether carrying propionibacterium acnes in sample, it is characterised in that: comprise the steps:
(1) genomic DNA of testing sample is extracted；
(2) genomic DNA extracted with step (1) is as template, uses primer described in claim 1 to carrying out PCR amplification；Amplification
Product meets at least one condition following, i.e. i.e. contains propionibacterium acnes genome in sample to be tested；
When using the primer pair of sequence shown in SEQ ID NO:1 and SEQ ID NO:2, obtain the expansion between 180bp and 200bp
Volume increase thing；
When using the primer pair of sequence shown in SEQ ID NO:3 and SEQ ID NO:4, obtain the expansion between 120bp and 140bp
Volume increase thing；
When using the primer pair of sequence shown in SEQ ID NO:5 and SEQ ID NO:6, obtain the expansion between 180bp and 200bp
Volume increase thing.
6. the detection as described in claim 5 is from the method whether carrying propionibacterium acnes in sample, it is characterised in that: institute
State the response procedures of PCR amplification concretely: 94 DEG C of denaturations 3min；94 DEG C of degeneration 30s, 60 DEG C-65 DEG C annealing 30s, 72 DEG C
Extend 45s, 35 circulations.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610340522.5A CN106167820A (en) | 2016-05-19 | 2016-05-19 | Identify or assist primer special and the application thereof identifying propionibacterium acnes |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610340522.5A CN106167820A (en) | 2016-05-19 | 2016-05-19 | Identify or assist primer special and the application thereof identifying propionibacterium acnes |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108078879A (en) * | 2017-12-11 | 2018-05-29 | 华南农业大学 | For preventing the fragrant chinaberry-Bulbus Lilii extract of the Huang of propionibacterium acnes and its application |
| CN110951854A (en) * | 2020-01-04 | 2020-04-03 | 广州中鑫基因医学科技有限公司 | Kit and method for detecting skin based on microbial PCR technology |
| WO2022021927A1 (en) * | 2020-07-27 | 2022-02-03 | 天津科技大学 | Metagenomics-based method for sampling microorganism on facial skin surface, and application |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001081581A2 (en) * | 2000-04-21 | 2001-11-01 | Corixa Corporation | Compositions and methods for the therapy and diagnosis of acne vulgaris |
| CN101177704A (en) * | 2006-11-10 | 2008-05-14 | 佳能株式会社 | Probe, probe set, probe-immobilized carrier, and genetic testing method |
| CN104364394A (en) * | 2012-03-17 | 2015-02-18 | 加州大学评议会 | Fast diagnosis and personalized treatments for acne |
-
2016
- 2016-05-19 CN CN201610340522.5A patent/CN106167820A/en not_active Withdrawn
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001081581A2 (en) * | 2000-04-21 | 2001-11-01 | Corixa Corporation | Compositions and methods for the therapy and diagnosis of acne vulgaris |
| CN101177704A (en) * | 2006-11-10 | 2008-05-14 | 佳能株式会社 | Probe, probe set, probe-immobilized carrier, and genetic testing method |
| CN104364394A (en) * | 2012-03-17 | 2015-02-18 | 加州大学评议会 | Fast diagnosis and personalized treatments for acne |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108078879A (en) * | 2017-12-11 | 2018-05-29 | 华南农业大学 | For preventing the fragrant chinaberry-Bulbus Lilii extract of the Huang of propionibacterium acnes and its application |
| CN110951854A (en) * | 2020-01-04 | 2020-04-03 | 广州中鑫基因医学科技有限公司 | Kit and method for detecting skin based on microbial PCR technology |
| WO2022021927A1 (en) * | 2020-07-27 | 2022-02-03 | 天津科技大学 | Metagenomics-based method for sampling microorganism on facial skin surface, and application |
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Application publication date: 20161130 |