CN110184371A - A kind of specific primer and its methods and applications detecting skin spy acinetobacter calcoaceticus - Google Patents
A kind of specific primer and its methods and applications detecting skin spy acinetobacter calcoaceticus Download PDFInfo
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- CN110184371A CN110184371A CN201910637631.7A CN201910637631A CN110184371A CN 110184371 A CN110184371 A CN 110184371A CN 201910637631 A CN201910637631 A CN 201910637631A CN 110184371 A CN110184371 A CN 110184371A
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- 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 invention discloses a kind of specific primers and its methods and applications for detecting skin spy acinetobacter calcoaceticus, the specific primer is as shown in NO.1~2 SEQ ID, by extracting sample to be tested genomic DNA, PCR amplification is carried out using above-mentioned primer, detected through gel electrophoresis, it takes pictures under gel imaging system detection, there are the DNA specific bands of 485bp, it is determined that contain skin spy acinetobacter calcoaceticus in sample.Short, high specificity, sensitivity are higher the time required to using detection method detection skin spy acinetobacter calcoaceticus of the invention.The invention avoids use the disadvantages such as conventional identification method is cumbersome, time-consuming, accuracy is low, recall rate is low.
Description
Technical field
The invention belongs to microorganisms technical fields, and in particular to it is a kind of detect skin spy acinetobacter calcoaceticus specific primer and its
Methods and applications.
Background technique
Skin spy acinetobacter calcoaceticus (Acinetobacter pittii) belongs to acinetobacter, is a kind of strictly aerobic, non-dairy
The gram-negative coccobacillus of sugar fermentation, is widespread in nature, is also distributed in the respiratory tract of humans and animals, to strong
Health individual is general not pathogenic, belongs to often in flora.Acinetobacter calcoaceticus is endangered due to being largely distributed and having in hospital environment at present,
Paid much attention to by researcher, is one of essential condition pathogenic bacteria of hospital acquired infections, respiratory system, uropoiesis can be caused
The infection of the whole bodies multisystem such as system, haematogenous, skin and wound.And it with the abuse of antibiotic, gradually appears multiple
Antibody-resistant bacterium and general antibody-resistant bacterium, harmfulness constantly rise, and have the possibility as superbacteria.Skin spy acinetobacter calcoaceticus and Bao Man are not
Lever bacterium (Acinetobacter baumanii) and hospital's acinetobacter calcoaceticus (Acinetobacter nosocomialis) belong to
Bao Man-Acinetobacter calcoaceticus complex, in mankind hospital recall rate with higher and harmfulness, skin spy acinetobacter calcoaceticus is
It is proved fish can be caused to fall ill, case fatality rate is higher, and it is motionless that the disease incidence and the death rate in patient in hospital are slightly less than Bao Man
Bacillus, but the disease incidence of skin spy's acinetobacter calcoaceticus and the death rate have the tendency that going up year by year, and researcher has been caused to pay attention to.And skin
Special acinetobacter calcoaceticus, Acinetobacter bauamnnii and hospital's acinetobacter calcoaceticus have similar phenotype and genetic correlation, it is difficult to pass through routine
Means are distinguished, and need to be identified by molecular biology method.
Since not only time-consuming for traditional method for determining bacteria such as biochemical test, accuracy rate is low, general by biochemical test
It can determine that category.And effectively and reliably area cannot be carried out to skin spy's acinetobacter calcoaceticus, Acinetobacter bauamnnii and hospital's acinetobacter calcoaceticus
Point, so needing to be detected by molecular biology for detection.And the means such as 16S rRNA sequence, for big
It measures for sample, testing cost is higher, is not suitable for conventional detection.Therefore, being badly in need of one kind has fastly skin spy's acinetobacter calcoaceticus
Speed, specificity, the PCR detection method of sensibility.
Summary of the invention
The purpose of the present invention is to provide a kind of skin spy acinetobacter calcoaceticus PCR detection primer and methods.The primer can be used for skin
The PCR of special acinetobacter calcoaceticus is detected, and has detection time short, at low cost, and testing result specificity is high, as a result easy interpretation, practicability
By force.
The present invention is realized especially by following technical scheme:
A kind of specific primer detecting skin spy acinetobacter calcoaceticus, the specific primer includes PARC-F and PARC-R,
Specifically:
PARC-F:CTCTCAGCAAAAGCAGCG；
PARC-R:CCTGATGAGCTAGCAGCAATAAG.
In another aspect of this invention, a kind of method for detecting skin spy acinetobacter calcoaceticus is provided, comprising the following steps:
1) sample to be tested genomic DNA is extracted；
2) PCR amplification is carried out using above-mentioned primer；
3) detected through gel electrophoresis, detection of taking pictures under gel imaging system, there are the DNA specific bands of 485bp, then really
Contain skin spy acinetobacter calcoaceticus in random sample product.
Further, the sample gene group is extracted using Ezup column bordetella gene group DNA extraction agent box.
Further, the pcr amplification reaction system is 10 μ L:2 × TSINGKE Master Mix (blue) 5 μ L,
PARC-F 0.5 μ L, PARC-R 0.5 μ L, 1 μ L of template DNA, distilled water are mended to 10 μ L.
Further, the pcr amplification reaction program are as follows: 95 DEG C of initial denaturation 5min；Into circulation, 95 DEG C of denaturation 30s,
55 DEG C of annealing 30s, 72 DEG C of extension 30s, 35 recycle；72 DEG C of extension 10min, 4 DEG C of preservation PCR products terminate.
In addition, the application the present invention also provides the specific primer in the detection of skin spy's acinetobacter calcoaceticus.
Meanwhile the present invention also provides a kind of for detecting the kit of skin spy's acinetobacter calcoaceticus, the kit includes
Above-mentioned primer pair.
The invention has the benefit that
Due to skin spy acinetobacter calcoaceticus and Acinetobacter bauamnnii, there is similar phenotype, conventional biochemical between hospital's acinetobacter calcoaceticus
Test can not be distinguished effectively, and after the amplification of 16S rRNA universal primer, it is also necessary to it is sequenced and determines bacterium kind, be not suitable for conventional inspection
It surveys.Specific fragment using the specific primer for the highly conserved PARC gene of skin spy acinetobacter calcoaceticus expands, can
Directly according to the electrophoresis result of PCR product to determine whether being skin spy acinetobacter calcoaceticus.Both with the specificity of molecular biology, phase
Time and cost have been saved again for the method for 16S rRNA sequencing.
Detailed description of the invention
Fig. 1 is PCR detection method annealing temperature evaluation test gel electrophoresis result figure in embodiment；M:Maker；1-7 is moved back
Fiery temperature is respectively 55 DEG C, 56 DEG C, 57 DEG C, 58 DEG C, 59 DEG C, 60 DEG C, 61 DEG C 8: negative control；
Fig. 2 is PCR detection method specificity evaluation test gel electrophoresis result figure in embodiment；M:Maker；1: Pi Te is not
Lever bacterium, 2: skin spy's acinetobacter calcoaceticus, 3: Acinetobacter johnsonii, 4: acinetobacter baumannii, 5: Acinetobacter bauamnnii 6: Tuo Neili
Acinetobacter calcoaceticus, 7: escherichia coli, 8: Pseudomonas aeruginosa, 9: Friedlander's bacillus, 10: staphylococcus aureus, 11: Mo Shi rubs
Bacillus radicosus, 12: serratia marcescens, 13: negative control；
Fig. 3 is that PCR detection method sensitivity evaluation tests gel electrophoresis result figure in embodiment；M:Maker；1:7.2ng/
uL；2:7.2 × 10-1ng/uL；3:7.2 × 10-2ng/uL；4:7.2 × 10-3ng/uL；5:7.2 × 10-4ng/uL；6:7.2 ×
10-5ng/uL；7:7.2 × 10-6ng/uL；8:7.2 × 10-7ng/uL；9: negative control；
Fig. 4 is that clinical sample detects gel electrophoresis result figure, M:Maker in embodiment；1: positive control；2-9: doubtful bacterium
Strain；10: negative control.
Specific embodiment
Below in conjunction with specific embodiment of the present invention, technical solution of the present invention is clearly and completely described, is shown
So, described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.Based on the reality in the present invention
Example is applied, every other embodiment obtained by those of ordinary skill in the art without making creative efforts all belongs to
In the scope of protection of the invention.
1 skin spy's acinetobacter calcoaceticus PCR method for detecting specificity of embodiment is established
(1) design of skin spy acinetobacter calcoaceticus specific primer
The present embodiment is directed to the house-keeping gene (ParC gene) of skin spy acinetobacter calcoaceticus, and designing has skin spy's acinetobacter calcoaceticus
High specificity and high a pair of of the specific primer of sensitivity, the specific primer are as follows to specifying information:
Primer sequence are as follows:
F:5 '-CTCTCAGCAAAAGCAGCG-3 '；
R:5 '-CCTGATGAGCTAGCAGCAATAAG-3 '.
(2) extraction of DNA of bacteria
Using Ezup column bordetella gene group DNA extraction agent box (bacterium), the DNA of bacterium is extracted, the specific steps are as follows:
1) bacterial solution of 1mL37 DEG C of constant incubator culture for 24 hours is taken, is added in 1.5mL centrifuge tube, room temperature 8000rmp
It is centrifuged 1min, abandons supernatant, collects thallus, 180 μ L Buffer Digestion are added, it is molten to add 20 μ LProteinase K
Liquid, concussion mix, and 56 DEG C of water-bath 1h are cracked completely to cell.During water-bath, it is mixed by inversion within every 10 minutes primary, promotion cell
Cracking.
2) 200 μ L Buffer BD are added, are sufficiently mixed by inversion.After Buffer BD is added, generated if any precipitating, it can 70
DEG C water-bath 10 minutes.
3) 200 μ L dehydrated alcohols are added, are sufficiently mixed by inversion, it is outstanding that there may be translucent fibre shapes after addition dehydrated alcohol
Floating object does not influence DNA and extracts and apply.
4) adsorption column is put into collecting pipe, the solution and translucent fibre shape for being obtained step (3) with pipettor suspend
Object does not influence the extraction and application of DNA.
5) adsorption column is recycled again in collecting pipe, 500 μ L PW Solution10000rmp is added and are centrifuged 30s, outwell
Collecting pipe solution.
6) adsorption column is placed back in into collecting pipe, 500 μ L Wash Solution, 10000rmp centrifugation 30s is added, outwell
Filtrate.
7) adsorption column is placed back in collecting pipe, is centrifuged 2min in 12000rmp room temperature, discard residual Wash
Solution.Adsorption column is opened into lid in being placed at room temperature for several minutes, thoroughly to dry remaining Wash in adsorbent material
The residual of Solution, Wash Solution will affect the yield and subsequent test of genomic DNA.
8) adsorption column is taken out, is put into a new 1.5mL centrifuge tube, 50-200 μ L CE Buffer is added and stands
3min, 12000rmp room temperature are centrifuged 2min, collect DNA solution, as DNA of bacteria.The DNA of extraction can carry out next step examination immediately
It tests or -20 DEG C saves.
(3) the best annealing temperature of skin spy acinetobacter calcoaceticus primer
Skin spy acinetobacter calcoaceticus is inoculated in 1mL nutrient broth fluid nutrient medium, is placed in 37 DEG C of constant incubators and cultivates for 24 hours
After increasing bacterium, genomic DNA is extracted by embodiment 1.Using skin spy's acinetobacter calcoaceticus DNA as pcr template, carried out using purpose primer
PCR reaction, reaction annealing temperature are respectively set to 55 DEG C, 56 DEG C, 57 DEG C, 58 DEG C, 59 DEG C, 60 DEG C, and 61 DEG C, remaining reaction condition
Without change.Detected through gel electrophoresis amplified production, observation gel electrophoresis result is according to the gel electrophoresis after PCR under ultraviolet light
As a result the optimum annealing temperature that 58 DEG C are skin spy acinetobacter calcoaceticus specific primer is determined.
As a result as shown in Figure 1, the specific band of No. 4 swimming lanes is most obvious, which is 58 DEG C.
(4) skin spy acinetobacter calcoaceticus PCR method for detecting specificity is established
Pcr amplification reaction system is 10 μ L:2 × TSINGKE Master Mix (blue), 5 0.5 μ L of μ L, PARC-F,
0.5 μ L of PARC-R, 1 μ L of template DNA, distilled water are mended to 10 μ L.
Pcr amplification reaction program are as follows: 95 DEG C of initial denaturation 5min；Into circulation, 95 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 72
DEG C extend 30s, 35 circulation；72 DEG C of extension 10min, 4 DEG C of preservation PCR products terminate.
(5) gel electrophoresis of PCR amplification result determines
1% agarose gel electrophoresis is carried out with PCR product, examines PCR amplification result.Judgement specifically: 1% gel electrophoresis
Amplified production is detected, electrophoresis result is observed under ultraviolet light irradiation, if there is the single amplified band of 485bp, then illustrates sample
In contain skin spy acinetobacter calcoaceticus；Conversely, being then free of skin spy acinetobacter calcoaceticus in sample.
The specific evaluation test of embodiment 2
Respectively with skin spy acinetobacter calcoaceticus (Acinetobacter pittii), Acinetobacter johnsonii (Acinetobacter
Johnsonii), acinetobacter baumannii (Acinetobacter baylyi), Acinetobacter bauamnnii (Acinetobacter
Baumanii), Tuo Neili acinetobacter calcoaceticus (Acinetobacter towneri), escherichia coli (Escherichia coli),
The yellow Portugal Pseudomonas aeruginosa (Pseudomonas aeruginosa) Friedlander's bacillus (Klebsiella peneumoniae)
Grape coccus (Staphylococcus aureus), morganella morganii (Morganella morganii), serratia marcescens
The DNA of bacteria of (Serratia marcescens) is the specificity verification test that template carries out primer PARC-F/PARC-R.
The extracting method of DNA of bacteria template is as follows: by selected microbionation in 5mLNB nutrient broth fluid nutrient medium,
With cultivated in 37 DEG C of constant incubators for 24 hours increase bacterium after, take 1mL bacterium solution, be put into 1.5mL sterile centrifugation tube: 12000r/min from
Heart 15min, discards supernatant liquid, and 500 μ L are added and sterilize distilled water, are gently blown and beaten with pipettor, is resuspended thallus, 12000r/min from
Heart 15min discards supernatant liquid, collects bacterium；100 μ L sterilizing distilled water is added, is gently blown and beaten with pipettor, thallus is resuspended, until
15min is boiled in boiling water, is taken out immediately, in -20 DEG C of placement 30min.Subsequent 35 DEG C of defrostings, 12000r/min are centrifuged 15min, take
Supernatant places 4 DEG C of spare or -20 DEG C of preservations.
Pcr amplification reaction system is 10 μ L:2 × TSINGKE Master Mix (blue), 5 0.5 μ L of μ L, PARC-F,
0.5 μ L of PARC-R, 1 μ L of template DNA, distilled water are mended to 10 μ L.
Pcr amplification reaction program are as follows: 95 DEG C of initial denaturation 5min；Into circulation, 95 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 72
DEG C extend 30s, 35 circulation；72 DEG C of extension 10min, 4 DEG C of preservation PCR products terminate.
As a result as shown in Fig. 2, electrophoresis result is that only swimming lane 1-2 skin spy's acinetobacter calcoaceticus specific band occurs at 485bp,
And other strains do not occur specific band.
The test of 3 sensitivity evaluation of embodiment
Skin spy acinetobacter calcoaceticus is inoculated in 1mL nutrient broth fluid nutrient medium, is placed in 37 DEG C of constant incubators and cultivates for 24 hours
After increasing bacterium, 1mL bacterium solution is taken to extract genomic DNA by embodiment 1, measuring DNA concentration by micro-spectrophotometer is 7.2ng/ μ
L.10 are pressed with sterilizing distilled water0-10-710 times of gradient dilutions are carried out to extracted DNA, are carried out by template of 8 gradient dilution liquid
PCR amplification, detected through gel electrophoresis amplified production observe gel electrophoresis result under ultraviolet light.
From the figure 3, it may be seen that in swimming lane 5 it can be seen that clear band, corresponding detection DNA extension rate are 10-4It dilutes again, it is dense
Degree is 0.00072ng/ μ L, and this law has preferable sensitivity.
The detection of 4 clinical sample of embodiment
The skin spy's acinetobacter calcoaceticus PCR method for detecting specificity established using embodiment 1 is divided from beef cattle oral cavity and nasal cavity
From to 8 plants of doubtful bacterial strains detected.
Testing result is shown in Fig. 4, and swimming lane 4 and 8 is positive result.
It although an embodiment of the present invention has been shown and described, for the ordinary skill in the art, can be with
Understand without departing from the principles and spirit of the present invention can to these examples carry out it is a variety of variation, modification, replacement and
Modification, the scope of the present invention is defined by the appended.
Sequence table
<110>Sichuan Agricultural University
<120>a kind of specific primer and its methods and applications for detecting skin spy acinetobacter calcoaceticus
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
ctctcagcaa aagcagcg 18
<210> 2
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
cctgatgagc tagcagcaat aag 23
Claims (7)
1. a kind of specific primer for detecting skin spy acinetobacter calcoaceticus, which is characterized in that the specific primer such as SEQ ID
Shown in NO.1~2.
2. a kind of method for detecting skin spy acinetobacter calcoaceticus, which comprises the following steps:
1) sample to be tested genomic DNA is extracted；
2) PCR amplification is carried out using above-mentioned primer；
3) detected through gel electrophoresis, detection of taking pictures under gel imaging system, there are the DNA specific bands of 485bp, it is determined that sample
Contain skin spy acinetobacter calcoaceticus in product.
3. a kind of method for detecting skin spy acinetobacter calcoaceticus according to claim 2, which is characterized in that the sample gene
Group is extracted using Ezup column bordetella gene group DNA extraction agent box.
4. a kind of method for detecting skin spy acinetobacter calcoaceticus according to claim 2, which is characterized in that the PCR amplification
Reaction system is 10 μ L:2 × TSINGKE Master Mix (blue), 5 0.5 0.5 μ L of μ L, PARC-R of μ L, PARC-F, template
1 μ L of DNA, distilled water are mended to 10 μ L.
5. a kind of method for detecting skin spy acinetobacter calcoaceticus according to claim 2, which is characterized in that the PCR amplification
Response procedures are as follows: 95 DEG C of initial denaturation 5min；Into circulation, 95 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C of extension 30s, 35 are followed
Ring；72 DEG C of extension 10min, 4 DEG C of preservation PCR products terminate.
6. application of the specific primer described in claim 1 in the detection of skin spy's acinetobacter calcoaceticus.
7. a kind of for detecting the kit of skin spy's acinetobacter calcoaceticus, which is characterized in that the kit includes claim 1 institute
The specific primer stated.
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| CN201910637631.7A CN110184371B (en) | 2019-07-15 | 2019-07-15 | Specific primer for detecting acinetobacter cutaneus as well as method and application thereof |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2014207895A (en) * | 2013-03-25 | 2014-11-06 | 愛知県 | Genotypic typing method of acinetobacter bacteria and primer set using the same |
| CN106755552A (en) * | 2017-03-20 | 2017-05-31 | 辽宁大学 | The PCR detection method of Vibrio vulnificus in a kind of aquiculture animal or aquatic food |
| CN109266658A (en) * | 2018-10-17 | 2019-01-25 | 昆明理工大学 | The specific gene and its primer of a kind of Acinetobacter bauamnnii and application |
-
2019
- 2019-07-15 CN CN201910637631.7A patent/CN110184371B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2014207895A (en) * | 2013-03-25 | 2014-11-06 | 愛知県 | Genotypic typing method of acinetobacter bacteria and primer set using the same |
| CN106755552A (en) * | 2017-03-20 | 2017-05-31 | 辽宁大学 | The PCR detection method of Vibrio vulnificus in a kind of aquiculture animal or aquatic food |
| CN109266658A (en) * | 2018-10-17 | 2019-01-25 | 昆明理工大学 | The specific gene and its primer of a kind of Acinetobacter bauamnnii and application |
Non-Patent Citations (2)
| Title |
|---|
| HUJER K. M.等: "Rapid Determination of Quinolone Resistance in Acinetobacter spp.", 《JOURNAL OF CLINICAL MICROBIOLOGY》 * |
| LI X. M.等: "Development and Evaluation of Species-Specific PCR for Detection of Nine Acinetobacter Species", 《ANN CLIN LAB SCI.》 * |
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Effective date of registration: 20220713 Address after: 635200 group 3, Changchang village, Sanhui Town, Qu county, Dazhou City, Sichuan Province Patentee after: Quxian Caijiashan Livestock Breeding Co.,Ltd. Address before: 611130 Huimin Road, Wenjiang District, Chengdu, Sichuan Province, No. 211 Patentee before: Sichuan Agricultural University |