CN104293964A - Specificity detection method for pseudomonas aeruginosa - Google Patents
Specificity detection method for pseudomonas aeruginosa Download PDFInfo
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- CN104293964A CN104293964A CN201410564729.1A CN201410564729A CN104293964A CN 104293964 A CN104293964 A CN 104293964A CN 201410564729 A CN201410564729 A CN 201410564729A CN 104293964 A CN104293964 A CN 104293964A
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- 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 method for detecting pseudomonas aeruginosa by using a polymerase chain reaction (PCR) technology, and a new nucleic acid primer for PCR. By adopting the detection method disclosed by the invention, the identification time of the pseudomonas aeruginosa can be significantly shortened, moreover, the repeatability is good, the reliability is high, and a basis is provided for sources of the pseudomonas aeruginosa and relevant identifications.
Description
Technical field
The invention belongs to microbiologic inhibition tests technical field, be specifically related to a kind of method for detecting specificity of Pseudomonas aeruginosa.
Background technology
Bacterial contamination is one of main bugbear threatening chick embryo culture virus production mode.Because kind of egg is from production, be transported to and enter between hatching, bacterium enters into the related link of virus production along with kind of egg, this is because bacterium may be attached on kind of the ight soil on egg surface, or the time to contact with sterilizing agent is short or resistance etc. reason, cause bacterium all can not eliminate in sterilizing part, simultaneously because bacteria live conditional request is comparatively loose, can survive at conventional environment, and fecundity is strong especially, if in egg inoculation, allantoic fluid results etc. will cause bacterium to be propagated in different chicken embryo because inoculating needle wherein and results head are subject to bacterial contamination, easily cause bacterial contamination industrial accident, cause producing unsuccessfully, cause financial loss.In order to better pre-bacteriological protection causes chicken embryo to produce virus failure, screen the means that effective sterilizing agent or microbiotic become a kind of necessity, but prerequisite is the bacterium that qualification is polluted is which kind of bacterium, just can screen targetedly.Therefore bacterium precise Identification becomes the key link.
Current Bacteria Identification includes biochemical identification and PCR qualification, biochemical identification often cannot accurately judge because result is uncertain in actually operating, therefore the means determining bacterial species completely can not be become, and gene amplification qualification (PCR qualification) can be increased the gene 16SrDNA of bacterium targetedly, again by order-checking and alignment, thus determine the kind of bacterium, be generally acknowledged a kind of other Main Means of microbiology class of qualification.
Through research and qualification, failed bacterium mainly Pseudomonas aeruginosa is produced for often causing, Pseudomonas aeruginosa (Pseudomonas aeruginosa) is also known as Pseudomonas aeruginosa, it is a kind of single raw flagellum, motion is active, without brood cell, and obligate aerobic gram negative bacillus, extensively being present in water, soil, air and humans and animals body skin and enteron aisle, is one of primary pollution source in tap water.Pseudomonas aeruginosa can produce multiple interior extracellular toxin, causes acute intestinal disorders and skin inflammation, in addition, because it has multidrug resistant ability, under given conditions, this bacterium also can cause secondary infection or polyinfection, cause pneumonia, meningitis, septicemia etc., time serious, can death be caused.At present, in the detection technique of Pseudomonas aeruginosa, traditional Physico-chemical tests method is still continued to use in national and foreign standards, comprise coating method, many test tubes MPN method, filter membrane method etc., need multiple step such as pure culture and biochemical identification, whole sense cycle 3-5 days, sensitivity is low, and can not meet the needs of field quick detection.
And above-mentioned PCR qualification needs order-checking and comparison, time generally takes about 5 days, and set up specific detection method for this bacterium, just can direct judged result by Auele Specific Primer and amplified fragments size, within 1 day, just result can be obtained, this can gain time for the effective sterilizing agent of screening or microbiotic, can reduce or avoid bacterial contamination accident.
Therefore the method for detecting specificity of a kind of Pseudomonas aeruginosa timely, quick, reproducible, with a high credibility is provided, just becomes urgent problem.
Summary of the invention
In view of the deficiencies in the prior art, the object of this invention is to provide a kind of method for detecting specificity of Pseudomonas aeruginosa, quick diagnosis can not only go out Pseudomonas aeruginosa and reproducible, with a high credibility.
The concrete technical scheme of the present invention is as follows:
A specific detection primer for Pseudomonas aeruginosa, comprises upstream and downstream primer 5'-GACGGGTGAGTAATGCCTA-3' and 5'-CACTGGTGTTCCTTCCTATA-3'.
A detection method for Pseudomonas aeruginosa, adopts above-mentioned detection primer.
Described detection method gained specific fragment size is 616kb.
The detection method of described Pseudomonas aeruginosa, comprises the steps: a. extracting bacterial genomes DNA; B.16S rDNA molecular specificity PCR primer amplification; C. specific amplification products is identified.
Described in step b, the reaction system of PCR primer amplification forms totally 50 μ L reaction systems by 25 μ L PCR Mixture, 2 μ L upstream and downstream primer solution, 2 μ L DNA profilings, 21 μ LddH2O.
Described in step b, the reaction conditions of PCR primer amplification is: 94 DEG C of denaturation 5 min; 94 DEG C of sex change 1min, 53 DEG C of annealing 30s, 72 DEG C extend 1.5min, 30 circulations; Last 72 DEG C extend 10min again.
Identify described in step c that the method for specific amplification products is for getting pcr amplification product 5 μ L, in 1% sepharose, 120V electrophoresis 15min, utilizes gel imaging system to take.
The detection method of described Pseudomonas aeruginosa, concrete steps are:
(1) after not having the Pseudomonas aeruginosa Solution culture method of miscellaneous bacteria, extract part bacterium liquid, extracting bacterial genomes DNA is used for 16S rDNA molecular specificity PCR to be identified;
(2) form 50 μ L reaction systems by 25 μ L PCR Mixture, 2 μ L upstream and downstream primer solution, 2 μ L DNA profilings, 21 μ LddH2O, reaction conditions is: 94 DEG C of denaturation 5 min; 94 DEG C of sex change 1min, 53 DEG C of annealing 30s, 72 DEG C extend 1.5min, 30 circulations; Last 72 DEG C extend 10min again;
(3) get pcr amplification product 5 μ L, in 1% sepharose, 120V electrophoresis 15min, utilizes gel imaging system to take.
The conserved genetic sequences that the present invention is directed to the 16S rDNA of Pseudomonas aeruginosa have devised a pair specificity upstream and downstream primer, and be respectively 5'-GACGGGTGAGTAATGCCTA-3' and 5'-CACTGGTGTTCCTTCCTATA-3', specific fragment size is 616kb.The Pseudomonas aeruginosa determined with pseudomonas putida, intestinal bacteria, citric acid bacillus, Bacillus cereus and different sources is as test sample, above-mentioned primer is utilized to carry out augmentation detection, only have Pseudomonas aeruginosa to detect specific fragment, the results are shown in Figure 1.Demonstrate the method for the invention specificity high.
Detection method of the present invention significantly can shorten the qualification time of Pseudomonas aeruginosa, and reproducible, with a high credibility, provides foundation to Pseudomonas aeruginosa source and qualification of being correlated with.
Accompanying drawing explanation
fig. 1 isprimer amplified result, wherein M:DL2000 Mark er, a: Pseudomonas putida; B: intestinal bacteria; C: citric acid bacillus; D: Bacillus cereus; S1 to S4 is the Pseudomonas aeruginosa of different sources through making a definite diagnosis.
Embodiment
Below further describe embodiments of the invention.These embodiments should not be considered to limitation of the present invention.It will be understood by those skilled in the art that in form with the various change in detailed content and without prejudice to the spirit and scope of the invention indicated in detail in the following claims.
embodiment 1
Pcr amplification primer, comprises sequence 5'-GACGGGTGAGTAATGCCTA-3' and 5'-CACTGGTGTTCCTTCCTATA-3', and specific fragment size is 616kb.Operation steps:
(1) after not having the Pseudomonas aeruginosa Solution culture method of miscellaneous bacteria, extract part bacterium liquid, extracting bacterial genomes DNA is used for 16S rDNA molecular specificity PCR to be identified;
(2) form 50 μ L reaction systems by 25 μ L PCR Mixture, 2 μ L upstream and downstream primer solution, 2 μ L DNA profilings, 21 μ LddH2O, reaction conditions is: 94 DEG C of denaturation 5 min; 94 DEG C of sex change 1min, 53 DEG C of annealing 30s, 72 DEG C extend 1.5min, 30 circulations; Last 72 DEG C extend 10min again;
(3) get pcr amplification product 5 μ L, in 1% sepharose, 120V electrophoresis 15min, utilizes gel imaging system to take.
The Pseudomonas aeruginosa determined with pseudomonas putida, intestinal bacteria, citric acid bacillus, Bacillus cereus and different sources, as test sample, utilizes above-mentioned primer to increase.The results are shown in accompanying drawing 1.
<110> ZhaoQing DaHuaNong Biological medicine Co., Ltd
A kind of method for detecting specificity of <120> Pseudomonas aeruginosa
<160> 2
<210> 1
<211> 19
<212> DNA
<213> artificial sequence
<400> 1
GACGGGTGAG TAATGCCTA 19
<210> 2
<211> 20
<212> DNA
<213> artificial sequence
<400> 2
CACTGGTGTT CCTTCCTATA 20
Claims (8)
1. a specific detection primer for Pseudomonas aeruginosa, comprises upstream and downstream primer 5'-GACGGGTGAGTAATGCCTA-3' and 5'-CACTGGTGTTCCTTCCTATA-3'.
2. a detection method for Pseudomonas aeruginosa, is characterized in that, adopts described in claim 1 and detects primer.
3. the detection method of Pseudomonas aeruginosa according to claim 2, it is characterized in that, described detection method gained specific fragment size is 616kb.
4. the detection method of Pseudomonas aeruginosa according to claim 2, it is characterized in that, the method comprises: a. extracting bacterial genomes DNA; B.16S rDNA molecular specificity PCR primer amplification; C. specific amplification products is identified.
5. the detection method of Pseudomonas aeruginosa according to claim 4, it is characterized in that, described in step b, the reaction system of PCR primer amplification forms totally 50 μ L reaction systems by 25 μ L PCR Mixture, 2 μ L upstream and downstream primer solution, 2 μ L DNA profilings, 21 μ LddH2O.
6. the detection method of Pseudomonas aeruginosa according to claim 4, is characterized in that, described in step b, the reaction conditions of PCR primer amplification is: 94 DEG C of denaturation 5 min; 94 DEG C of sex change 1min, 53 DEG C of annealing 30s, 72 DEG C extend 1.5min, 30 circulations; Last 72 DEG C extend 10min again.
7. the detection method of Pseudomonas aeruginosa according to claim 4, it is characterized in that, step c identifies that the method for specific amplification products is for getting pcr amplification product 5 μ L, and in 1% sepharose, 120V electrophoresis 15min, utilizes gel imaging system to take.
8. the detection method of Pseudomonas aeruginosa according to claim 2, it is characterized in that, the method concrete steps are:
After not having the Pseudomonas aeruginosa Solution culture method of miscellaneous bacteria, extract part bacterium liquid, extracting bacterial genomes DNA is used for 16S rDNA molecular specificity PCR to be identified;
Form 50 μ L reaction systems by 25 μ L PCR Mixture, 2 μ L upstream and downstream primer solution, 2 μ L DNA profilings, 21 μ LddH2O, reaction conditions is: 94 DEG C of denaturation 5 min; 94 DEG C of sex change 1min, 53 DEG C of annealing 30s, 72 DEG C extend 1.5min, 30 circulations; Last 72 DEG C extend 10min again;
Get pcr amplification product 5 μ L, in 1% sepharose, 120V electrophoresis 15min, utilizes gel imaging system to take.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105671141A (en) * | 2015-04-30 | 2016-06-15 | 汕头国际旅行卫生保健中心 | Pseudomonas aeruginosa typing identification method based on 23S-5S rRNA gene sequencing spectrogram analysis method |
| CN106976990A (en) * | 2017-03-09 | 2017-07-25 | 浙江大学舟山海洋研究中心 | A kind of method of the pseudomonas aeruginosa degraded oil of utilization producing rhamnolipid with high yield |
| CN110106266A (en) * | 2019-05-09 | 2019-08-09 | 宁夏回族自治区食品检测研究院 | Identify the method for pseudomonas aeruginosa and pseudomonas putida |
| CN111154900A (en) * | 2020-01-19 | 2020-05-15 | 广东省微生物研究所（广东省微生物分析检测中心） | Pseudomonas aeruginosa specific new molecular target and rapid detection method thereof |
-
2014
- 2014-10-22 CN CN201410564729.1A patent/CN104293964A/en active Pending
Non-Patent Citations (1)
| Title |
|---|
| KHULOD I. HASSAN ET AL.: ""Molecular identification of Pseudomonas aeruginosa isolated from Hospitals in Kurdistan region"", 《JOURNAL OF ADVANCED MEDICAL RESEARCH》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105671141A (en) * | 2015-04-30 | 2016-06-15 | 汕头国际旅行卫生保健中心 | Pseudomonas aeruginosa typing identification method based on 23S-5S rRNA gene sequencing spectrogram analysis method |
| CN105671141B (en) * | 2015-04-30 | 2019-06-25 | 汕头国际旅行卫生保健中心 | A kind of pseudomonas aeruginosa Classification Identification method based on 23S-5S rRNA gene sequencing spectrum analysis method |
| CN106976990A (en) * | 2017-03-09 | 2017-07-25 | 浙江大学舟山海洋研究中心 | A kind of method of the pseudomonas aeruginosa degraded oil of utilization producing rhamnolipid with high yield |
| CN110106266A (en) * | 2019-05-09 | 2019-08-09 | 宁夏回族自治区食品检测研究院 | Identify the method for pseudomonas aeruginosa and pseudomonas putida |
| CN111154900A (en) * | 2020-01-19 | 2020-05-15 | 广东省微生物研究所（广东省微生物分析检测中心） | Pseudomonas aeruginosa specific new molecular target and rapid detection method thereof |
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