CN107164497A - Loop-mediated isothermal amplification technique detects the primer and kit of pseudomonas aeruginosa - Google Patents
Loop-mediated isothermal amplification technique detects the primer and kit of pseudomonas aeruginosa Download PDFInfo
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- CN107164497A CN107164497A CN201710439230.1A CN201710439230A CN107164497A CN 107164497 A CN107164497 A CN 107164497A CN 201710439230 A CN201710439230 A CN 201710439230A CN 107164497 A CN107164497 A CN 107164497A
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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 the primer and pseudomonas aeruginosa gene quick diagnosis reagent kit that one group of loop-mediated isothermal amplification technique detects pseudomonas aeruginosa.The A of prior art China patent CN 102134601 disclose ring mediated isothermal amplification detection primer, detection method and the detection kit of a kind pseudomonas aeruginosa, the invention is directed to the ecfX genes of pseudomonas aeruginosa, designs and screened a set of specific detection primer group and the detection kit containing the detection primer group and utilization detection of the detection kit by LAMP.Compared with prior art, the present invention on its basis, one group of new primer is devised also with ecfX genes.By the contrast experiment of two groups of primers, primer of the invention has more preferable sensitivity high.
Description
Technical field
The present invention relates to the primer and kit that one group of loop-mediated isothermal amplification technique detects pseudomonas aeruginosa, belong to raw
Analyte detection technical field.
Background technology
Pseudomonas aeruginosa (P.Aeruginosa) original claims Pseudomonas aeruginosa, is that a kind of high fatal rate, hemolytic, the resistance to the action of a drug are strong
Gram-Negative bacillus, in classification belong to pseudomonadaceae in pseudomonas, up to more than 200 kinds of this Pseudomonas species,
In distributed in nature extensively, it is one of most common bacterium present in soil.The topmost growth conditions of this bacterium is moist ring
Border, other conditions are less demanding.The textile easily moisture absorption, all factors for possessing P. aeruginosa growth, using the teaching of the invention it is possible to provide its
The ecotopia of breeding, including suitable pH, temperature and water nutrition, can also provide the high surface area needed for its breeding.Therefore spin
Once pollution pseudomonas aeruginosa, the bacterium tenaciously can be adsorbed on fiber in fabric, general cleaning, exposure are not eliminate
's.
Pseudomonas aeruginosa can temporary paraisitism in skin, therefore contact body surface infection rate is at a relatively high, can cause people's middle ear
Inflammation, keratitis, urethritis and ALRI, can also cause endocarditis, gastroenteritis, pyothorax, or even cause by blood flow
Septicemia, can cause patient death.
Pseudomonas aeruginosa detection has a variety of methods, and common mainly has traditional standard method, full automatic microorganism analysis
System analysis method, molecular biology method (Standard PCR method, Multiplex PCR, PCR-DHPLC methods, Real-time PCR methods, base
Because of chip technology), immunological method (immunomagnetic isolation method, fluorescence activated cell partition method, enzyme linked immunosorbent assay), albumen
Polypeptide detection methods etc..Traditional detection method program is cumbersome, cycle length, sensitivity are low.Immunological method prepare antibody it is more difficult,
Multiple components can not be detected simultaneously, and high is required to experimenter's craftsmenship, cross pollution easily occurs.Molecular biology method has
High sensitivity, it is accurate, quick the advantages of, but need specialized equipment equipment, easily pollution requires higher to reviewer.
The content of the invention
The technical problems to be solved by the invention are：A kind of loop-mediated isothermal amplification technique detection pseudomonas aeruginosa is provided
Primer and kit.
Loop-mediated isothermal amplification technique detects the primer of pseudomonas aeruginosa, and primer sequence is as follows：
Outside primer is to F3 and B3：
Sense primer F3：GCTCGTACGGAGATACAGCC；
Anti-sense primer B3：TGTTCACCGTTTGTATTGGCG；
Inner primer is to FIB and BIP：
Sense primer FIP：
GTCGAATCCCTTTAGGACGGAGACCCTTTCGAACGCGGGGAATGGCC；
Anti-sense primer BIP:
AAGTTCACACTGTGGAGGAGCAACCCAATAGACCCGAGCCATGTTTCC
Using above-mentioned primer, the present invention also provides a kind of pseudomonas aeruginosa gene quick diagnosis reagent kit, including BstDNA gathers
Synthase, reaction solution, sample pretreatment liquid, nitrite ion and positive control solution composition.
Reaction solution：In per 1L reaction solutions containing 1.6~2mmol dNTP, 20~25mmol Tris-HCl, 10~
12.5mmol potassium chloride, 10~12.5mmol ammonium sulfate, 8~10mmol magnesium sulfate, 1~1.25ml TritonX-100,0.8~
1mol glycine betaines, each 0.2~0.25mol of inner primer FIP/BIP each 1.6~2mol and outer primer F3/B3.
Tris-HCl containing 10~20mmol pH 8.0,1~2mmol EDTA and 10 in per 1L sample pretreatment liquid~
12ml Triton X-100。
Developer：For 10% fluorescent dye SYBR GREEN I.
Positive control solution：Pseudomonas aeruginosa gene group DNA.
Beneficial effects of the present invention：
The A of prior art China patent CN 102134601 disclose the ring mediated isothermal amplification inspection of a kind pseudomonas aeruginosa
Primer, detection method and detection kit are surveyed, the invention is directed to the ecfX genes of pseudomonas aeruginosa, designs and screened one
Set specific detection primer group and detection kit containing the detection primer group and pass through LAMP using the detection kit
Detection.
Compared with prior art, the present invention on its basis, one group of new primer is devised also with ecfX genes.
By the contrast experiment of two groups of primers, primer of the invention has more preferable sensitivity high.
Embodiment
Embodiment 1
Loop-mediated isothermal amplification technique detects the primer of pseudomonas aeruginosa, and primer sequence is as follows：
Outside primer is to F3 and B3：
Sense primer F3：GCTCGTACGGAGATACAGCC；
Anti-sense primer B3：TGTTCACCGTTTGTATTGGCG；
Inner primer is to FIB and BIP：
Sense primer FIP：
GTCGAATCCCTTTAGGACGGAGACCCTTTCGAACGCGGGGAATGGCC；
Anti-sense primer BIP:
AAGTTCACACTGTGGAGGAGCAACCCAATAGACCCGAGCCATGTTTCC
Using above-mentioned primer, the present invention also provides a kind of pseudomonas aeruginosa gene quick diagnosis reagent kit, including BstDNA gathers
Synthase, reaction solution, sample pretreatment liquid, nitrite ion and positive control solution composition.
Reaction solution：In per 1L reaction solutions containing 1.6~2mmol dNTP, 20~25mmol Tris-HCl, 10~
12.5mmol potassium chloride, 10~12.5mmol ammonium sulfate, 8~10mmol magnesium sulfate, 1~1.25ml TritonX-100,0.8~
1mol glycine betaines, each 0.2~0.25mol of inner primer FIP/BIP each 1.6~2mol and outer primer F3/B3.
Tris-HCl containing 10~20mmol pH 8.0,1~2mmol EDTA and 10 in per 1L sample pretreatment liquid~
12ml Triton X-100。
Developer：For 10% fluorescent dye SYBR GREEN I.
Positive control solution：Pseudomonas aeruginosa gene group DNA.
The specificity verification of embodiment 2
30 plants of 20 plants of pseudomonas aeruginosa and non-pseudomonas aeruginosa are collected, by these bacterial strains respectively in nutrient broth (secondary haemolysis
Property vibrios in 3.5% sodium chloride nutrient broth) after 37 DEG C of culture 24h, take 1mL bacterium solutions, draw according to the offer of embodiment 1
Thing and loop-mediated isothermal amplification technique of the prior art extract the DNA of each bacterium, and carry out LAMP amplifications and addition respectively
Developer is observed.
As a result it is as follows：The detection primer of the present invention has preferable specificity, only pseudomonas aeruginosa strains amplification sun
Property, other non-pseudomonas aeruginosa strains are feminine gender.
The Sensitivity comparison of embodiment 3 is verified
Tested according to detection method and detection primer disclosed in the Chinese A of patent CN 102134601 of prior art, and with
The primer that invention is provided carries out Sensitivity comparison experiment.
It is reference strain with pseudomonas aeruginosa Pseudomonas aeruginosa ATCC 27853, by it in LB
In culture medium after 37 DEG C of culture 24h, 1mL bacterium solutions are taken, 10 times of gradient dilutions are carried out with sterile saline, choose 5 dilutions
Degree carries out plate count.
Plate count bacterial population of learning from else's experience is respectively：
0.01cfu/mL、0.05cfu/mL、0.25cfu/mL、1.25cfu/mL、6.25 cfu/mL。
DNA of bacteria is extracted according to detection method disclosed in the Chinese A of patent CN 102134601 of prior art, LAMP is carried out
Amplification, adds chromogenic reagent observation, verifies sensitivity.
Tested by 5 repetitions, wherein detection method and inspection disclosed in the Chinese A of patent CN 102134601 of prior art
Primer is surveyed, 5 experimental result sensitivity is 1.25cfu/mL.
The primer that the present invention is provided, 3 experimental result sensitivity is 0.25cfu/mL, and 2 experimental result sensitivity is
1.25cfu/mL 。
Compared with the primer of prior art, sensitivity is higher.
SEQUENCE LISTING
<110>Zhao Jiguang
<120>Loop-mediated isothermal amplification technique detects the primer and kit of pseudomonas aeruginosa
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> primer F3
<400> 1
GCTCGTACGGAGATACAGCC 20
<210> 2
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> primer B3
<400> 2
TGTTCACCGTTTGTATTGGCG 21
<210> 3
<211> 47
<212> DNA
<213> Artificial Sequence
<220>
<223> primer FIP
<400> 3
GTCGAATCCCTTTAGGACGGAGACCCTTTCGAACGCGGGGAATGGCC 47
<210> 4
<211> 48
<212> DNA
<213> Artificial Sequence
<220>
<223> BIP
<400> 4
AAGTTCACACTGTGGAGGAGCAACCCAATAGACCCGAGCCATGTTTCC 48
Claims (2)
1. loop-mediated isothermal amplification technique detects the primer of pseudomonas aeruginosa, it is characterised in that primer sequence is as follows：
Outside primer is to F3 and B3：
Sense primer F3：GCTCGTACGGAGATACAGCC；
Anti-sense primer B3：TGTTCACCGTTTGTATTGGCG；
Inner primer is to FIB and BIP：
Sense primer FIP：
GTCGAATCCCTTTAGGACGGAGACCCTTTCGAACGCGGGGAATGGCC；
Anti-sense primer BIP:
AAGTTCACACTGTGGAGGAGCAACCCAATAGACCCGAGCCATGTTTCC
A kind of pseudomonas aeruginosa gene quick diagnosis reagent kit, it is characterised in that including BstDNA polymerases, reaction solution, sample
Savor pretreatment fluid, nitrite ion and positive control solution composition；
Reaction solution：Contain 1.6~2mmol dNTP, 20~25mmol Tris-HCl, 10~12.5mmol chlorine in per 1L reaction solutions
Change potassium, 10~12.5mmol ammonium sulfate, 8~10mmol magnesium sulfate, 1~1.25ml TritonX-100,0.8~1mol beets
Alkali, each 0.2~0.25mol of inner primer FIP/BIP each 1.6~2mol and outer primer F3/B3；
Tris-HCl containing 10~20mmol pH 8.0,1~2mmol EDTA and 10~12ml in per 1L sample pretreatment liquid
Triton X-100；
Developer：For 10% fluorescent dye SYBR GREEN I.
2. positive control solution：Pseudomonas aeruginosa gene group DNA.
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| CN201710439230.1A CN107164497A (en) | 2017-06-12 | 2017-06-12 | Loop-mediated isothermal amplification technique detects the primer and kit of pseudomonas aeruginosa |
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|---|---|---|---|
| CN201710439230.1A CN107164497A (en) | 2017-06-12 | 2017-06-12 | Loop-mediated isothermal amplification technique detects the primer and kit of pseudomonas aeruginosa |
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| Publication Number | Publication Date |
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| CN107164497A true CN107164497A (en) | 2017-09-15 |
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| CN201710439230.1A Withdrawn CN107164497A (en) | 2017-06-12 | 2017-06-12 | Loop-mediated isothermal amplification technique detects the primer and kit of pseudomonas aeruginosa |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107937577A (en) * | 2017-11-29 | 2018-04-20 | 中国海洋大学 | The LAMP primer group and method of the Pseudomonas fluorescens of thermostable lipase are produced in quick detection raw milk |
| CN109182464A (en) * | 2018-10-29 | 2019-01-11 | 漳州龙文琪睿生物科技有限公司 | Coronoid process dissipate capsule bacterium loop-mediated isothermal amplification detection kit and its detection method |
| CN109266762A (en) * | 2018-09-13 | 2019-01-25 | 昆明医科大学第附属医院 | Pseudomonas aeruginosa detection method and kit based on LAMP principle |
| CN110129316A (en) * | 2018-02-05 | 2019-08-16 | 北京智德医学检验所有限公司 | It is a kind of for detecting the LAMP primer composition and application of 4 kinds of gramnegative bacteriums in intraocular liquid |
| CN110257538A (en) * | 2019-07-04 | 2019-09-20 | 中国水产科学研究院黄海水产研究所 | A kind of aerobic denitrifiers screening primer and method |
-
2017
- 2017-06-12 CN CN201710439230.1A patent/CN107164497A/en not_active Withdrawn
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107937577A (en) * | 2017-11-29 | 2018-04-20 | 中国海洋大学 | The LAMP primer group and method of the Pseudomonas fluorescens of thermostable lipase are produced in quick detection raw milk |
| CN110129316A (en) * | 2018-02-05 | 2019-08-16 | 北京智德医学检验所有限公司 | It is a kind of for detecting the LAMP primer composition and application of 4 kinds of gramnegative bacteriums in intraocular liquid |
| CN109266762A (en) * | 2018-09-13 | 2019-01-25 | 昆明医科大学第附属医院 | Pseudomonas aeruginosa detection method and kit based on LAMP principle |
| CN109182464A (en) * | 2018-10-29 | 2019-01-11 | 漳州龙文琪睿生物科技有限公司 | Coronoid process dissipate capsule bacterium loop-mediated isothermal amplification detection kit and its detection method |
| CN110257538A (en) * | 2019-07-04 | 2019-09-20 | 中国水产科学研究院黄海水产研究所 | A kind of aerobic denitrifiers screening primer and method |
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Application publication date: 20170915 |