CN104164499B - A kind of for expanding the specific primer that Psychrobacter belongs to - Google Patents
A kind of for expanding the specific primer that Psychrobacter belongs to Download PDFInfo
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- CN104164499B CN104164499B CN201410369525.2A CN201410369525A CN104164499B CN 104164499 B CN104164499 B CN 104164499B CN 201410369525 A CN201410369525 A CN 201410369525A CN 104164499 B CN104164499 B CN 104164499B
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Abstract
A kind of for expanding the specific primer that Psychrobacter belongs to: in 5 ' AAGACTCTACGGTTAATACCCA 3 ', with universal primer, 5 ' GGTTACCTTGTTACGACTT 3 ' pairing uses can directly amplify length from all kinds of sample DNAs and be about the Psychrobacter purpose fragment of 1078p.The present invention solves and lacks the problem that primer directly amplifies Psychrobacter target sequence from hybrid dna sample at present.
Description
Technical field
The present invention relates to a kind of for expanding the specific primer that Psychrobacter belongs to, can be used in sample
The Molecular Detection of Psychrobacter.
Background technology
Psychrobacter belongs to (Psychrobacter) antibacterial, classifies in Moraxella Cordycepps (Moraxellaceae),
Including Psychrobacter immobilis (P.immobilis), excrement Psychrobacter (P.faecalis), occupy cold Psychrobacter
(P.frigidicola), totally 9 kinds of antibacterials such as cold Psychrobacter of dwelling (P.glacincola).The bacterial strain of this genus is
Gram-negative need to support bacillus or coccus, its size be (0.9-1.3) um × (1.5-3.8) um without
Power, aerobic, it is possible to grow at 5 DEG C, typically can not grow in the range of 35-37 DEG C, oxidase
It is the positive with catalase, gains the name because of well-grown at low temperatures.It is distributed mainly on the environment of Chang Leng
In the low temperature environments such as polar regions, north and south, high mountain, glacier, freezer, deep-sea and soil, it can lead to
Cross special physiological mechanism and adapt to the change of ambient temperature, to things such as Oil in Sewage Water hydro carbons, chlorophenols
The biodegradation of matter, catalysis low temperature fermentation, expression thermally labile protein and production freezeproof protectant
Etc. aspect have a wide range of applications.
The separation of Psychrobacter relies primarily on the classical biology side of selective medium separation and Culture
Method, then carries out taxonomic identification by Bacterial Physiological biochemical characteristic.But this method separation cycle is long, work
Amount is big and is difficult to obtain purpose function stem and pure bacterial strain, and this makes the discovery of Psychrobacter and separates tool
There are occasionality and randomness, greatly reduce the probability excavating this specific kind strain resource.Along with
Developing rapidly of molecular biology, the molecular detection technology of PCR-based is studied at environmental microorganism
Middle application is more and more universal.By bacterial strain carries out DNA extraction, use antibacterial 16srDNA general
Primer 2 7F/1492R expands purpose fragment, and order-checking can learn its gene order, but the specificity of this method
Not strong, the hybrid dna sample that the most multiple bacterium coexists, there is no the existing primer of method to from mixed
Close DNA sample directly amplifies and there is Psychrobacter (Psychrobacter) inherited character
16srDNA V3 Variable Area partial sequence, brings tired to the detection research of Psychrobacter in soil
Difficult.
Summary of the invention
It is an object of the invention to provide a kind of specific primer for expanding Psychrobacter, to improve public affairs
Know defect present in technology.
For achieving the above object, the specific primer for expanding Psychrobacter genus that the present invention provides
For: 5 '-AAGACTCTACGGTTAATACCCA-3 '.
In described specific primer, 5 '-AAGACTCTACGGTTAATACCCA-3 ' are forward
Primer；5 '-GGTTACCTTGTTACGACTT-3 ' are reverse primer.
The present invention for the specific primer expanding Psychrobacter can solve to lack at present primer from
Hybrid dna sample directly amplifies there is Psychrobacter (Psychrobacter) inherited character
The problem of 16srDNA V3 Variable Area partial sequence.
Accompanying drawing explanation
Fig. 1 is that in embodiment 1,6 kind DNA of bacteria amplified productions are coagulated by PsyF/1492R primer
Gel electrophoresis figure.
Fig. 2 is the PsyF/1492R primer gel to pedotheque DNA cloning product in embodiment 2
Electrophoretogram.
Detailed description of the invention
The present invention is (named with specific primer 5 '-AAGACTCTACGGTTAATACCCA-3 '
PsyF) as forward primer, with 5 '-GGTTACCTTGTTACGACTT-3 ' (in universal primer
1492R) be reverse primer pairing use, can directly amplify length from all kinds of sample DNAs
Degree is about the Psychrobacter purpose fragment of 1078bp.
Illustrate below in conjunction with embodiment, but technical scheme is not limited to act set forth below
Embodiment, also include the combination in any between specific embodiment.
Embodiment one
Respectively with Psychrobacter (Psychrobacter), Flavobacterium (Flavobacterium), intestinal bar
Bacterium (Enterobacter), arthrobacterium (Arthrobacter), bacillus cereus (Bacillus) and vacation list
It is that template carries out the special of primer PsyF/1492R that born of the same parents bacterium (Pseudomoans) 6 belongs to DNA of bacteria
Property checking test.
PCR amplification system is: 25 μ L are drawn by 0.5 μ L DNA profiling (about 10ng), 2.5 μ L forwards
Thing PsyF (10pM), 2.5 μ L reverse primer 1492R (10pM), 2.5 μ L 10 × PCR buffer (contain
Mg2+), 2.0 μ L dNTP s (2.5mmol/L), 0.1 μ L Taq archaeal dna polymerase (5U/ μ L), sterilizing
Distilled water supplies 25 μ L.Pcr amplification reaction condition is: denaturation 95 DEG C, 5min, degeneration
94 DEG C of 1min, anneal 50 DEG C of 50s, extends 72 DEG C of 1min, totally 30 circulations, and 72 DEG C extend 10min,
4 DEG C of preservations.
The sequence amplified by the present embodiment carries out agarose gel electrophoresis detection, testing result such as Fig. 1
Shown in, in Fig. 1, M swimming lane is standard DL2000, and 1-6 swimming lane is respectively containing Psychrobacter
(Psychrobacter), Flavobacterium (Flavobacterium), enterobacteria (Enterobacter),
Arthrobacterium (Arthrobacter), bacillus cereus (Bacillus) and pseudomonas (Pseudomoans)
The amplification of DNA, No. 7 swimming lanes are the amplification without DNA blank.From Fig. 1
Can be seen that present embodiment can be accurate for the specific primer PsyF/1492R expanding Psychrobacter
The true target sequence fragment amplifying Psychrobacter, and fail from nearly edge DNA of bacteria and blank right
Nucleic acid fragment is amplified according to.Convert big after sequence fragment corresponding for No. 1 swimming lane is connected with carrier T
Enterobacteria DH5 α competent cell checks order, and sequencing result is: sequence length is 1078bp, and warp
Blast analyzes, and the distinguished sequence for Psychrobacter (Psychrobacter) (sees Psychrobacter order-checking
Result).
Embodiment two
Apply primer PsyF/1492R to 6 parts of low temperature soil samples (by the micro-life of Northeast Agricultural University's environment
Thing laboratory provide) DNA carry out PCR amplification test.PCR amplification system is: 25 μ L are by 0.5 μ L
DNA profiling (about 10ng), 2.5 μ L forward primer PsyF (10pM), 2.5 μ L reverse primers
1492R (10pM), 2.5 μ L 10 × PCR buffer (containing Mg2+), 2.0 μ L dNTP s (2.5mmol/L),
0.1 μ L Taq archaeal dna polymerase (5U/ μ L), sterilizing distilled water supplies 25 μ L.Pcr amplification reaction bar
Part is: 95 DEG C of 5min of denaturation, 94 DEG C of 1min of degeneration, and anneal 50 DEG C of 50s, extends 72 DEG C of 1min,
Totally 30 circulations, 72 DEG C extend 10min, 4 DEG C of preservations.
Sequence present embodiment amplified carries out agarose gel electrophoresis detection, testing result such as figure
Shown in 2, in Fig. 2, M swimming lane is standard DL2000, and 1-6 swimming lane is respectively low temperature soil sample
The amplification of DNA, No. 7 swimming lanes are the amplification without DNA blank.From Fig. 2
Can be seen that present embodiment can be accurate for the specific primer PsyF/1492R expanding Psychrobacter
The true target sequence fragment amplifying Psychrobacter from all low temperature soil sample DNAs.At random
Select after No. 1 sequence fragment corresponding with No. 6 swimming lanes is connected with carrier T respectively and convert escherichia coli
DH5 α competent cell checks order, and sequencing result is: sequence length is 1078bp, and divides through blast
Analysis, for the distinguished sequence (seeing Psychrobacter sequencing result) of Psychrobacter (Psychrobacter).
Psychrobacter sequencing result
ACACCACCGTGGTGAGCGCCATCCTAAAAGGTTAGGCTACCCACTTCTGGTGCAATCAACTCC
CATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATTCTGATCCGC
GATTACTAGCGATTCCTACTTCATGGAGTCGAGTTGCAGACTCCAATCTGGACTACGATAGGC
TTTTTGAGATTCGCATCACATCGCTGTGTAGCTGCCCTCTGTACCTACCATTGTAGCACGTGT
GTAGCCCTGGTCGTAAGGGCCATGATGACTTGACGTCGTCCCCGCCTTCCTCCAGTTTGTCAC
TGGCAGTATCCTTAGAGTTCCCGGCCAAACCGCTGGTAACTAAGGACAAGGGTTGCGCTCGTT
GCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAGCCATGCAGCACCTGTATTCTA
ATTCCCGAAGGCACTCCCGCATCTCTGCAGGATTCTAGATATGTCAAGACCAGGTAAGGTTCT
TCGCGTTGCATCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCATTTG
AGTTTTAACCTTGCGGCCGTACTCCCCAGGCGGTCTACTTATTGCGTTAGCTGCGTCACTAAG
TCCTCAAGGGACCCAACGACTAGTAGACATCGTTTACGGCGTGGACTACCAGGGTATCTAATC
CTGTTTGCTACCCACGCTTTCGAACCTCAGTGTCAGTATGATGCCAGAAGGCTGCCTTCGCCA
TCGGTATTCCTCCAGATCTCTACGCATTTCACCGCTACACCTGGAATTCTACCTTCCTCTCAC
CTACTCTAGCCTAACAGTATCAGATGCCGTTCCCAGGTTAAGCCCGGGGCTTTCACATCTGAC
TTATCAAGCCACCTACGCTCGCTTTACGCCCAGTAATTCCGATTAACGCTTGCACCCTCTGTA
TTACCGCGGCTGCTGGCACAGAGTTAGCCGGTGCTTATTCTGCAGCTAATGTCATCGTCTATG
GGTATTAACCATAGAGTCTTCTTCACTGCTTAAAGTGCTTTACAACCAAAAGGCCTTCTTCAC
ACACGCG
Claims (1)
1. one kind is used for expanding the specific primer pair that Psychrobacter belongs to, and it by sequence is
The forward primer of 5'-AAGACTCTACGGTTAATACCCA-3' and sequence are
The reverse primer composition of 5'-GGTTACCTTGTTACGACTT-3'.
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|---|---|---|---|
| CN201410369525.2A CN104164499B (en) | 2014-07-30 | 2014-07-30 | A kind of for expanding the specific primer that Psychrobacter belongs to |
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|---|---|---|---|
| CN201410369525.2A CN104164499B (en) | 2014-07-30 | 2014-07-30 | A kind of for expanding the specific primer that Psychrobacter belongs to |
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| CN104164499A CN104164499A (en) | 2014-11-26 |
| CN104164499B true CN104164499B (en) | 2016-08-24 |
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