CN104531887B - The authentication method of Klebsiella pneumonia and the primer - Google Patents
The authentication method of Klebsiella pneumonia and the primer Download PDFInfo
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- CN104531887B CN104531887B CN201510020845.1A CN201510020845A CN104531887B CN 104531887 B CN104531887 B CN 104531887B CN 201510020845 A CN201510020845 A CN 201510020845A CN 104531887 B CN104531887 B CN 104531887B
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Abstract
The present invention relates to authentication method and the primer of a kind of Klebsiella pneumonia, belong to biological technical field.The present invention obtains two pairs of primers (synthetic gene sequence) by creating work, uses this primer that Klebsiella pneumonia, escherichia coli and bacillus pyocyaneus are carried out PCR amplification, and amplified production is the specific sequence of Klebsiella pneumonia；It is thus possible to Klebsiella pneumonia is made a distinction with escherichia coli and bacillus pyocyaneus.The primer for identifying Klebsiella pneumonia of the present invention, comprises primer to K1 or/and primer is to K2；Wherein, K1 includes that K1 F and K1 R, K2 include K2 F and K2 R；SEQ ID NO.1 that described K1 F, K1 R, K2 F, the nucleotide sequence of K2 R are respectively in sequence table, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4.
Description
Technical field
The present invention relates to authentication method and the primer of a kind of Klebsiella pneumonia, belong to biological technical field.
Background technology
Klebsiella pneumonia (K.pnenmoniae) is a kind of G-bacillus, belongs to enterobacteriaceae, conditionality pathogenic bacterium.Lung
Scorching klebsiella has high similarity with escherichia coli in the culture medium such as common Nutrient agar, maconkey agar,
The form of antibacterial, color, abnormal smells from the patient are the most close, and Gram’s staining mirror is all short and small G-dialister bacterium in picking up.On conventional antibacterial
Cultivation is difficult to Klebsiella pneumonia and escherichia coli are distinguished, and does biochemical identification and the 16sRNA qualification need of antibacterial
Want long time, the time of about 3-4 days.If doing 16sRNA qualification to also need to check order, cost is higher.And fluorescence
Real-time quantitative polymerase chain reaction (real time PCR) although technology simplifies operating process, shorten detection time
Between, but needing more complicated quantitative determination instrument, general laboratory is not equipped with.It addition, fur-bearing animal klebsiellosis Pnemoniae
With bronchitis, pneumonia, septicemia, meningitis, peritonitis, diarrhoea waits as cardinal symptom；Also the pneumonia caused with bacillus pyocyaneus
Symptom identical.The mink pneumonia that annual bacillus pyocyaneus and Klebsiella pneumonia cause is countless, in veterinary clinic ten
Divide common.Visible more quickly being distinguished with escherichia coli, bacillus pyocyaneus by Klebsiella pneumonia is highly important.Institute
With, it is badly in need of at present a kind of method wanting fairly simple feasible test in laboratory by Klebsiella pneumonia and escherichia coli, green
Bacillus makes a distinction pus.
Summary of the invention
The present invention obtains two pairs of primers (synthetic gene sequence) by creating work, uses this primer to pneumonia gram
The primary Salmonella of thunder, escherichia coli and bacillus pyocyaneus carry out PCR amplification, and amplified production is the specific sequence of Klebsiella pneumonia；
It is thus possible to Klebsiella pneumonia is distinguished with escherichia coli and bacillus pyocyaneus.
An object of the present invention be to provide a kind of for PCR amplification, can by Klebsiella pneumonia and escherichia coli,
The primer that bacillus pyocyaneus distinguishes.
A kind of primer for identifying Klebsiella pneumonia, comprises primer to K1 or/and primer is to K2；
Wherein, K1 includes that K1-F and K1-R, K2 include K2-F and K2-R；
SEQ ID NO.1, the SEQ ID that the nucleotide sequence of described K1-F, K1-R, K2-F, K2-R is respectively in sequence table
NO.2、SEQ ID NO.3、SEQ ID NO.4。
K1-F is: 5-CGATGCTACTTATCCCGACA-3, as shown in SEQ ID NO.1,
K1-R is: 5-ACCACCAGCAGACGAACTT-3, as shown in SEQ ID NO.2；
K2-F is: 5-CGGAGCGTTTTTCAATCGG-3, as shown in SEQ ID NO.3,
K2-R is: 5-TGAGCGGGTAATAAATGCGG-3, as shown in SEQ ID NO.4.
Described primer can independent packaging to K2 to K1 and described primer, it is possible to pack after mixing.
With above-mentioned primer, testing sample is carried out PCR amplification, obtain and uniquely obtain the PCR amplification that size is 303bp and produce
Thing, sequencing result is as shown in SEQ ID NO.5.Comparison finds after deliberation, and amplified production is Klebsiella pneumonia dissolved blood protein
In gene order wherein one section.Although, dissolved blood protein gene order does not the most possess specificity, but by the amplification of the present invention
Product finds with the gene order contrast of escherichia coli, bacillus pyocyaneus, and amplified production is relative to escherichia coli, the base of bacillus pyocyaneus
Because sequence possesses specificity, it is possible to Klebsiella pneumonia is distinguished with escherichia coli, bacillus pyocyaneus.
Second object of the present invention is to provide one and can be distinguished with escherichia coli, bacillus pyocyaneus by Klebsiella pneumonia
The detectable opened.
The detectable of the present invention, comprises above-mentioned primer；Also comprise PCR reagent.
Described PCR reagent refers in polymerase chain reaction dNTP, Taq archaeal dna polymerase used, MgCl2And PCR
Reaction buffer etc..
The authentication method of Klebsiella pneumonia, carries out PCR amplification by above-mentioned primer or detectable to testing sample,
To pcr amplification product；PCR amplified production is detected by agarose gel electrophoresis or sequencing.
Concrete is: take 4 μ L testing samples, 4 μ L primers to K1 or/and each 2 μ L of primer primer upper and lower to K2() and 25 μ L
PCR reagent, is supplemented to 50 μ L with sterilizing distilled water；The first step, 94 DEG C of denaturations 5min；Second step, 94 DEG C of degeneration 30s；3rd
Step, 58 DEG C of annealing 30s；4th step, 72 DEG C extend 1min；Second step carries out 35 circulations altogether to the 4th step；5th step, 72 DEG C of ends
Extend 7min, obtain pcr amplification product；Take 5ul amplified production through 1.5%(W/V) agarose gel electrophoresis, electrophoresis result is at BIO-
Take a picture on RAD ultraviolet gel imaging system.If band clearly occurring at about 300bp, then explanation testing sample contains
Klebsiella pneumonia.
This method is used for non-diagnostic and therapeutic purposes.
Beneficial effect
The primer of the present invention, relative to escherichia coli, the high specificity of the gene order of bacillus pyocyaneus, it is possible to by pneumonia gram
Primary Salmonella distinguishes thunder with escherichia coli, bacillus pyocyaneus；
Whether primer of the present invention is when existing Klebsiella pneumonia for qualification, highly sensitive, can be detected by pneumonia
The least concentration of klebsiella is 0.0001Mcf；
The authentication method of the present invention, starts to agarose gel electrophoresis from PCR amplification, and the time is about 3 hours, significantly
Save the time, and test procedure is simple to operation, improves detection efficiency.
Accompanying drawing explanation
Fig. 1, the primer of the present invention agarose gel electrophoresis figure to Klebsiella Pneumoniae pcr amplification product；In figure, M:DNA
Maker DL2000,1-7 swimming lane and 9-15 swimming lane are the amplified production of Klebsiella pneumonia, and 8 and 16 swimming lanes are negative control；
1-8 swimming lane pair of primers, 9-16 swimming lane second is to primer；
Fig. 2, the primer of the present invention agarose gel electrophoresis figure to colibacillus PCR amplified production；In figure, M1, M2:DNA
maker DL2000；1-10 swimming lane and 12-21 swimming lane are the 10 colibacillary amplified productions of strain；11 and 22 swimming lanes are negative right
According to；1-11 swimming lane is pair of primers；2-22 swimming lane is second pair of primer；
Fig. 3, the primer of the present invention agarose gel electrophoresis figure to bacillus pyocyaneus pcr amplification product；In figure, M1, M2:DNA
maker DL2000；1-10 swimming lane and 12-21 swimming lane are the 10 colibacillary amplified productions of strain；11 and 22 swimming lanes are negative right
According to；1-11 swimming lane is pair of primers；2-22 swimming lane is second pair of primer；
Fig. 4, primer of the present invention is to Klebsiella Pneumoniae, escherichia coli and the pcr amplification product of bacillus pyocyaneus mixed bacteria liquid
Agarose gel electrophoresis figure；In figure, M:DNA maker DL2000；1-3 swimming lane is that pair of primers detects kerekou pneumonia primary
Salmonella, escherichia coli, the result of three kinds of mixed bacteria liquids of bacillus pyocyaneus；4-6 swimming lane is that pair of primers detects e coil k 1 pneumonia
Bacterium, the result of two kinds of mixed bacteria liquids of bacillus pyocyaneus；7-9 swimming lane is that pair of primers detects Klebsiella pneumonia, escherichia coli
The result of two kinds of mixed bacteria liquids；10 swimming lanes are positive control, the result of detection Klebsiella pneumonia；11 swimming lanes are negative right
According to；12-14 swimming lane is second pair of primer detection Klebsiella pneumonia, escherichia coli, the knot of three kinds of mixed bacteria liquids of bacillus pyocyaneus
Really；15-17 swimming lane is second pair of primer detection Klebsiella pneumonia, the result of two kinds of mixed bacteria liquids of bacillus pyocyaneus；18-20 swims
Road is second pair of primer detection Klebsiella pneumonia, the result of two kinds of mixed bacteria liquids of escherichia coli；21 swimming lanes are positive control,
The result of detection Klebsiella pneumonia；22 swimming lanes are negative control；
Fig. 5, the primer sensitivity tests result to Klebsiella Pneumoniae (single bacterium colony)；In figure, M:DNA maker
DL2000,1-7 swimming lane, 9-15 swimming lane are kerekou pneumonia primary, and size is 300bp, and 8 and 16 swimming lanes are negative control.1-8 swimming lane
Pair of primers, 9-16 swimming lane second is to primer；
Fig. 6, primer is to the K1 sensitivity tests result to Klebsiella Pneumoniae；In figure, M:DNA maker DL2000,1,
2 swimming lane bacterium solution masterplate concentration are 0.002Mcf, and 3,4 swimming lane bacterium solution masterplate concentration are 0.001Mcf, 5,6 swimming lane bacterium solution masterplate concentration
For 0.0001Mcf, 7 swimming lane bacterium solution masterplate concentration be 0.1Mcf as positive control, 8 swimming lanes are negative control；
Fig. 7, primer is to the K2 sensitivity tests result to Klebsiella Pneumoniae；In figure, M:DNA maker DL2000,1,
2 swimming lane bacterium solution masterplate concentration are 0.002Mcf, and 3,4 swimming lane bacterium solution masterplate concentration are 0.001Mcf, 5,6 swimming lane bacterium solution masterplate concentration
For 0.0001Mcf, 7 swimming lane bacterium solution masterplate concentration be 0.1Mcf as positive control, 8 swimming lanes are negative control；
In Fig. 1-7, M swimming lane, from top to bottom the length of band be respectively 2000bp, 1000bp, 750bp, 500bp,
250bp、100bp；It is distilled water that negative control refers to masterplate.
Detailed description of the invention
Experimental technique used in following embodiment if no special instructions, is conventional method.
Material used in following embodiment, reagent etc., if no special instructions, the most commercially obtain.
In following embodiment, the seven strain Klebsiella Pneumoniaes (K.pnenmoniae) that this laboratory preserves, it is recorded in 2012
Year January in July, 2014 is isolated from the fur-bearing animal (mainly mink) of morbidity；Now it is saved in Shandong Province's animal epidemic disease
Sick prevention and control centre.
The 10 strain escherichia coli (E.coli) that this laboratory preserves, are recorded in January, 2012 in July, 2014 from morbidity
Isolated in fur-bearing animal (mainly mink)；Now it is saved in Shandong Animal Disease Prevention and Control Center.
The 10 strain bacillus pyocyaneus (Pseudomonas aeruginosa) that this laboratory preserves, are recorded in January, 2012 extremely
In July, 2014 is isolated from the fur-bearing animal (mainly mink) of morbidity；Now it is saved in Shandong Province's animal epidemic prevention
With control centre.
Embodiment 1
Synthetic primer is to K1, K2；
K1:
K1-F is: 5-CGATGCTACTTATCCCGACA-3, as shown in SEQ ID NO.1,
K1-R is: 5-ACCACCAGCAGACGAACTT-3, as shown in SEQ ID NO.2；
K2:
K2-F is: 5-CGGAGCGTTTTTCAATCGG-3, as shown in SEQ ID NO.3,
K2-R is: 5-TGAGCGGGTAATAAATGCGG-3, as shown in SEQ ID NO.4.
Embodiment 2
Klebsiella Pneumoniae is recovered on ordinary nutrient agar, choose single bacterium colony, shake bacterium overnight, by bacterium solution as masterplate
Carry out PCR reaction.PCR reaction system: 2 × Taq PCR MasterMix 25 upper and lower primer of μ L, K1-F and K1-R() each 2 μ L
(or each 2 μ L of K2-F and K2-R), are masterplate 4 μ L by bacterium solution, are finally supplemented to 50 μ L with sterilizing distilled water.Reaction condition is:
The first step, denaturation 5min under the temperature conditions of 94 DEG C；Second step, degeneration 30s under the temperature conditions of 94 DEG C；3rd step,
Anneal under the temperature conditions of 58 DEG C 30s；4th step, extends 1min under the temperature conditions of 72 DEG C；Second step is to the 4th step altogether
Carry out 35 circulations；5th step: extend 7min under the temperature conditions of 72 DEG C eventually；PCR amplification terminates.Amplified production is checked order,
Shown in its sequence equal SEQ ID NO.5, for the specific gene sequences of Klebsiella pneumonia.Visible, this primer can be by pneumonia
Klebsiella detects.Wherein, 2 × Taq PCR MasterMix；Comprise Taq archaeal dna polymerase, dNTPs, MgCl2, reaction
The reinforcing agent of buffer, PCR reaction and optimize agent and stabilizer, concentration is 2 ×；It is purchased from day root biochemical technology (Beijing) limited
Company, catalog number is KT201.
Replica test:
Specific test to Klebsiella pneumonia
7 Klebsiella pneumoniaes preserved by this laboratory are respectively adopted embodiment 2 method and carry out pcr amplification reaction
(masterplate of PCR reaction is single bacterium colony).After pcr amplification reaction terminates, take 5ul pcr amplification product respectively through 1.5%(W/V)
Agarose gel electrophoresis, electrophoresis result is taken a picture on BIO-RAD ultraviolet gel imaging system；Result is as it is shown in figure 1, at 303bp
All there is clear band in place.Checking order amplified production, its sequence is all as shown in SEQ ID NO.5, for Klebsiella pneumonia
Specific gene sequences.Visible, determination of Klebsiella pneumoniae can be arrived by this primer.
Embodiment 3
To colibacillary specific test
The 10 strain escherichia coli preserved by this laboratory are respectively adopted the method for embodiment 2 and carry out pcr amplification reaction (PCR
Masterplate be single bacterium colony).After pcr amplification reaction terminates, take 5ul pcr amplification product respectively through 1.5%(W/V) agarose coagulate
Gel electrophoresis, electrophoresis result is taken a picture on BIO-RAD ultraviolet gel imaging system；Result is not as in figure 2 it is shown, go out at 303bp
Existing band.Visible, E. coli detection can not be arrived by this primer.
Embodiment 4
Specific test to bacillus pyocyaneus
The 10 strain escherichia coli preserved by this laboratory are respectively adopted the method for embodiment 2 and carry out pcr amplification reaction (PCR
Masterplate be single bacterium colony).After pcr amplification reaction terminates, take 5ul pcr amplification product respectively through 1.5%(W/V) agarose coagulate
Gel electrophoresis, electrophoresis result is taken a picture on BIO-RAD ultraviolet gel imaging system；Result is not as it is shown on figure 3, all have at 303bp
Band occurs.Visible, bacillus pyocyaneus can not be detected by this primer.
Embodiment 5
Klebsiella pneumonia (strain), escherichia coli (strain) and the escherichia coli (strain) that this laboratory is preserved point
Do not recover on ordinary nutrient agar, choose single bacterium colony, shake bacterium overnight.Three kinds of bacterium solution mixing, any two kinds of bacterium solution are mixed it
After as masterplate, use the method for embodiment 2 to carry out pcr amplification reaction.After pcr amplification reaction terminates, take 5ul PCR amplification and produce
Thing is through 1.5%(W/V) agarose gel electrophoresis, electrophoresis result is taken a picture on BIO-RAD ultraviolet gel imaging system；Result such as Fig. 4
Shown in.Any one strain in 6 Klebsiella pneumoniaes additionally is big with any one strain in other 9 strain escherichia coli and 9 strains
Enterobacteria, carries out pcr amplification reaction as masterplate, the method for employing embodiment 2 after using aforesaid way mixing.PCR amplification is anti-
After should terminating, take 5ul pcr amplification product through 1.5%(W/V) agarose gel electrophoresis, electrophoresis result is at BIO-RAD ultraviolet gel
Take a picture in imaging system；Result is identical with Fig. 4.Visible, these two pairs of primers have good specificity to Klebsiella pneumonia,
Escherichia coli the biggest with obscuring degree for Klebsiella pneumonia and bacillus pyocyaneus can be distinguished.
Embodiment 6
Sensitivity tests:
(1) Klebsiella pneumonia 7 strain is drawn on ordinary nutrient agar, take single bacterium colony and make the masterplate of PCR reaction, its
His reaction system is constant, is finally supplemented to 50 μ L with sterilizing distilled water.Reaction condition is with replica test and specific test.Can
Seeing, single bacterium colony just can detect with this primer, illustrates that this primer sensitivity is good.Result such as Fig. 5；
(2) the single bacterium colony of the Klebsiella pneumonia having cultivated 12 hours is taken, in 3ml 85%Nacl normal saline,
Bacterial concentration (wheat formula concentration) is adjusted to 0.1 Mcf, and carries out 50 times on this basis, 100 times, 1000 times of dilutions, the dilutest
Bacterial concentration after releasing is 0.002Mcf, 0.001Mcf, 0.0001Mcf.Respectively take its 4ul and do the masterplate of PCR reaction.Other PCR
Reaction condition and reaction system are with embodiment 2.Wherein, primer to the sensitivity tests result of K1 as shown in Figure 6, primer is to K2's
Sensitivity tests result is as shown in Figure 7.Visible, when bacterial concentration is 0.0001Mcf still seen from band clearly, illustrate that this draws
The sensitivity of thing is higher.
<110>Shandong Animal Disease Prevention and Control Center
<120>authentication method of Klebsiella pneumonia and the primer
<160>5
<210>1
<211>20
<212>DNA
<213>synthetic
<400>1
CGATGCTACT TATCCCGACA 20
<210>2
<211>19
<212>DNA
<213>artificial sequence
<400>2
ACCACCAGCA GACGAACTT 19
<210>3
<211>19
<212>DNA
<213>artificial sequence
<400>3
CGGAGCGTTT TTCAATCGG 19
<210>4
<211>20
<212>DNA
<213>artificial sequence
<400>4
TGAGCGGGTA ATAAATGCGG 20
<210>5
<211>303
<212>DNA
<213>artificial sequence
<400>5
CGATGCTACT TATCCCGACA GCCCGGAGCG TTTTTCAATC GGCGCGCCGC TGGGGCGAGG 60
TTTACGTCTC AACCGGCTGG GGATCCACCA CGAGCGGCTG CCGCCCGGGC GGCGCACCTC 120
TTATCCACAC GCGGAGAGCG ATGAGGAAGA GTTCATCTAC GTGCTGGAGG GCTATCCGGA 180
AGTGTGGATA AACGGCTATC TCTGGAAGCT GGAGCCCGGC GACAGCGTGG GTTTTCCCGC 240
TGGTACCGGC ATCTGCCACA CCTTTCTCAA TAACACCGAG CAGGAAGTTC GTCTGCTGGT 300
GGT 303
Claims (6)
1. the primer being used for identifying Klebsiella pneumonia, it is characterised in that comprise primer to K1 or/and primer is to K2；
Wherein, K1 includes that K1-F and K1-R, K2 include K2-F and K2-R；
SEQ ID NO.1, the SEQ ID that the nucleotide sequence of described K1-F, K1-R, K2-F, K2-R is respectively in sequence table
NO.2、SEQ ID NO.3、SEQ ID NO.4。
2. the detectable being used for identifying Klebsiella pneumonia, it is characterised in that comprise the primer described in claim 1
To K1 or/and primer is to K2.
Detectable the most according to claim 2, it is characterised in that also comprise PCR reagent.
Detectable the most according to claim 3, it is characterised in that described PCR reagent comprises in polymerase chain reaction
Used dNTP, Taq archaeal dna polymerase, MgCl2With PCR reaction buffer.
5. the authentication method of the Klebsiella pneumonia of a non-diseases diagnostic purpose, it is characterised in that with described in claim 1
Primer or the detectable of claim 2,3 or 4 testing sample is carried out PCR amplification, obtain pcr amplification product；Use agar
Sugar gel electrophoresis or sequencing detection pcr amplification product.
Authentication method the most according to claim 5, it is characterised in that take 4 μ L testing samples, 4 μ L primers to K1 or/and draw
Thing, to K2 and 25 μ L PCR reagent, is supplemented to 50 μ L with sterilizing distilled water；The first step, 94 DEG C of denaturations 5min；Second step, 94 DEG C
Degeneration 30s；3rd step, 58 DEG C of annealing 30s；4th step, 72 DEG C extend 1min；Second step carries out 35 circulations altogether to the 4th step；
5th step, 72 DEG C extend 7min eventually, obtain pcr amplification product；Take 5 μ l amplified productions to tie through 1.5% agarose gel electrophoresis, electrophoresis
Fruit is taken a picture on BIO-RAD ultraviolet gel imaging system.
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| CN110499374A (en) * | 2019-08-12 | 2019-11-26 | 山东农业大学 | A kind of triple PCR primer sets, kit and application detecting Klebsiella Pneumoniae |
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| CN104762392A (en) * | 2015-04-09 | 2015-07-08 | 山东省动物疫病预防与控制中心 | Triple fluorogenic quantitative PCR detection method of escherichia coli, Klebsiella pneumoniae and acinetobacter |
| CN104946762B (en) * | 2015-06-17 | 2018-02-06 | 中生北控生物科技股份有限公司 | Detect the kit of Friedlander's bacillus |
| CN105112505A (en) * | 2015-07-23 | 2015-12-02 | 南开大学 | Nucleotides with specificity on Klebsiella K44, K59, K61, and K63 serotypes, and applications thereof |
| CN105132555B (en) * | 2015-09-08 | 2018-05-29 | 重庆文理学院 | The PCR detection kit and detection method of a kind of pneumonia infection Klebsiella of Chinese giant salamander, Andrias davidianus |
| CN107365869B (en) * | 2017-09-08 | 2021-03-19 | 江苏农林职业技术学院 | Method and primer for detecting Klebsiella pneumoniae by loop-mediated isothermal amplification technology |
| CN108531629A (en) * | 2018-05-31 | 2018-09-14 | 广西壮族自治区兽医研究所 | A kind of PCR amplification primer of quick detection Friedlander's bacillus and its application |
| CN112143821A (en) * | 2020-09-30 | 2020-12-29 | 北京师范大学 | Reagent for detecting candida albicans, cryptococcus neoformans and klebsiella pneumoniae by FRET and application thereof |
| CN113621722A (en) * | 2021-08-17 | 2021-11-09 | 汉唐和元(武汉)科技有限公司 | Porcine Klebsiella pneumoniae detection kit and application thereof |
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