CN104531887A - Identification method for Klebsiella pneumoniae and adopted primers - Google Patents
Identification method for Klebsiella pneumoniae and adopted primers Download PDFInfo
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- 241000588747 Klebsiella pneumoniae Species 0.000 title claims abstract description 59
- 238000000034 method Methods 0.000 title claims abstract description 18
- 238000012408 PCR amplification Methods 0.000 claims abstract description 31
- 201000008225 Klebsiella pneumonia Diseases 0.000 claims description 45
- 206010035717 Pneumonia klebsiella Diseases 0.000 claims description 45
- 238000012360 testing method Methods 0.000 claims description 20
- 238000000246 agarose gel electrophoresis Methods 0.000 claims description 14
- 238000004519 manufacturing process Methods 0.000 claims description 13
- 238000001514 detection method Methods 0.000 claims description 10
- 239000003153 chemical reaction reagent Substances 0.000 claims description 8
- 238000001962 electrophoresis Methods 0.000 claims description 7
- 239000000499 gel Substances 0.000 claims description 7
- 238000003384 imaging method Methods 0.000 claims description 7
- 239000012807 PCR reagent Substances 0.000 claims description 6
- 239000012153 distilled water Substances 0.000 claims description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
- 230000001954 sterilising effect Effects 0.000 claims description 4
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 claims description 3
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 claims description 3
- 230000004087 circulation Effects 0.000 claims description 3
- 238000004925 denaturation Methods 0.000 claims description 3
- 230000036425 denaturation Effects 0.000 claims description 3
- 239000011535 reaction buffer Substances 0.000 claims description 3
- 238000012163 sequencing technique Methods 0.000 claims description 3
- 238000000137 annealing Methods 0.000 claims description 2
- 239000002773 nucleotide Substances 0.000 claims description 2
- 125000003729 nucleotide group Chemical group 0.000 claims description 2
- 238000003752 polymerase chain reaction Methods 0.000 claims description 2
- 241000589517 Pseudomonas aeruginosa Species 0.000 abstract description 26
- 108090000623 proteins and genes Proteins 0.000 abstract description 6
- 241000588724 Escherichia coli Species 0.000 abstract description 4
- 108091028043 Nucleic acid sequence Proteins 0.000 abstract 1
- 241000894006 Bacteria Species 0.000 description 59
- 230000009182 swimming Effects 0.000 description 41
- 230000000968 intestinal effect Effects 0.000 description 26
- 239000007788 liquid Substances 0.000 description 19
- 239000013642 negative control Substances 0.000 description 9
- 230000035945 sensitivity Effects 0.000 description 6
- 208000031295 Animal disease Diseases 0.000 description 4
- 241000772415 Neovison vison Species 0.000 description 4
- 206010035664 Pneumonia Diseases 0.000 description 4
- 230000006806 disease prevention Effects 0.000 description 4
- 239000006916 nutrient agar Substances 0.000 description 4
- 239000013641 positive control Substances 0.000 description 4
- 230000001580 bacterial effect Effects 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 108010017384 Blood Proteins Proteins 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000012797 qualification Methods 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- 206010012735 Diarrhoea Diseases 0.000 description 1
- 241000588921 Enterobacteriaceae Species 0.000 description 1
- 238000003794 Gram staining Methods 0.000 description 1
- 201000009906 Meningitis Diseases 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- 206010040047 Sepsis Diseases 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 108700005078 Synthetic Genes Proteins 0.000 description 1
- 241000209140 Triticum Species 0.000 description 1
- 235000021307 Triticum Nutrition 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 206010006451 bronchitis Diseases 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 238000009629 microbiological culture Methods 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 206010034674 peritonitis Diseases 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 238000012207 quantitative assay Methods 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 208000013223 septicemia Diseases 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 239000012745 toughening agent Substances 0.000 description 1
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- 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 relates to an identification method for Klebsiella pneumoniae and adopted primers, and belongs to the field of biotechnology. The two pairs of primers (artificially synthesized gene sequences) are acquired through creative labor and used for conducting PCR amplification on the Klebsiella pneumoniae, escherichia coli and pseudomonas aeruginosa, and an amplified product is a specificity sequence of the Klebsiella pneumoniae. Thus, the Klebsiella pneumoniae can be distinguished from the escherichia coli and the pseudomonas aeruginosa. The primers for identifying the Klebsiella pneumoniae comprise the primer pair K1 or/and the primer pair K2, wherein the primer pair K1 comprises K1-F and K1-R, and the primer pair K2 comprises K2-F and K2-R. Nucleotide sequences of K1-F, K1-R, K2-F and K2-R are SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4 in a sequence table.
Description
Technical field
The present invention relates to a kind of authentication method and the primer 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.Klebsiella pneumonia and intestinal bacteria have high similarity on the substratum such as common nutrient agar medium, maconkey agar, and the form of bacterium, color, smell are all very close, and gramstaining mirror is all short and small G-dialister bacterium in picking up.In the microbial culture of routine, be difficult to Klebsiella pneumonia and intestinal bacteria difference to come, and do the biochemical identification of bacterium and 16sRNA qualification needs long time, the time of about 3-4 days.If do 16sRNA qualification also to need to check order, cost compare is high.And Fluorescence quantitative real-time polymerase chain reaction (real time PCR) is although technology simplifies operating process, shorten the time of detection, need more complicated quantitative assay instrument, general laboratory is not equipped with.In addition, furbearer klebsiellosis Pnemoniae with bronchitis, pneumonia, septicemia, meningitis, peritonitis, diarrhoea waits as cardinal symptom; Identical with the symptom of the pneumonia that Pseudomonas aeruginosa also causes.The mink pneumonia that annual Pseudomonas aeruginosa and Klebsiella pneumonia cause is countless, very common in veterinary clinic.Visible Klebsiella pneumonia and intestinal bacteria, Pseudomonas aeruginosa being distinguished more fast is very important.So, be badly in need of at present wanting a kind of method of fairly simple feasible test in laboratory Klebsiella pneumonia and intestinal bacteria, Pseudomonas aeruginosa to be made a distinction.
Summary of the invention
the present invention obtains two pairs of primers (synthetic gene sequence) by creating work, and adopt this primer pair Klebsiella pneumonia, intestinal bacteria and Pseudomonas aeruginosa to carry out pcr amplification, amplified production is the specific sequence of Klebsiella pneumonia; Thus Klebsiella pneumonia and intestinal bacteria and Pseudomonas aeruginosa can be distinguished.
An object of the present invention is to provide a kind of for pcr amplification, the primer that Klebsiella pneumonia and intestinal bacteria, Pseudomonas aeruginosa can be distinguished.
For the identification of a primer for Klebsiella pneumonia, comprise primer pair K1 or/and primer pair K2;
Wherein, K1 comprises K1-F and K1-R, and K2 comprises K2-F and K2-R;
The nucleotide sequence of described K1-F, K1-R, K2-F, K2-R is respectively SEQ ID NO.1, SEQ ID NO.2 in sequence table, 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 pair K1 and described primer pair K2 can independent packaging, packs after also can mixing.
Carry out pcr amplification with above-mentioned primer pair testing sample, obtain and uniquely obtain the pcr amplification product that size is 303bp, sequencing result is as shown in SEQ ID NO.5.Comparison finds after deliberation, and amplified production is wherein one section in Klebsiella pneumonia dissolved blood protein gene order.Although, dissolved blood protein gene order does not possess specificity usually, but the gene order of amplified production of the present invention and intestinal bacteria, Pseudomonas aeruginosa is contrasted and finds, amplified production possesses specificity relative to the gene order of intestinal bacteria, Pseudomonas aeruginosa, Klebsiella pneumonia and intestinal bacteria, Pseudomonas aeruginosa can be distinguished.
Second object of the present invention is to provide a kind of detection reagent that Klebsiella pneumonia and intestinal bacteria, Pseudomonas aeruginosa can be distinguished.
Detection reagent of the present invention, comprises above-mentioned primer; Also comprise PCR reagent.
Described PCR reagent refers to dNTP, Taq archaeal dna polymerase, MgCl used in polymerase chain reaction
2with PCR reaction buffer etc.
The authentication method of Klebsiella pneumonia, carries out pcr amplification by above-mentioned primer or detection reagent to testing sample, obtains pcr amplification product; PCR amplified production is detected by agarose gel electrophoresis or sequencing.
Concrete is: get 4 μ L testing samples, 4 μ L primer pair K1 or/and each 2 μ L of the upper and lower primer of primer pair K2() and 25 μ L PCR reagent, be supplemented to 50 μ L with sterilizing distilled water; The first step, 94 DEG C of denaturation 5min; Second step, 94 DEG C of sex change 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; Get 5ul amplified production through 1.5%(W/V) agarose gel electrophoresis, electrophoresis result is taken a picture on BIO-RAD ultraviolet gel imaging system.If there is band clearly at about 300bp, then illustrate in testing sample containing Klebsiella pneumonia.
Present method is used for non-diagnostic and therapeutic purpose.
Beneficial effect
Primer of the present invention, relative to the high specificity of the gene order of intestinal bacteria, Pseudomonas aeruginosa, can distinguish Klebsiella pneumonia and intestinal bacteria, Pseudomonas aeruginosa;
Whether for the identification of when there is Klebsiella pneumonia in primer of the present invention, highly sensitive, can detect that the minimum concentration of Klebsiella pneumonia is 0.0001Mcf;
Authentication method of the present invention, to agarose gel electrophoresis from pcr amplification, the time is approximately 3 hours, greatly saves the time, and testing sequence is simple to operation, improves detection efficiency.
Accompanying drawing explanation
Fig. 1, the agarose gel electrophoresis figure of primer pair Klebsiella Pneumoniae pcr amplification product of the present invention; 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 pair of primer;
Fig. 2, the agarose gel electrophoresis figure of primer pair colibacillus PCR amplified production of the present invention; In figure, M1, M2:DNA maker DL2000; 1-10 swimming lane and 12-21 swimming lane are the colibacillary amplified production of 10 strain; 11 and 22 swimming lanes are negative control; 1-11 swimming lane is pair of primers; 2-22 swimming lane is second pair of primer;
Fig. 3, the agarose gel electrophoresis figure of primer pair Pseudomonas aeruginosa pcr amplification product of the present invention; In figure, M1, M2:DNA maker DL2000; 1-10 swimming lane and 12-21 swimming lane are the colibacillary amplified production of 10 strain; 11 and 22 swimming lanes are negative control; 1-11 swimming lane is pair of primers; 2-22 swimming lane is second pair of primer;
Fig. 4, the agarose gel electrophoresis figure of the pcr amplification product of primer pair Klebsiella Pneumoniae of the present invention, intestinal bacteria and Pseudomonas aeruginosa mixed bacteria liquid; In figure, M:DNA maker DL2000; 1-3 swimming lane is the result that pair of primers detects Klebsiella pneumonia, intestinal bacteria, Pseudomonas aeruginosa three kinds of mixed bacteria liquids; 4-6 swimming lane is the result that pair of primers detects Klebsiella pneumonia, Pseudomonas aeruginosa two kinds of mixed bacteria liquids; 7-9 swimming lane is the result that pair of primers detects Klebsiella pneumonia, intestinal bacteria two kinds of mixed bacteria liquids; 10 swimming lanes are positive control, detect the result of Klebsiella pneumonia; 11 swimming lanes are negative control; 12-14 swimming lane is the result that second pair of primer detects Klebsiella pneumonia, intestinal bacteria, Pseudomonas aeruginosa three kinds of mixed bacteria liquids; 15-17 swimming lane is the result that second pair of primer detects Klebsiella pneumonia, Pseudomonas aeruginosa two kinds of mixed bacteria liquids; 18-20 swimming lane is the result that second pair of primer detects Klebsiella pneumonia, intestinal bacteria two kinds of mixed bacteria liquids; 21 swimming lanes are positive control, detect the result of Klebsiella pneumonia; 22 swimming lanes are negative control;
Fig. 5, the sensitivity test result of primer pair Klebsiella Pneumoniae (single bacterium colony); In figure, M:DNA maker DL2000,1-7 swimming lane, 9-15 swimming lane are kerekou pneumonia the 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 pair of primer;
Fig. 6, primer pair K1 are to the sensitivity test result of Klebsiella Pneumoniae; In figure, M:DNA maker DL2000,1,2 swimming lane bacterium liquid masterplate concentration are 0.002Mcf, 3,4 swimming lane bacterium liquid masterplate concentration are 0.001Mcf, 5,6 swimming lane bacterium liquid masterplate concentration are 0.0001Mcf, 7 swimming lane bacterium liquid masterplate concentration be 0.1Mcf as positive control, 8 swimming lanes are negative control;
Fig. 7, primer pair K2 are to the sensitivity test result of Klebsiella Pneumoniae; In figure, M:DNA maker DL2000,1,2 swimming lane bacterium liquid masterplate concentration are 0.002Mcf, 3,4 swimming lane bacterium liquid masterplate concentration are 0.001Mcf, 5,6 swimming lane bacterium liquid masterplate concentration are 0.0001Mcf, 7 swimming lane bacterium liquid masterplate concentration be 0.1Mcf as positive control, 8 swimming lanes are negative control;
In Fig. 1-7, M swimming lane, the length of band is respectively 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp from top to bottom; It is distilled water that negative control refers to masterplate.
Embodiment
The experimental technique used in following embodiment if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
In following embodiment, the seven strain Klebsiella Pneumoniaes (K.pnenmoniae) that this laboratory is preserved, are recorded in separation from the furbearer (mainly mink) of morbidity in January, 2012 to 2014 year July and obtain; Now be kept at Shandong Animal Disease Prevention and Control Center.
The 10 strain intestinal bacteria (E.coli) that this laboratory is preserved, are recorded in separation from the furbearer (mainly mink) of morbidity in January, 2012 to 2014 year July and obtain; Now be kept at Shandong Animal Disease Prevention and Control Center.
The 10 strain Pseudomonas aeruginosas (Pseudomonas aeruginosa) that this laboratory is preserved, are recorded in separation from the furbearer (mainly mink) of morbidity in January, 2012 to 2014 year July and obtain; Now be kept at Shandong Animal Disease Prevention and Control Center.
embodiment 1
Synthetic primer pair 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 and spend the night, bacterium liquid is used as masterplate and carries out PCR reaction.PCR reaction system: 2 × Taq PCR MasterMix 25 μ L, the upper and lower primer of K1-F and K1-R() each 2 μ L of each 2 μ L(or K2-F and K2-R), be masterplate 4 μ L with bacterium liquid, be finally supplemented to 50 μ L with sterilizing distilled water.Reaction conditions is: the first step, denaturation 5min under the temperature condition of 94 DEG C; Second step, sex change 30s under the temperature condition of 94 DEG C; 3rd step, anneal 30s under the temperature condition of 58 DEG C; 4th step, at the temperature condition downward-extension 1min of 72 DEG C; Second step carries out 35 circulations altogether to the 4th step; 5th step: extend 7min eventually under the temperature condition of 72 DEG C; PCR increases end.To amplified production order-checking, shown in its sequence equal SEQ ID NO.5, be the specific gene sequences of Klebsiella pneumonia.Visible, determination of Klebsiella pneumoniae can arrive by this primer.Wherein, 2 × Taq PCR MasterMix; Comprise Taq archaeal dna polymerase, dNTPs, MgCl
2, reaction buffer, PCR reaction toughener and optimize agent and stablizer, concentration is 2 ×; Be purchased from TIANGEN Biotech (Beijing) Co., Ltd., catalog number is KT201.
replica test:
to the specific test of Klebsiella pneumonia
7 Klebsiella pneumoniaes preserved in this laboratory adopt embodiment 2 method to carry out pcr amplification reaction (masterplate of PCR reaction is single bacterium colony) respectively.After pcr amplification reaction terminates, get 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; As shown in Figure 1, all there is clear band at 303bp place in result.To amplified production order-checking, its sequence, all as shown in SEQ ID NO.5, is the specific gene sequences of Klebsiella pneumonia.Visible, determination of Klebsiella pneumoniae can arrive by this primer.
embodiment 3
to colibacillary specific test
The 10 strain intestinal bacteria preserved in this laboratory adopt the method for embodiment 2 to carry out pcr amplification reaction (masterplate of PCR is single bacterium colony) respectively.After pcr amplification reaction terminates, get 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; As shown in Figure 2, not there is band at 303bp place in result.Visible, E. coli detection can not arrive by this primer.
embodiment 4
to the specific test of Pseudomonas aeruginosa
The 10 strain intestinal bacteria preserved in this laboratory adopt the method for embodiment 2 to carry out pcr amplification reaction (masterplate of PCR is single bacterium colony) respectively.After pcr amplification reaction terminates, get 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; As shown in Figure 3, all not there is band at 303bp place in result.Visible, Pseudomonas aeruginosa can not detect by this primer.
embodiment 5
The Klebsiella pneumonia (strain) preserved in this laboratory, intestinal bacteria (strain) and intestinal bacteria (strain) recover respectively on ordinary nutrient agar, choose single bacterium colony, shake bacterium and spend the night.Be used as masterplate by after three kinds of bacterium liquid mixing, any two kinds of bacterium liquid mixing, adopt the method for embodiment 2 to carry out pcr amplification reaction.After pcr amplification reaction terminates, get 5ul pcr amplification product through 1.5%(W/V) agarose gel electrophoresis, electrophoresis result is taken a picture on BIO-RAD ultraviolet gel imaging system; Result as shown in Figure 4.Any strain in 6 Klebsiella pneumoniaes in addition, with any strain in other 9 strain intestinal bacteria and 9 strain intestinal bacteria, be used as masterplate after adopting aforesaid way mixing, adopt the method for embodiment 2 to carry out pcr amplification reaction.After pcr amplification reaction terminates, get 5ul pcr amplification product through 1.5%(W/V) agarose gel electrophoresis, electrophoresis result is taken a picture on BIO-RAD ultraviolet gel imaging system; Result is identical with Fig. 4.Visible, these two pairs of primer pair Klebsiella pneumonia have good specificity, can by Klebsiella pneumonia with obscure the very large intestinal bacteria of degree and Pseudomonas aeruginosa distinguishes.
embodiment 6
sensitivity test:
(1) draw on ordinary nutrient agar by Klebsiella pneumonia 7 strain, get the masterplate that single bacterium colony does PCR reaction, other reaction systems are constant, are finally supplemented to 50 μ L with sterilizing distilled water.Reaction conditions is with replica test and specific test.Visible, single bacterium colony just can detect with this primer, illustrates that this primer susceptibility is good.Result is as Fig. 5;
(2) the single bacterium colony of the Klebsiella pneumonia having cultivated 12 hours is got, in 3ml 85%Nacl physiological saline, bacterial concentration (wheat formula concentration) is adjusted to 0.1 Mcf, and carry out 50 times on this basis, 100 times, 1000 times of dilutions, the bacterial concentration namely after dilution is 0.002Mcf, 0.001Mcf, 0.0001Mcf.Respectively get the masterplate that its 4ul does PCR reaction.Other PCR reaction conditionss and reaction system are with embodiment 2.Wherein, the sensitivity test result of primer pair K1 as shown in Figure 6, the sensitivity test result of primer pair K2 as shown in Figure 7.Visible, the still visible band clearly when bacterial concentration is 0.0001Mcf, illustrates that the susceptibility of this primer is higher.
<110> Shandong Animal Disease Prevention and Control Center
The authentication method of <120> 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 (5)
1. for the identification of a primer for Klebsiella pneumonia, it is characterized in that, comprise primer pair K1 or/and primer pair K2;
Wherein, K1 comprises K1-F and K1-R, and K2 comprises K2-F and K2-R;
The nucleotide sequence of described K1-F, K1-R, K2-F, K2-R is respectively SEQ ID NO.1, SEQ ID NO.2 in sequence table, SEQ ID NO.3, SEQ ID NO.4.
2. for the identification of a detection reagent for Klebsiella pneumonia, it is characterized in that, comprise primer pair K1 or/and primer pair K2.
3. detection reagent according to claim 2, is characterized in that, also comprises PCR reagent.
4. detection reagent according to claim 3, is characterized in that, described PCR reagent comprises dNTP, Taq archaeal dna polymerase, MgCl used in polymerase chain reaction
2with PCR reaction buffer.
5. an authentication method for Klebsiella pneumonia, is characterized in that, carries out pcr amplification, obtain pcr amplification product by the detection reagent of primer according to claim 1 or claim 2,3 or 4 to testing sample; Pcr amplification product is detected by agarose gel electrophoresis or sequencing.
6.authentication method according to claim 5, is characterized in that, gets 4 μ L testing samples, 4 μ L primer pair K1 or/and primer pair K2 and 25 μ L PCR reagent, is supplemented to 50 μ L with sterilizing distilled water; The first step, 94 DEG C of denaturation 5min; Second step, 94 DEG C of sex change 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; Get 5ul amplified production through 1.5% agarose gel electrophoresis, electrophoresis result is taken a picture on BIO-RAD ultraviolet gel imaging system.
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| 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 |
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