CN106609305A - Primers and method for detection of enterobacter cloacae O21 type - Google Patents
Primers and method for detection of enterobacter cloacae O21 type Download PDFInfo
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- CN106609305A CN106609305A CN201510713266.5A CN201510713266A CN106609305A CN 106609305 A CN106609305 A CN 106609305A CN 201510713266 A CN201510713266 A CN 201510713266A CN 106609305 A CN106609305 A CN 106609305A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- 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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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
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Abstract
The invention discloses primers and a method for detection of enterobacter cloacae O21 type, wherein the primers comprise a forward primer 5'-TGTCTTGACCGCCTAT-3' and a reverse primer of 5'-AAACCGAATGATAAGC-3'. During the detection, DNA of a sample is extracted, and the PCR amplification is performed by using the primers, and the amplified product is detected by 1% agarose gel electrophoresis. The detection method has the advantages of short required time, high specificity and high sensitivity for detecting the enterobacter cloacae O21 type, and the disadvantages of complicated operation, long time consuming, low accuracy, low detection rate and the like of a traditional identification method are avoided.
Description
Technical field
The present invention relates to technical field of biological, and in particular to a kind of primer and method of detection enterobacter cloacae O21 types.
Background technology
Enterobacter cloacae (Enterobacter cloacae) is widely present in nature as a kind of Gram-negative tubbiness bacillus
In boundary, can detect in the feces of soil, plant, insecticide and humans and animals, be the normal strain of humans and animals intestinal it
One, while it is also a kind of important conditioned pathogen.In recent years, due to third and fourth generation cephalosporin, carbon green grass or young crops enzyme alkene
The super extensive pedigree antibiotic such as class widely using clinically, the Resistant strain of the bacterium is increasing so that clinically to this
The treatment of microbial infectious disease tends to complicating.The main cause of the appearance of Resistant strain was the failure in the very first time
Pathogen is distinguished, in the case of not clear pathogen, Drug abuse.Since the eighties, there is document report successively to the moon
Enterobacter cloacae carries out Serotypes, bacteriophage typing, bacteriocin typing, and the means such as biotype typing.But more than
Classifying method can only be used in combination, and due to being used alone, a kind of repeatability of method typing is undesirable and typing ability is weak, no
Well enterobacter cloacae can be carried out into accurate typing.Therefore, clinically in the urgent need to a kind of fast and accurate detection handss
Section, recognizes pathogen, so as to carry out immunotherapy targeted autoantibody.
The content of the invention
It is an object of the invention to overcome the deficiencies in the prior art, there is provided a kind of detection for enterobacter cloacae O21 types is drawn
Thing and method.The method can be used for the PCR detections of enterobacter cloacae O21 types, low cost short with detection time, inspection
Survey result specificity high, as a result easily judge, it is practical.
The technical purpose of the present invention is realized by following technical scheme.
A kind of primer of detection enterobacter cloacae O21 types, by forward primer 5 '-TGTCTTGACCGCCTAT -3 '
Constitute with reverse primer 5 '-AAACCGAATGATAAGC -3 '.SEQ ID No.1 and SEQ ID i.e. in sequence table
The nucleotide sequence of No.2.
Application of the above-mentioned primer in detection enterobacter cloacae O21 types, that is, the method for detecting enterobacter cloacae O21 types is first
The DNA of detected sample is first extracted, and with sample DNA as template, using by forward primer
What 5 '-TGTCTTGACCGCCTAT -3 ' and reverse primer 5 '-AAACCGAATGATAAGC -3 ' were constituted draws
Whether thing enters performing PCR amplification, then pcr amplification product is entered into row agarose gel electrophoresis detection, contain in judgement sample
There are enterobacter cloacae O21 types.
In above-mentioned detection method, if electrophoresis result single band occurs in 264bp positions, illustrate to contain in sample
Enterobacter cloacae O21 types；Conversely, then not containing enterobacter cloacae O21 types in sample.
In above-mentioned detection method, the reaction system for entering performing PCR amplification is the reaction system of 30 μ L, specially：10×
PCR Buffer (contain Mg2+)2.5μL；The μ L of dNTP 1 of 2.5mmol/L；Taq enzyme 1.5-2.25U；10
The primer of μm ol/L (detects the primer of enterobacter cloacae O21 types, by forward primer
What 5 '-TGTCTTGACCGCCTAT -3 ' and reverse primer 5 '-AAACCGAATGATAAGC -3 ' were constituted draws
Thing to) 0.1-0.3 μ L；The μ L of sample DNA (i.e. template DNA) 1 of 10-300ng/ μ L；Finally with sterilizing ultra-pure water
Mend to 30 μ L.
In above-mentioned detection method, when entering performing PCR amplification, PCR response procedures are：95 DEG C of 5min, 1 circulation；94
DEG C 30s, 58 DEG C of -65 DEG C of 45s, 68 DEG C of 1min, totally 35 circulations；72 DEG C of 7min, 1 circulation；4 DEG C of preservations.
In above-mentioned detection method, 10 μ L pcr amplification products carry out 1% agarose gel electrophoresiies detection, detection when from
Detection is sampled in pcr amplification product, i.e. the reaction system of PCR amplifications is PCR detection system.
Compared with prior art, the invention has the advantages that：Using the detection method detection enterobacter cloacae O21 of the present invention
Short, high specificity, sensitivity are high the time required to type.Present invention, avoiding cumbersome, time-consuming using conventional identification method
Long, accuracy is low, the low shortcoming of recall rate.Meanwhile, instrument and equipment, reagent involved in the present invention etc. are more commonly used,
General laboratories can carry out detection work, and practicality is higher.The target spot that the present invention is detected has unicity, examines
Survey result special, it is easy to judge.
Description of the drawings
Fig. 1 is PCR detection method specificity assessment preformed gel electrophoresis result figure in embodiment.
Fig. 2 is PCR detection method sensitivity evaluation preformed gel electrophoresis result figure in embodiment.
Fig. 3 is clinical sample detection Gel electrophoresis results figure in embodiment.
Specific embodiment
Technical scheme is further illustrated with reference to specific embodiment.The reality of unreceipted actual conditions in embodiment
Applying method, generally according to normal condition such as Sambrook equimoleculars clone, laboratory manual (New York:Cold Spring
Habor Laboratory Press, 1989) described in condition, or according to the condition proposed by manufacturer.
Experiment reagent
| Distilled water | Milli-Q systems, Millipore Co., USA |
| 10 × PCR Buffer (contain Mg2+) | Precious biological engineering (Dalian) company limited |
| The dNTP of 2.5mmol/L | Precious biological engineering (Dalian) company limited |
| Taq enzyme | Precious biological engineering (Dalian) company limited |
| DL1000 molecular weight standards | Precious biological engineering (Dalian) company limited |
| 100bp DNA Ladder molecular weight standards | Precious biological engineering (Dalian) company limited |
| 1% agarose gel | Genview Co.,USA |
Experimental apparatus
| Constant-temperature table | Shanghai Shiping Experiment Equipment Co., Ltd. |
| Spectrophotometer | German Eppendorf companies, BioPhotometer |
| Gel imaging system | Shanghai Tanon companies, GIS 2010 |
| Ultra-pure water instrument | Millipore Co.,USA |
| Centrifuge | Hunan company of Xiang Yi Laboratory Instruments development corporation, Ltd. H1650-W |
Experimental strain
| Bacterium name | Source | Strain original number |
| Enterobacter cloacae Enterobacter cloacae O21 strains | NCTC | 11590 |
| Enterobacter cloacae Enterobacter cloacae O1 strains | NCTC | 11570 |
| Escherichia coli Escherichia coli O157 strains | CMCC | 44752-5 |
| The typhi O67 strains of Salmonella Salmonella | CMCC | 50071-9 |
| Shigella dysenteriae Shigella dysenteriae O1 strains | CMCC | 51491 |
| Shigella bogdii Shigella boydii O2 strains | CMCC | C 2 |
CMCC：Chinese medicine Microbiological Culture Collection administrative center, i.e. Chinese medicine antibacterial preservation administrative center, Beijing east
City the Temple of Heaven West 2；NCTC：National Collection of Type Cultures,Central Public Health
State-run standard strain collecting center of Laboratory, London, United Kingdom Britain.
Step one, design is directed to the specific primer pair of enterobacter cloacae O21 types, and by Invitrogen Invitrogens (Shanghai)
Trade Co., Ltd synthesizes.
Primer sequence is：Forward primer：5’—TGTCTTGACCGCCTAT—3’；
Reverse primer：5’—AAACCGAATGATAAGC—3’.
It is prepared by step 2, DNA profiling
Enterobacter cloacae O21 types are inoculated in into 5mL nutrient broths fluid medium, and (preparation method refers to J.Sambrook
Deng《Molecular Cloning:A Laboratory guide (second edition)》Page 909) in, concussion and cultivate 12h increases bacterium in 35 DEG C of constant-temperature tables
Afterwards, 1mL bacterium solutions are taken, in being put into 1.5mL sterile centrifugation tubes；12000r/min is centrifuged 15min, abandons most supernatant, plus
Enter 500 μ L sterilizing ultra-pure waters, gently blown and beaten with pipettor, resuspended thalline, 12000r/min centrifugation 15min are abandoned on to the greatest extent
Clear liquid, collects antibacterial；100 μ L sterilizing ultra-pure waters are added, is gently blown and beaten with pipettor, resuspended thalline, in being placed in boiling water
15min is boiled, is taken out immediately, at -20 DEG C 30min is placed.Subsequent 35 DEG C of defrostings, 12000r/min centrifugation 15min, take
Supernatant places 4 DEG C of standby or -20 DEG C of preservations.
Step 3, enterobacter cloacae O21 type specificities PCR detection method is set up
PCR detection system is 30 μ L (entering the reaction system of performing PCR amplification), and reaction system is specially：10×PCR
Buffer (contains Mg2+) 2.5 μ L, the μ L of dNTP 1 of 2.5mmol/L, Taq enzyme 1.5U, the primer pair of 10 μm of ol/L
The μ L of template DNA 1 of 0.3 μ L, 100ng/ μ L, are finally mended to 30 μ L with sterilizing ultra-pure water；PCR detects that response procedures are：
95 DEG C of denaturations 5min, 1 circulation；94 DEG C of degeneration 30s, 60 DEG C of annealing 45s, 68 DEG C of extension 1min, carry out 35 and follow
Ring；72 DEG C of extension 7min, 1 circulation；4 DEG C of preservations.
Step 4, PCR amplifications gel electrophoresiss judge
The decision method is specially：10 μ L pcr amplification products are taken, 1% agarose gel electrophoresiies detection, uviol lamp shines
Lower observation electrophoresis result is penetrated, if there is single band in 264bp positions, illustrates to contain enterobacter cloacae O21 in sample
Type；Conversely, then not containing enterobacter cloacae O21 types in sample.
The assessment experiment of PCR detection method specificity
Prepare by above-mentioned DNA profiling and PCR detection method, to the enterobacter cloacae, escherichia coli, the Salmonella that preserve
Bacterium, shigella dysenteriae, Shigella bogdii carry out pcr amplification reaction, and carry out 1% agarose gel to amplified production
Electrophoresis detection, specific detection result is shown in Fig. 1.In figure：Swimming lane M is DL1000 molecular weight standards；Swimming lane 1 is mould
Plate is the negative control of sterilizing ultra-pure water；Swimming lane 2-7 be respectively enterobacter cloacae Enterobacter cloacae O21 strains,
Enterobacter cloacae Enterobacter cloacae O1 strains, escherichia coli Escherichia coli O157 strains, Salmonella
Salmonella typhi O67 strains, shigella dysenteriae Shigella dysenteriae O1 strains, Shigella bogdii Shigella
Boydii O2 strains.Electrophoresis result is only enterobacter cloacae Enterobacter cloacae O21 strains at 264bp in Fig. 1
There is specific band, and there is not specific band in other strains, show for enterobacter cloacae Enterobacter cloacae
The specific detection of O21 strains.
PCR detection method sensitivity evaluation is tested
The sensitivity of PCR detection method (is being carried out by the most low DNA quality that can be detected in each reaction to represent
After PCR amplifications in reaction system sample DNA quality).The extracting genome DNA liquid of pure culture is taken, light splitting is used
Photometric determination DNA concentration, with the gradient dilution (i.e. concentration dilution) that the ultra-pure water that sterilizes carries out different multiples, obtains
DNA (the i.e. enterobacter cloacae Enterobacter of 1000ng/ μ L, 100ng/ μ L, 10ng/ μ L, 1ng/ μ L gradients
Cloacae O21 strains), the DNA of above-mentioned variable concentrations is entered under the same conditions performing PCR amplification (dilute with 1 μ L gradients
Release liquid and enter performing PCR amplification for template), detected through gel electrophoresis amplified production observes Gel electrophoresis results under ultra violet lamp,
As shown in Figure 2.In Fig. 2：Swimming lane M is DL1000 molecular weight standards；Swimming lane 1-4 is enterobacter cloacae O21 patterns
The PCR amplifications of plate amount 1000ng, 100ng, 10ng, 1ng.As shown in Figure 2, detection method of the invention can
The most low DNA quality of detection is 10ng, with preferable sensitivity.
Clinical doubtful bacterial strain detection
The enterobacter cloacae O21 type specificities PCR detection method set up using said method is to from being clinically separated2 plants
Doubtful bacterial strain is detected.Testing result is shown in Fig. 3, and swimming lane M is in figureDL1000Molecular weight standard；Swimming lane1For the moon
Enterobacter cloacae O21 type positive controls, swimming lane 2-3 be 2 plants of doubtful bacterial strains, swimming lane 4 for template be sterilize ultra-pure water the moon
Property control.Understand in figure, the doubtful bacterial strain of 2 plants of clinics is negative result.
Do exemplary description to the present invention above, it should explanation, in the case of the core without departing from the present invention,
Any simple deformation, modification or other skilled in the art can not spend the equivalent of creative work to fall
Enter protection scope of the present invention.
Claims (6)
1. it is a kind of detection enterobacter cloacae O21 types primer, it is characterised in that by forward primer
5 '-TGTCTTGACCGCCTAT -3 ' and reverse primer 5 '-AAACCGAATGATAAGC -3 ' are constituted.
2. the method for detecting enterobacter cloacae O21 types, it is characterised in that extract the DNA of detected sample first, and
With sample DNA as template, using by forward primer 5 '-TGTCTTGACCGCCTAT -3 ' and reverse primer
The primer of the compositions of 5 '-AAACCGAATGATAAGC -3 ' enters performing PCR amplification, then carries out pcr amplification product
Whether agarose gel electrophoresiies are detected, containing enterobacter cloacae O21 types in judgement sample, if electrophoresis result is in 264bp positions
The single band of appearance is put, then illustrates to contain enterobacter cloacae O21 types in sample；Conversely, then not containing cloaca intestinal bar in sample
Bacterium O21 types.
3. method according to claim 2, it is characterised in that the reaction system for entering performing PCR amplification is 30 μ L
Reaction system, specially：10 × PCR Buffer (contain Mg2+)2.5μL；The μ L of dNTP 1 of 2.5mmol/L；Taq
Enzyme 1.5-2.25U；The primer 0.1-0.3 μ L of 10 μm of ol/L, the primer is to detect drawing for enterobacter cloacae O21 types
Thing, by forward primer 5 '-TGTCTTGACCGCCTAT -3 ' and reverse primer
The primer pair of the compositions of 5 '-AAACCGAATGATAAGC -3 '；The μ L of sample DNA 1 of 10-300ng/ μ L；Finally
Mended to 30 μ L with sterilizing ultra-pure water.
4. method according to claim 2, it is characterised in that when entering performing PCR and expanding, PCR response procedures are：
95 DEG C of 5min, 1 circulation；94 DEG C of 30s, 58 DEG C of -65 DEG C of 45s, 68 DEG C of 1min, totally 35 circulations；72 DEG C of 7min,
1 circulation；4 DEG C of preservations.
5. method according to claim 2, it is characterised in that 10 μ L pcr amplification products carry out 1% agarose and coagulate
Gel electrophoresis detect that be sampled detection from pcr amplification product in detection, i.e. the reaction system of PCR amplifications is PCR
Detection system.
6. application of the primer as claimed in claim 1 in detection enterobacter cloacae O21 types.
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| CN106609305B CN106609305B (en) | 2020-02-28 |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102952881A (en) * | 2012-10-25 | 2013-03-06 | 浙江省淡水水产研究所 | Enterobacter cloacae specific PCR (polymerase chain reaction) detection primer |
| CN103305627A (en) * | 2013-07-16 | 2013-09-18 | 中国农业科学院上海兽医研究所 | Escherichia coli O1, O2, O18 and O78 serotype detection kit and detection method thereof |
| CN103305626A (en) * | 2013-07-08 | 2013-09-18 | 江苏省农业科学院 | Primer for detecting escherichia coli pathogenic serum type and detection kit |
| CN103540666A (en) * | 2013-10-22 | 2014-01-29 | 宁波大学 | Multiplex PCR (Polymerase Chain Reaction) detection method of enterobacter cloacae in sewage outlet water body |
| CN107164453A (en) * | 2017-04-25 | 2017-09-15 | 北京农业职业学院 | A kind of escherichia coli of piglets Serotype Identification and its virulence factor detection method |
-
2015
- 2015-10-27 CN CN201510713266.5A patent/CN106609305B/en not_active Expired - Fee Related
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102952881A (en) * | 2012-10-25 | 2013-03-06 | 浙江省淡水水产研究所 | Enterobacter cloacae specific PCR (polymerase chain reaction) detection primer |
| CN103305626A (en) * | 2013-07-08 | 2013-09-18 | 江苏省农业科学院 | Primer for detecting escherichia coli pathogenic serum type and detection kit |
| CN103305627A (en) * | 2013-07-16 | 2013-09-18 | 中国农业科学院上海兽医研究所 | Escherichia coli O1, O2, O18 and O78 serotype detection kit and detection method thereof |
| CN103540666A (en) * | 2013-10-22 | 2014-01-29 | 宁波大学 | Multiplex PCR (Polymerase Chain Reaction) detection method of enterobacter cloacae in sewage outlet water body |
| CN107164453A (en) * | 2017-04-25 | 2017-09-15 | 北京农业职业学院 | A kind of escherichia coli of piglets Serotype Identification and its virulence factor detection method |
Non-Patent Citations (2)
| Title |
|---|
| ANDREI V. FILATOV ET AL.: ""Structure and genetics of the O-antigen of Enterobacter cloacae C6285 containing di-N-acetyllegionaminic acid"", 《CARBOHYDRATE RESEARCH》 * |
| YAN REN ET AL.: ""Complete Genome Sequence of Enterobacter cloacae subsp. cloacae Type Strain ATCC 13047"", 《JOURNAL OF BACTERIOLOGY》 * |
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