CN107419013A - Detect pseudomonas aeruginosa and plcH primer, kit and method in water - Google Patents
Detect pseudomonas aeruginosa and plcH primer, kit and method in water Download PDFInfo
- Publication number
- CN107419013A CN107419013A CN201710579952.7A CN201710579952A CN107419013A CN 107419013 A CN107419013 A CN 107419013A CN 201710579952 A CN201710579952 A CN 201710579952A CN 107419013 A CN107419013 A CN 107419013A
- Authority
- CN
- China
- Prior art keywords
- pseudomonas aeruginosa
- plch
- primer
- pcr
- water
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- 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
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
-
- 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
-
- 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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/16—Primer sets for multiplex assays
Abstract
The invention discloses pseudomonas aeruginosa and plcH primer, kit and method in detection water, the sequence and probe sequence of the primer are respectively：Sense primer P.AF, anti-sense primer P.AR, probe P.AP, sense primer plcHF, anti-sense primer plcHR, probe plcHP.The invention aims to overcome weak point of the prior art, there is provided a kind of primer and component and reasonable mixture ratio, easy to use, detection is quick, accurately, the kit of pseudomonas aeruginosa and plcH genes water is detected suitable for dual ddPCR；Another object of the present invention is to provide a kind of method of pseudomonas aeruginosa and plcH genes in detection water using mentioned reagent box, and this method is easy to operate, quick, and testing result is accurate.
Description
Technical field
Pseudomonas aeruginosa and its important virulence factor alkali protease PlcH pair in water are detected the present invention relates to a kind of
Weight droplet type digital pcr detection kit, the invention further relates to one kind using P. aeruginosa in mentioned reagent box detection drinking water
Bacterium and plcH method.
Background technology
Pseudomonas aeruginosa Pseudomonas aeruginosa are former to claim Pseudomonas aeruginosa.It is extensive in distributed in nature, for soil
One of most common bacterium present in earth.Various water, air, the skin of normal person, respiratory tract and enteron aisle etc. have this bacterium to deposit
.Essential condition existing for this bacterium is moist environment.The bacterium is a kind of common environmental microorganism, because to nutritional requirement not
Height, excel at leveraging various carbon sources and ammonification compound as nitrogen source, so in the environment such as water, soil, food and hospital
It is widely present.The U.S., Canada, Europe, Japan, Brazil and the World Health Organization are to the green pseudomonad content of copper in drinking water
Restriction MPN<3/L must not detect per 250mL.China《National food safety standard packs drinking water》(GB19298-
2014), then provide in 5 water samples, 250mL must not detect pseudomonas aeruginosa in each sample.
In October, 2009,《Natural mineral water》Etc. aqueous phase close national food safety standard successively all by bacterium
Fall this total index to delete.Although this reform has practical significance, objectively so that water manufacturing enterprise simplifies
Disinfectant program, substantially increase the exceeded probability of pseudomonas aeruginosa.In recent years, various regions are even more that frequency shows the green false list of copper in drinking water
The exceeded situation of born of the same parents.
There are some researches show the plcH genes of pseudomonas aeruginosa may play unusual effect, such as by acting on
Cell surface active material significantly affects cell function, changes innate immune response in many ways, plcH is in Human Umbilical Vein Endothelial Cells
Selective apoptosis work.
, quick detection accurate to pseudomonas aeruginosa that the strict control of the drinking water quality of production needs, at the same it is false to verdigris
Monad is propagated risk and assessed.Special primer, probe of the invention by designing pin pseudomonas aeruginosa and plcH genes
Combination, it may be appreciated that in water in the distribution of pseudomonas aeruginosa and bacterial strain plcH genes popularity.
At present, the related many food security standards of drinking water require carries out quantitative detection to pseudomonas aeruginosa,
But the quantitative detection for pseudomonas aeruginosa mainly or after being cultivated by conventional method is counted, but conventional method is deposited
It is cumbersome in process, the shortcomings of cycle is long.It is not only cumbersome and Standard PCR method needs PCR to carry out electrophoresis after expanding, and
Quantitative detection can not be realized.At present, Southern blot and real-time fluorescence quantitative PCR are conventional two kinds of foreign genes copies
Number analytical technology, it is widely used in copy number of foreign gene analysis.But there is also certain defect for both approaches.For example,
Workload is big, the cycle is long when Southern blot methods are analyzed, operation requires that high, accuracy is poor, especially for copying more
The analysis of shellfish gene, it is as a result easily less than normal.Quantitative fluorescent PCR is necessarily dependent upon standard song when analyzing copy number of foreign gene
The gene of line and known copy number, simply a kind of relative quantitation method, and the quality of standard curve is vulnerable to DNA purity, primer
The factors such as concentration, the response inhabitation factor with probe influence；In addition, standard curve must be based on standard substance foundation, and
The limitednumber and expensive price of standard substance are not applied for all research.
Droplet digital pcr is a kind of new absolute quantitation technology of rising in recent years, it be based on single-molecule PCR method come
The nucleic acid quantification counted, it is a kind of method of absolute quantitation.It is main to use the micro- of the popular research field of present analysis chemistry
Stream control or droplet method, the nucleic acid solution after Macrodilution is dispersed in the microreactor or droplet of chip, each reaction
The nucleic acid-templated number of device is less than or equal to 1.So pass through after PCR cycle, there is the reactor of a nucleic acid templates
Fluorescence signal will be provided, does not just have fluorescence signal without the reactor of template.According to relative scale and the volume of reactor,
Can extrapolates the gene copy number of original solution.
But the analysis work report of the copy number based on droplet digital pcr platform is less, for pseudomonas aeruginosa and
The detection of plcH genes, which just more has no, to be reported.The present invention be based on droplet type ddPCR platforms, establish pseudomonas aeruginosa and
Its plcH gene copy number analysis method, result of study carry for the quantitative detection of analysis food security biogenic risks and assumptions
New method and reference have been supplied, has also provided a technical support means for food security quality control.
The content of the invention
The invention aims to overcome weak point of the prior art, there is provided a kind of primer and component and proportioning
Rationally, easy to use, detection is quick, accurately, pseudomonas aeruginosa and plcH genes water is detected suitable for dual ddPCR
Kit；
Another object of the present invention is to provide pseudomonas aeruginosa and plcH bases in a kind of detection water using mentioned reagent box
The method of cause, this method is easy to operate, quick, and testing result is accurate.
In order to achieve the above object, the present invention uses following scheme：
Pseudomonas aeruginosa and plcH gene primers in a kind of water, it is characterised in that the sequence and probe sequence of the primer
Respectively：
Sense primer P.AF:TGGTAGTCCACGCCGTAAA
Anti-sense primer P.AR:CAGACTGCGATCCGGACTACG
Probe P.AP:TCGACCGCCTGGGGAGTACG
Sense primer plcHF：TACTGGTCCTACGAGCCCAA
Anti-sense primer plcHR：CCTGGTTCGGTTCGAGTTCA
Probe plcHP：TCCGCGAGTTCCACGGCAAC
The kit of pseudomonas aeruginosa and plcH genes in a kind of ddPCR detections water, it is characterised in that in the kit
20.0 μ L reaction systems include following components：The μ L of wherein 2 × ddPCR Super Mix 10.0, pseudomonas aeruginosa and plcH are just
Each 0.1-1.0 μ L of reverse primer, each 0.1-1.0 μ L of probe, the μ L of DNA profiling 4.0.
A kind of ddPCR detection pseudomonas aeruginosas and the method for PlcH genes, it is characterised in that comprise the following steps：
A, sample DNA is extracted；
B, each reactive component is added into above-mentioned reaction system, then adds 70.0 μ l mineral oil, droplet is transferred to after mixing
Droplet is automatically generated on generator；
C, caused droplet is carefully transferred completely into 96 hole reaction plate PCR reaction tubes；Again by 96 hole reaction plates in sealer
Instrument upper sealing film, it is placed in regular-PCR instrument and enters performing PCR reaction.
D, droplet fluorescence detector application software is opened, 96 hole reaction plates after PCR reactions are terminated, which are inserted directly into, to be set
It is standby, the PCR response situations per droplet in PCR reaction tubes are detected, the copy of testing gene is finally calculated according to amber pine distribution law
Number.
The method of detection pseudomonas aeruginosa and plcH genes as described above, it is characterised in that PCR is expanded in step B
Response procedures are carried out according to the following steps：
(1) 94 DEG C of pre-degeneration 3min；
(2) 94 DEG C of denaturation 10s, 60 DEG C of annealing 1min, carry out 40 circulations altogether；
(3) 98 DEG C of enzyme heat inactivation 10min；
(4) 4 DEG C stop reaction.
The method of detection pseudomonas aeruginosa and plcH genes as described above, it is characterised in that extract sample described in step A
Product DNA's comprises the following steps that：
50mL water samples 10000rpm is centrifuged 10 minutes, or takes 50mL water samples to grip filter membrane with tweezers by membrane filtration
In 50mL centrifuge tube, with 10000rpm is centrifuged 10 minutes again after 2ml ultrapure waters, supernatant is removed；5mL CTAB are added to carry
Liquid is taken, is fully mixed.65 DEG C of incubation 30min, vibrate frequently；8000rpm room temperatures centrifugation 10min afterwards, takes 1ml supernatants to enter
In 2ml centrifuge tubes；Add after 700 μ L chloroforms and use forced oscillation, 13000rpm centrifugations 10min, the transfer μ L of supernatant 600 are to newly
In 2ml centrifuge tubes；Add 350 μ L NaCl solutions precipitation is suspended, add 350 μ L chloroforms, vortex oscillation is mixed
Even, 13000rpm centrifuges 10min, and the isopropanol that 0.8 times of volume is added after transfer supernatant is used for precipitate nucleic acids, and room temperature is placed
20min, 13000rpm centrifuge 10min, supernatant discarding, add the ethanol solutions of 500 μ L 70% washing precipitation, are dissolved in 50 μ L
In TE solution.
Sensitivity and Specificity is tested in the present invention：
Sequence verification is carried out to positive amplification product using the methods of sequencing, as a result positive amplification Product Sequence is carried out
When Blast compares, sequence with Genbank aim sequence very high homologies.The reference strain genomic DNA of 10 times of dilutions is added
Enter previous reaction system, repeat that there is repeatability very well in experiment display detection sample.Dilute template concentrations logarithm value and
Also it is in good linear relationship R between copy numerical value2≥0.95.Illustrate that this method has preferable accuracy and well steady
It is qualitative.
The present invention compared with prior art, has advantages below:
1) reagent constituents and reasonable mixture ratio of the present invention, easy to use, detection is quick, accurately, is quantified suitable for ddPCR
Detect pseudomonas aeruginosa and plcH genes；
2) detection method simplifies testing process, and need not make standard curve, substantially reduces detection week
Phase, detection time shorten two days or so than tradition culture method of counting；
3) detection method whole process is without using standard curve, and direct with new-generation sequencing slitless connection,
Absolute quantification analysis can perform to gene copy number.
4) digital pcr detecting system is handled by droplet, can greatly reduce the interference of background and matrix, sensitivity
Can be with as little as 1 copy, therefore, the slight change to low concentration mrna concentration carries out detection accurate and that repeatability is good.
5) pseudomonas aeruginosa and plcH genes simultaneously can be achieved in same reaction system using detection method
Precisely detected, it is easy to operate, quick.With preferable industrialization prospect.
Embodiment
The present invention is described further with reference to embodiment：
Embodiment 1
The primer of pseudomonas aeruginosa and plcH genes in present invention detection water, sequence are respectively：
Sense primer P.AF:TGGTAGTCCACGCCGTAAA
Anti-sense primer P.AR:CAGACTGCGATCCGGACTACG
Probe P.AP:TCGACCGCCTGGGGAGTACG
Sense primer plcHF：TACTGGTCCTACGAGCCCAA
Anti-sense primer plcHR：CCTGGTTCGGTTCGAGTTCA
Probe plcHP：TCCGCGAGTTCCACGGCAAC
Embodiment 2
The kit of pseudomonas aeruginosa and plcH genes in a kind of detection water of the present invention, wherein 20 μ L reaction system bags
Include following components：
The μ L of wherein 2 × ddPCR Super Mix 10.0, pseudomonas aeruginosa and each 1.0 μ L of the forward and reverse primers of PlcH, visit
Each 1.0 μ L of pin, the μ L of DNA profiling 4.0.
Wherein primer sequence is as follows:
Sense primer P.AF:TGGTAGTCCACGCCGTAAA
Anti-sense primer P.AR:CAGACTGCGATCCGGACTACG
Probe P.AP:TCGACCGCCTGGGGAGTACG
Sense primer plcHF：TACTGGTCCTACGAGCCCAA
Anti-sense primer plcHR：CCTGGTTCGGTTCGAGTTCA
Probe plcHP：TCCGCGAGTTCCACGGCAAC.
Embodiment 3
The present invention it is a kind of detection water in the kit of pseudomonas aeruginosa and plcH genes, wherein reaction system include with
Lower component：
The μ L of wherein 2 × ddPCR Super Mix 10.0, pseudomonas aeruginosa and each 0.1 μ L of the forward and reverse primers of PlcH, visit
Each 0.1 μ L of pin, the μ L of DNA profiling 4.0.
Wherein primer sequence is as follows:
Sense primer P.AF:TGGTAGTCCACGCCGTAAA
Anti-sense primer P.AR:CAGACTGCGATCCGGACTACG
Probe P.AP:TCGACCGCCTGGGGAGTACG
Sense primer plcHF：TACTGGTCCTACGAGCCCAA
Anti-sense primer plcHR：CCTGGTTCGGTTCGAGTTCA
Probe plcHP：TCCGCGAGTTCCACGGCAAC.
Embodiment 4
Pseudomonas aeruginosa and plcH genetic methods in present invention detection water, comprise the following steps：
A, sample DNA is extracted；
B, the 20.0 μ l reaction systems for adding each reactive component in above-described embodiment 2,70.0 μ l mineral are then added
Oil, it is transferred to after mixing on drop generator and automatically generates droplet；
C, caused droplet is carefully transferred completely into 96 hole reaction plate PCR reaction tubes；96 hole reaction plates are being sealed again
Film instrument upper sealing film, it is placed in regular-PCR instrument and enters performing PCR reaction.
D, droplet fluorescence detector application software is opened, 96 hole reaction plates after PCR reactions are terminated, which are inserted directly into, to be set
It is standby, the PCR response situations per droplet in PCR reaction tubes are detected, the copy of testing gene is finally calculated according to amber pine distribution law
Number.
Described fluorescent PCR amplification is carried out according to the following steps：
(1) 94 DEG C of pre-degeneration 3min；
(2) 94 DEG C of denaturation 10s, 60 DEG C of annealing 1min, carry out 40 circulations altogether；
(3) 98 DEG C of enzyme heat inactivation 10min；
(4) 4 DEG C stop reaction.
Embodiment 5
Pseudomonas aeruginosa and plcH genetic methods in present invention detection water, comprise the following steps：
50mL water samples 10000rpm is centrifuged 10 minutes, or takes 50mL water samples to grip filter membrane with tweezers by membrane filtration
In 50mL centrifuge tube, with 10000rpm is centrifuged 10 minutes again after 2ml ultrapure waters, supernatant is removed；5mL CTAB are added to carry
Liquid is taken, is fully mixed.65 DEG C of incubation 30min, vibrate frequently；8000rpm room temperatures centrifugation 10min afterwards, takes 1ml supernatants to enter
In 2ml centrifuge tubes；Add after 700 μ L chloroforms and use forced oscillation, 13000rpm centrifugations 10min, the transfer μ L of supernatant 600 are to newly
In 2ml centrifuge tubes；Add 350 μ L NaCl solutions precipitation is suspended, add 350 μ L chloroforms, vortex oscillation is mixed
Even, 13000rpm centrifuges 10min, and the isopropanol that 0.8 times of volume is added after transfer supernatant is used for precipitate nucleic acids, and room temperature is placed
20min, 13000rpm centrifuge 10min, supernatant discarding, add the ethanol solutions of 500 μ L 70% washing precipitation, are dissolved in 50 μ L
In TE solution.
The μ L of wherein 2 × ddPCR Super Mix 10.0, pseudomonas aeruginosa and each 1.0 μ L of the forward and reverse primers of plcH, visit
Each 1.0 μ L of pin, the μ L of DNA profiling 4.0.Wherein primer sequence is as follows:
Sense primer P.AF:TGGTAGTCCACGCCGTAAA
Anti-sense primer P.AR:CAGACTGCGATCCGGACTACG
Probe P.AP:TCGACCGCCTGGGGAGTACG
Sense primer plcHF：TACTGGTCCTACGAGCCCAA
Anti-sense primer plcHR：CCTGGTTCGGTTCGAGTTCA
Probe plcHP：TCCGCGAGTTCCACGGCAAC.
Enter performing PCR amplification, carry out according to the following steps：
(1) 94 DEG C of pre-degeneration 3min；
(2) 94 DEG C of denaturation 10s, 60 DEG C of annealing 1min, carry out 40 circulations altogether；
(3) 98 DEG C of enzyme heat inactivation 10min；
(4) 4 DEG C stop reaction.
Embodiment 6
Pseudomonas aeruginosa and plcH genetic methods in present invention detection water, comprise the following steps：
50mL water samples 10000rpm is centrifuged 10 minutes, or takes 50mL water samples to grip filter membrane with tweezers by membrane filtration
In 50mL centrifuge tube, with 10000 rpm are centrifuged 10 minutes again after 2ml ultrapure waters, supernatant is removed；5mL CTAB are added to carry
Liquid is taken, is fully mixed.65 DEG C of incubation 30min, vibrate frequently；8000rpm room temperatures centrifugation 10min afterwards, takes 1ml supernatants to enter
In 2ml centrifuge tubes；Add after 700 μ L chloroforms and use forced oscillation, 13000rpm centrifugations 10min, the transfer μ L of supernatant 600 are to newly
In 2ml centrifuge tubes；Add 350 μ L NaCl solutions precipitation is suspended, add 350 μ L chloroforms, vortex oscillation is mixed
Even, 13000rpm centrifuges 10min, and the isopropanol that 0.8 times of volume is added after transfer supernatant is used for precipitate nucleic acids, and room temperature is placed
20min, 13000rpm centrifuge 10min, supernatant discarding, add the ethanol solutions of 500 μ L 70% washing precipitation, are dissolved in 50 μ L
In TE solution.
The μ L of wherein 2 × ddPCR Super Mix 10.0, pseudomonas aeruginosa and each 0.1 μ L of the forward and reverse primers of plcH, visit
Each 0.1 μ L of pin, the μ L of DNA profiling 4.0.Wherein primer sequence is as follows:
Sense primer P.AF:TGGTAGTCCACGCCGTAAA
Anti-sense primer P.AR:CAGACTGCGATCCGGACTACG
Probe P.AP:TCGACCGCCTGGGGAGTACG
Sense primer plcHF：TACTGGTCCTACGAGCCCAA
Anti-sense primer plcHR：CCTGGTTCGGTTCGAGTTCA
Probe plcHP：TCCGCGAGTTCCACGGCAAC.
Enter performing PCR amplification, carry out according to the following steps：
(1) 94 DEG C of pre-degeneration 3min；
(2) 94 DEG C of denaturation 10s, 60 DEG C of annealing 1min, carry out 40 circulations altogether；
(3) 98 DEG C of enzyme heat inactivation 10min；
(4) 4 DEG C stop reaction.
Interpretation of result
In development process of the present invention, while using the classical culture protocols of culture medium, carry out pseudomonas aeruginosa quantity survey
Fixed control.Test in triplicate, pseudomonas aeruginosa results averaged.As a result show, the present invention uses digital pcr side
Method is to the copy number of pseudomonas aeruginosa in water, the total plate count result coefficient R detected with classical culture protocols2≥
99%.In addition, digital pcr of the present invention is enjoyed oneself to the full, single sample whole process detection time of method is 4 hours, considerably shorter than existing traditional
Flat board culture method of counting, and detect pseudomonas aeruginosa simultaneously, moreover it is possible to quantitative detection, Neng Gouzhun are carried out to its plcH gene
Really evaluation pathogenic bacteria and pathogenic bacteria carry the propagation risk of drug resistant gene, therefore are sent out with good technical advantage and industrialization
Exhibition prospect.The general principle and principal character and advantages of the present invention of the present invention has been shown and described above.The skill of the industry
For art personnel it should be appreciated that the present invention is not limited to the above embodiments, described in above-described embodiment and specification is to say
Bright principle of the invention, without departing from the spirit and scope of the present invention, various changes and modifications of the present invention are possible,
These changes and improvements all fall within the protetion scope of the claimed invention.The claimed scope of the invention will by appended right
Ask book and its equivalent thereof.
SEQUENCE LISTING
<110>Cai is first complete
<120>Detect pseudomonas aeruginosa and plcH primer, kit and method in water
<130>Detect pseudomonas aeruginosa and plcH primer, kit and method in water
<160> 6
<170> PatentIn version 3.3
<210> 1
<211> 19
<212> DNA
<213> Pseudomonas aeruginosa
<400> 1
tggtagtcca cgccgtaaa 19
<210> 2
<211> 21
<212> DNA
<213> Pseudomonas aeruginosa
<400> 2
cagactgcga tccggactac g 21
<210> 3
<211> 20
<212> DNA
<213> Pseudomonas aeruginosa
<400> 3
tcgaccgcct ggggagtacg 20
<210> 4
<211> 20
<212> DNA
<213> Pseudomonas aeruginosa
<400> 4
tactggtcct acgagcccaa 20
<210> 5
<211> 20
<212> DNA
<213> Pseudomonas aeruginosa
<400> 5
cctggttcgg ttcgagttca 20
<210> 6
<211> 20
<212> DNA
<213> Pseudomonas aeruginosa
<400> 6
tccgcgagtt ccacggcaac 20
Claims (5)
1. pseudomonas aeruginosa and drug resistant gene plcH primer in a kind of detection water, it is characterised in that the primer and probe combines
Sequence be respectively：
Sense primer P.AF:TGGTAGTCCACGCCGTAAA
Anti-sense primer P.AR:CAGACTGCGATCCGGACTACG
Probe P.AP:TCGACCGCCTGGGGAGTACG
Sense primer plcHF：TACTGGTCCTACGAGCCCAA
Anti-sense primer plcHR：CCTGGTTCGGTTCGAGTTCA
Probe plcHP：TCCGCGAGTTCCACGGCAAC.
2. pseudomonas aeruginosa and drug resistant gene plcH digital pcr detection kit in a kind of detection water, it is characterised in that should
Reaction system includes following components in kit：The μ L of wherein 2 × ddPCR Super Mix 10.0, pseudomonas aeruginosa and plcH
Each 0.1-1.0 μ L of forward and reverse primer, each 0.1-1.0 μ L of probe, the μ L of DNA profiling 4.0.
A kind of 3. pseudomonas aeruginosa and drug resistant gene plcH method in detection water, it is characterised in that comprise the following steps：
A, detect in water and extract sample DNA；
B, each reactive component is added into reaction system as claimed in claim 2, then adds 70.0 μ l mineral oil, turned after mixing
Move on to and droplet is automatically generated on drop generator；Take positive quality control and the negative Quality Control in the kit respectively simultaneously, according to
With the processing of step A identicals method, corresponding DNA profiling is obtained；
C, digital pcr mixed liquor will be prepared and be made as the micro- reactions of Water-In-Oil PCR, and be transferred completely into 96 hole reaction plate PCR reactions
Guan Zhong；Again by 96 hole reaction plates in sealer instrument upper sealing film, it is placed in regular-PCR instrument and enters performing PCR reaction；
D, droplet fluorescence detector application software is opened, 96 hole reaction plates after PCR reactions are terminated are inserted directly into equipment, detect
The PCR response situations of droplet in per PCR reaction tubes, the copy number of testing gene is finally calculated according to amber pine distribution law.
4. pseudomonas aeruginosa and drug resistant gene plcH method in detection water according to claim 3, it is characterised in that
The response procedures that PCR is expanded in step C are carried out according to the following steps：
(1) 94 DEG C of pre-degeneration 3min；
(2) 94 DEG C of denaturation 10s, 59 DEG C of annealing 1min, carry out 40 circulations altogether；
(3) 98 DEG C of enzyme heat inactivation 10min；
(4) 4 DEG C stop reaction.
5. pseudomonas aeruginosa and drug resistant gene plcH method in detection water according to claim 3, it is characterised in that
Comprising the following steps that for sample DNA is extracted described in step A：
The direct 10000rpm of 50mL water samples founds the heart 10 minutes, or takes 50mL water samples to grip filter membrane with tweezers by membrane filtration
50mL centrifuge tube with after 2ml ultrapure waters again 10000rpm centrifuge 10 minutes, remove supernatant；Add 5mL CTAB extractions
Liquid, fully mix；65 DEG C of incubation 30min, vibrate frequently；8000r/min room temperatures centrifugation 10min afterwards, takes 1ml supernatants to enter 2ml
In centrifuge tube；Forced oscillation, 13000g centrifugations 10min, the transfer μ L of supernatant 600 to new 2ml centrifugations are used after adding 700 μ L chloroforms
Guan Zhong；The CTAB precipitation solutions of 2 times of volumes of addition are reverse to be stored at room temperature 60min afterwards for several times, 13000 × g centrifugation 10min, discards
Supernatant, add 350 μ L NaCl solutions and precipitation is suspended, add 350 μ L chloroforms, vortex oscillation is mixed, 13000g
Centrifuge 10min, shift the isopropanol of 0.8 times of volume is added after supernatant be used for precipitate nucleic acids, room temperature placement 20min, 13000g from
Heart 10min, supernatant discarding, the ethanol solutions of 500 μ L 70% washing precipitation is added, is dissolved in 50 μ L TE solution.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710579952.7A CN107419013A (en) | 2017-07-17 | 2017-07-17 | Detect pseudomonas aeruginosa and plcH primer, kit and method in water |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710579952.7A CN107419013A (en) | 2017-07-17 | 2017-07-17 | Detect pseudomonas aeruginosa and plcH primer, kit and method in water |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107419013A true CN107419013A (en) | 2017-12-01 |
Family
ID=60429907
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710579952.7A Pending CN107419013A (en) | 2017-07-17 | 2017-07-17 | Detect pseudomonas aeruginosa and plcH primer, kit and method in water |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107419013A (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101429539A (en) * | 2007-11-09 | 2009-05-13 | 中山大学达安基因股份有限公司 | Reagent kit for real time fluorescence quantitative PCR detection of bacillus pyocyaneus |
| CN105950732A (en) * | 2016-05-25 | 2016-09-21 | 中国农业大学 | Animal-derived food pathogen identification and drug-resistant and toxic gene detection composite chip |
| CN106048052A (en) * | 2016-07-29 | 2016-10-26 | 中山出入境检验检疫局检验检疫技术中心 | Kit and method for detecting staphylococcus aureus and integron in food |
| CN106520984A (en) * | 2016-12-06 | 2017-03-22 | 湖南圣湘生物科技有限公司 | Pseudomonas aeruginosa nucleic acid fluorescent PCR (polymerase chain reaction) detection kit and detection method |
-
2017
- 2017-07-17 CN CN201710579952.7A patent/CN107419013A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101429539A (en) * | 2007-11-09 | 2009-05-13 | 中山大学达安基因股份有限公司 | Reagent kit for real time fluorescence quantitative PCR detection of bacillus pyocyaneus |
| CN105950732A (en) * | 2016-05-25 | 2016-09-21 | 中国农业大学 | Animal-derived food pathogen identification and drug-resistant and toxic gene detection composite chip |
| CN106048052A (en) * | 2016-07-29 | 2016-10-26 | 中山出入境检验检疫局检验检疫技术中心 | Kit and method for detecting staphylococcus aureus and integron in food |
| CN106520984A (en) * | 2016-12-06 | 2017-03-22 | 湖南圣湘生物科技有限公司 | Pseudomonas aeruginosa nucleic acid fluorescent PCR (polymerase chain reaction) detection kit and detection method |
Non-Patent Citations (2)
| Title |
|---|
| FATEMEH FARAJI等: "Molecular detection of virulence genes in Pseudomonas aeruginosa isolated from children with Cystic Fibrosis and burn wounds in Iran", 《MICROBIAL PATHOGENESIS》 * |
| 宋月等: "绿脓杆菌SYBR Green I 实时定量PCR检测方法的建立及初步应用", 《华北农学报》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107513561A (en) | Detect primer, kit and the method for P. aeruginosa and C.perfringens | |
| CN106048052A (en) | Kit and method for detecting staphylococcus aureus and integron in food | |
| CN104946795B (en) | Primer, probe and kit for Site Detection various serotype foot and mouth disease virus | |
| CN108977529A (en) | It is a kind of to utilize newborn's TRECs and KRECs gene copy number detection kit of digital pcr technology and its application | |
| CN107513562A (en) | Detect pseudomonas aeruginosa and ExoU primer, kit and method in water | |
| CN106086208A (en) | For detecting vibrio parahaemolyticus and the test kit of integron and method in food | |
| CN105112558B (en) | The triple real time fluorescent quantitative RT PCR detection kits of the type of foot and mouth disease virus O, A and Asia I | |
| CN104513857A (en) | Loop-mediated isothermal amplification detection primer group, detection method and kit of vibrio parahaemolyticus | |
| CN101570780A (en) | Detection kit and detection method for brucellae in meat products | |
| CN101565753A (en) | Rapid diagnostic kit for staphylococcus aureus gene based on loop-mediated isothermal amplification technology and detecting method thereof | |
| CN107287328A (en) | Detect primer, kit and the method for pseudomonas aeruginosa and toxA in water | |
| CN100386442C (en) | Prime and probe sequence for detecting nucleotide fregment of 01 Group comma bacillus | |
| CN102978282B (en) | Typhoid fever salmonella and salmonella paratyphi fluorescent quantitative polymerase chain reaction (PCR) detection kit and application thereof | |
| CN104878116A (en) | Quantitative determination method for pathogenic bacteria in food | |
| CN106148534A (en) | For detecting shigella and the test kit of integron and method in food | |
| CN108913770A (en) | It is a kind of to utilize newborn's TRECs and KRECs gene copy number detection kit of digital pcr technology and its application | |
| CN107653331A (en) | Detect pseudomonas aeruginosa and ExoY primer, kit and method in water | |
| CN100395350C (en) | Prime and probe sequence for detecting nucleotide fregment of comma bacillus | |
| CN107447001A (en) | Detect pseudomonas aeruginosa and ExoS primer, kit and method in water | |
| CN100386443C (en) | Primer and probe sequence for detecting nucleotide fragment of 0139 group choleraic vibrio | |
| CN107419013A (en) | Detect pseudomonas aeruginosa and plcH primer, kit and method in water | |
| CN107419012A (en) | Detect pseudomonas aeruginosa and lasB primer, kit and method in water | |
| CN109971886A (en) | A kind of kit for detecting CPV nucleic acid, RPA primer pair, probe and method | |
| CN100395347C (en) | Primer for detecting salmonella nucleotide fragment and probe sequence | |
| CN100395346C (en) | Primer for detecting Listern nucleotide segment of monocellular hyperplasia and probe sequence |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20171201 |